A tandem affinity purification tag of TGA2 for isolation of interacting proteins in Arabidopsis thaliana

PubMed Central

Stotz, Henrik U; Findling, Simone; Nukarinen, Ella; Weckwerth, Wolfram; Mueller, Martin J; Berger, Susanne

2014-01-01

Tandem affinity purification (TAP) tagging provides a powerful tool for isolating interacting proteins in vivo. TAP-tag purification offers particular advantages for the identification of stimulus-induced protein interactions. Type II bZIP transcription factors (TGA2, TGA5 and TGA6) play key roles in pathways that control salicylic acid, ethylene, xenobiotic and reactive oxylipin signaling. Although proteins interacting with these transcription factors have been identified through genetic and yeast 2-hybrid screening, others are still elusive. We have therefore generated a C-terminal TAP-tag of TGA2 to isolate additional proteins that interact with this transcription factor. Three lines most highly expressing TAP-tagged TGA2 were functional in that they partially complemented reactive oxylipin-responsive gene expression in a tga2 tga5 tga6 triple mutant. TAP-tagged TGA2 in the most strongly overexpressing line was proteolytically less stable than in the other 2 lines. Only this overexpressing line could be used in a 2-step purification process, resulting in isolation of co-purifying bands of larger molecular weight than TGA2. TAP-tagged TGA2 was used to pull down NPR1, a protein known to interact with this transcription factor. Mass spectrometry was used to identify peptides that co-purified with TAP-tagged TGA2. Having generated this TGA2 TAP-tag line will therefore be an asset to researchers interested in stimulus-induced signal transduction processes. PMID:25482810

Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures

PubMed Central

2010-01-01

Background Papaver somniferum (opium poppy) is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies. Results A cDNA library was prepared for opium poppy cell cultures treated with a fungal elicitor for 10 h. Using 454 GS-FLX Titanium pyrosequencing, 427,369 expressed sequence tags (ESTs) with an average length of 462 bp were generated. Assembly of these sequences yielded 93,723 unigenes, of which 23,753 were assigned Gene Ontology annotations. Transcripts encoding all known sanguinarine biosynthetic enzymes were identified in the EST database, 5 of which were represented among the 50 most abundant transcripts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of total protein extracts from cell cultures treated with a fungal elicitor for 50 h facilitated the identification of 1,004 proteins. Proteins were fractionated by one-dimensional SDS-PAGE and digested with trypsin prior to LC-MS/MS analysis. Query of an opium poppy-specific EST database substantially enhanced peptide identification. Eight out of 10 known sanguinarine biosynthetic enzymes and many relevant primary metabolic enzymes were represented in the peptide database. Conclusions The integration of deep transcriptome and proteome analyses provides an effective platform to catalogue the components of secondary metabolism, and to identify genes encoding uncharacterized enzymes. The establishment of corresponding transcript and protein databases generated by next-generation technologies in a system with a well-defined metabolite profile facilitates an improved linkage between genes, enzymes, and pathway components. The proteome database represents the most relevant alkaloid-producing enzymes, compared with the much deeper and more complete transcriptome library. The transcript database contained full-length mRNAs encoding most alkaloid biosynthetic enzymes, which is a key requirement for the functional characterization of novel gene candidates. PMID:21083930

Development of novel metabolite-responsive transcription factors via transposon-mediated protein fusion.

PubMed

Younger, Andrew K D; Su, Peter Y; Shepard, Andrea J; Udani, Shreya V; Cybulski, Thaddeus R; Tyo, Keith E J; Leonard, Joshua N

2018-02-01

Naturally evolved metabolite-responsive biosensors enable applications in metabolic engineering, ranging from screening large genetic libraries to dynamically regulating biosynthetic pathways. However, there are many metabolites for which a natural biosensor does not exist. To address this need, we developed a general method for converting metabolite-binding proteins into metabolite-responsive transcription factors-Biosensor Engineering by Random Domain Insertion (BERDI). This approach takes advantage of an in vitro transposon insertion reaction to generate all possible insertions of a DNA-binding domain into a metabolite-binding protein, followed by fluorescence activated cell sorting to isolate functional biosensors. To develop and evaluate the BERDI method, we generated a library of candidate biosensors in which a zinc finger DNA-binding domain was inserted into maltose binding protein, which served as a model well-studied metabolite-binding protein. Library diversity was characterized by several methods, a selection scheme was deployed, and ultimately several distinct and functional maltose-responsive transcriptional biosensors were identified. We hypothesize that the BERDI method comprises a generalizable strategy that may ultimately be applied to convert a wide range of metabolite-binding proteins into novel biosensors for applications in metabolic engineering and synthetic biology. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

The Use of Transcription Terminators to Generate Transgenic Lines of Chinese Hamster Ovary Cells (CHO) with Stable and High Level of Reporter Gene Expression.

PubMed

Gasanov, N B; Toshchakov, S V; Georgiev, P G; Maksimenko, O G

2015-01-01

Mammalian cell lines are widely used to produce recombinant proteins. Stable transgenic cell lines usually contain many insertions of the expression vector in one genomic region. Transcription through transgene can be one of the reasons for target gene repression after prolonged cultivation of cell lines. In the present work, we used the known transcription terminators from the SV40 virus, as well as the human β- and γ-globin genes, to prevent transcription through transgene. The transcription terminators were shown to increase and stabilize the expression of the EGFP reporter gene in transgenic lines of Chinese hamster ovary (CHO) cells. Hence, transcription terminators can be used to create stable mammalian cells with a high and stable level of recombinant protein production.

Transcriptional Profiling of Human Endogenous Retrovirus Group HERV-K(HML-2) Loci in Melanoma

PubMed Central

Schmitt, Katja; Reichrath, Jörg; Roesch, Alexander; Meese, Eckart; Mayer, Jens

2013-01-01

Recent studies suggested a role for the human endogenous retrovirus (HERV) group HERV-K(HML-2) in melanoma because of upregulated transcription and expression of HERV-K(HML-2)-encoded proteins. Very little is known about which HML-2 loci are transcribed in melanoma. We assigned >1,400 HML-2 cDNA sequences generated from various melanoma and related samples to genomic HML-2 loci, identifying a total of 23 loci as transcribed. Transcription profiles of loci differed significantly between samples. One locus was found transcribed only in melanoma-derived samples but not in melanocytes and might represent a marker for melanoma. Several of the transcribed loci harbor ORFs for retroviral Gag and/or Env proteins. Env-encoding loci were transcribed only in melanoma. Specific investigation of rec and np9 transcripts indicated transcription of protein encoding loci in melanoma and melanocytes hinting at the relevance of Rec and Np9 in melanoma. UVB irradiation changed transcription profiles of loci and overall transcript levels decreased in melanoma and melanocytes. We further identified transcribed HML-2 loci formed by reverse transcription of spliced HML-2 transcripts by L1 machinery or in a retroviral fashion, with loci potentially encoding HML-2-like proteins. We reveal complex, sample-specific transcription of HML-2 loci in melanoma and related samples. Identified HML-2 loci and proteins encoded by those loci are particularly relevant for further studying the role of HML-2 in melanoma. Transcription of HERVs appears as a complex mechanism requiring specific studies to elucidate which HERV loci are transcribed and how transcribed HERVs may be involved in disease. PMID:23338945

Comparative vesicle proteomics reveals selective regulation of protein expression in chestnut blight fungus by a hypovirus.

PubMed

Wang, Jinzi; Wang, Fangzhen; Feng, Youjun; Mi, Ke; Chen, Qi; Shang, Jinjie; Chen, Baoshan

2013-01-14

The chestnut blight fungus (Cryphonectria parasitica) and hypovirus constitute a model system to study fungal pathogenesis and mycovirus-host interaction. Knowledge in this field has been gained largely from investigations at gene transcription level so far. Here we report a systematic analysis of the vesicle proteins of the host fungus with/without hypovirus infection. Thirty-three differentially expressed protein spots were identified in the purified vesicle protein samples by two-dimensional electrophoresis and mass spectrometry. Down-regulated proteins were mostly cargo proteins involved in primary metabolism and energy generation and up-regulated proteins were mostly vesicle associated proteins and ABC transporter. A virus-encoded protein p48 was found to have four forms with different molecular mass in vesicles from the virus-infected strain. While a few of the randomly selected differentially expressed proteins were in accordance with their transcription profiles, majority were not in agreement with their mRNA accumulation patterns, suggesting that an extensive post-transcriptional regulation may have occurred in the host fungus upon a hypovirus infection. Copyright © 2012 Elsevier B.V. All rights reserved.

The strategy of fusion genes construction determines efficient expression of introduced transcription factors.

PubMed

Adamus, Tomasz; Konieczny, Paweł; Sekuła, Małgorzata; Sułkowski, Maciej; Majka, Marcin

2014-01-01

The main goal in gene therapy and biomedical research is an efficient transcription factors (TFs) delivery system. SNAIL, a zinc finger transcription factor, is strongly involved in tumor, what makes its signaling pathways an interesting research subject. The necessity of tracking activation of intracellular pathways has prompted fluorescent proteins usage as localization markers. Advanced molecular cloning techniques allow to generate fusion proteins from fluorescent markers and transcription factors. Depending on fusion strategy, the protein expression levels and nuclear transport ability are significantly different. The P2A self-cleavage motif through its cleavage ability allows two single proteins to be simultaneously expressed. The aim of this study was to compare two strategies for introducing a pair of genes using expression vector system. We have examined GFP and SNAI1 gene fusions by comprising common nucleotide polylinker (multiple cloning site) or P2A motif in between them, resulting in one fusion or two independent protein expressions respectively. In each case transgene expression levels and translation efficiency as well as nuclear localization of expressed protein have been analyzed. Our data showed that usage of P2A motif provides more effective nuclear transport of SNAIL transcription factor than conventional genes linker. At the same time the fluorescent marker spreads evenly in subcellular space.

Identification of Putative Olfactory Genes from the Oriental Fruit Moth Grapholita molesta via an Antennal Transcriptome Analysis

PubMed Central

Li, Yiping; Wu, Junxiang

2015-01-01

Background The oriental fruit moth, Grapholita molesta, is an extremely important oligophagous pest species of stone and pome fruits throughout the world. As a host-switching species, adult moths, especially females, depend on olfactory cues to a large extent in locating host plants, finding mates, and selecting oviposition sites. The identification of olfactory genes can facilitate investigation on mechanisms for chemical communications. Methodology/Principal Finding We generated transcriptome of female antennae of G.molesta using the next-generation sequencing technique, and assembled transcripts from RNA-seq reads using Trinity, SOAPdenovo-trans and Abyss-trans assemblers. We identified 124 putative olfactory genes. Among the identified olfactory genes, 118 were novel to this species, including 28 transcripts encoding for odorant binding proteins, 17 chemosensory proteins, 48 odorant receptors, four gustatory receptors, 24 ionotropic receptors, two sensory neuron membrane proteins, and one odor degrading enzyme. The identified genes were further confirmed through semi-quantitative reverse transcription PCR for transcripts coding for 26 OBPs and 17 CSPs. OBP transcripts showed an obvious antenna bias, whereas CSP transcripts were detected in different tissues. Conclusion Antennal transcriptome data derived from the oriental fruit moth constituted an abundant molecular resource for the identification of genes potentially involved in the olfaction process of the species. This study provides a foundation for future research on the molecules involved in olfactory recognition of this insect pest, and in particular, the feasibility of using semiochemicals to control this pest. PMID:26540284

Second-generation inhibitors demonstrate the involvement of p38 mitogen-activated protein kinase in post-transcriptional modulation of inflammatory mediator production in human and rodent airways.

PubMed

Birrell, Mark A; Wong, Sissie; McCluskie, Kerryn; Catley, Matthew C; Hardaker, Elizabeth L; Haj-Yahia, Saleem; Belvisi, Maria G

2006-03-01

The exact role of p38 mitogen-activated protein kinase (MAPK) in the expression of inflammatory cytokines is not clear; it may regulate transcriptionally, post-transcriptionally, translationally, or post-translationally. The involvement of one or more of these mechanisms has been suggested to depend on the particular cytokine, the cell type studied, and the specific stimulus used. Interpretation of some of the published data is further complicated by the use of inhibitors such as 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB 203580) used at single, high concentrations. The aim of this study was to determine the impact of two second-generation p38 MAPK inhibitors on the expression of a range of inflammatory cytokines at the gene and protein levels in human cultured cells. Similar assessment of the impact of these compounds on inflammatory cytokine expression in a preclinical in vivo model of airway inflammation was performed. The results in THP-1 cells and primary airway macrophages clearly show that protein expression is inhibited at much lower concentrations of inhibitor than are needed to impact on gene expression. In the rodent model, both compounds, at doses that cause maximal inhibition of cellular recruitment, inhibit tumor necrosis factor alpha (TNFalpha) protein production without impacting on nuclear factor kappaB pathway activation or TNFalpha gene expression. In summary, the data shown here demonstrate that, although at high compound concentrations there is some level of transcriptional regulation, the predominant role of p38 MAPK in cytokine production is at the translational level. These data question whether the effect of p38 inhibitors on gene transcription is related to their potential therapeutic role as anti-inflammatory compounds.

Deubiquitylating enzymes as cancer stem cell therapeutics.

PubMed

Haq, Saba; Suresh, Bharathi; Ramakrishna, Suresh

2018-01-01

The focus of basic and applied research on core stem cell transcription factors has paved the way to initial delineation of their characteristics, their regulatory mechanisms, and the applicability of their regulatory proteins for protein-induced pluripotent stem cells (protein-IPSC) generation and in further clinical settings. Striking parallels have been observed between cancer stem cells (CSCs) and stem cells. For the maintenance of stem cells and CSC pluripotency and differentiation, post translational modifications (i.e., ubiquitylation and deubiquitylation) are tightly regulated, as these modifications result in a variety of stem cell fates. The identification of deubiquitylating enzymes (DUBs) involved in the regulation of core stem cell transcription factors and CSC-related proteins might contribute to providing novel insights into the implications of DUB regulatory mechanisms for governing cellular reprogramming and carcinogenesis. Moreover, we propose the novel possibility of applying DUBs coupled with core transcription factors to improve protein-iPSC generation efficiency. Additionally, this review article further illustrates the potential of applying DUB inhibitors as a novel therapeutic intervention for targeting CSCs. Thus, defining DUBs as core pharmacological targets implies that future endeavors to develop their inhibitors may revolutionize our ability to regulate stem cell maintenance and differentiation, somatic cell reprogramming, and cancer stem cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Alternatively Spliced Homologous Exons Have Ancient Origins and Are Highly Expressed at the Protein Level

PubMed Central

Abascal, Federico; Ezkurdia, Iakes; Rodriguez-Rivas, Juan; Rodriguez, Jose Manuel; del Pozo, Angela; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L.

2015-01-01

Alternative splicing of messenger RNA can generate a wide variety of mature RNA transcripts, and these transcripts may produce protein isoforms with diverse cellular functions. While there is much supporting evidence for the expression of alternative transcripts, the same is not true for the alternatively spliced protein products. Large-scale mass spectroscopy experiments have identified evidence of alternative splicing at the protein level, but with conflicting results. Here we carried out a rigorous analysis of the peptide evidence from eight large-scale proteomics experiments to assess the scale of alternative splicing that is detectable by high-resolution mass spectroscopy. We find fewer splice events than would be expected: we identified peptides for almost 64% of human protein coding genes, but detected just 282 splice events. This data suggests that most genes have a single dominant isoform at the protein level. Many of the alternative isoforms that we could identify were only subtly different from the main splice isoform. Very few of the splice events identified at the protein level disrupted functional domains, in stark contrast to the two thirds of splice events annotated in the human genome that would lead to the loss or damage of functional domains. The most striking result was that more than 20% of the splice isoforms we identified were generated by substituting one homologous exon for another. This is significantly more than would be expected from the frequency of these events in the genome. These homologous exon substitution events were remarkably conserved—all the homologous exons we identified evolved over 460 million years ago—and eight of the fourteen tissue-specific splice isoforms we identified were generated from homologous exons. The combination of proteomics evidence, ancient origin and tissue-specific splicing indicates that isoforms generated from homologous exons may have important cellular roles. PMID:26061177

Ligand-independent activation of the oestrogen receptor by mutation of a conserved tyrosine.

PubMed Central

White, R; Sjöberg, M; Kalkhoven, E; Parker, M G

1997-01-01

The oestrogen receptor is a member of the nuclear receptor family of transcription factors which, on binding the steroid hormone 17beta-oestradiol, interacts with co-activator proteins and stimulates gene expression. Replacement of a single tyrosine in the hormone-binding domain generated activated forms of the receptor which stimulated transcription in the absence of hormone. This increased activation is related to a decrease in hydrophobicity and a reduction in size of the side chain of the amino acid with which the tyrosine is replaced. Ligand-independent, in common with ligand-dependent transcriptional activation, requires an amphipathic alpha-helix at the C-terminus of the ligand-binding domain which is essential for the interaction of the receptor with a number of potential co-activator proteins. In contrast to the wild-type protein, constitutively active receptors were able to bind both the receptor-interacting protein RIP-140 and the steroid receptor co-activator SRC-1 in a ligand-independent manner, although in the case of SRC-1 this was only evident when the receptors were prebound to DNA. We propose, therefore, that this tyrosine is required to maintain the receptor in a transcriptionally inactive state in the absence of hormone. Modification of this residue may generate a conformational change in the ligand-binding domain of the receptor to form an interacting surface which allows the recruitment of co-activators independent of hormone binding. This suggests that this tyrosine may be a target for a different signalling pathway which forms an alternative mechanism of activating oestrogen receptor-mediated transcription. PMID:9135157

Imaging Erg and Jun transcription factor interaction in living cells using fluorescence resonance energy transfer analyses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Camuzeaux, Barbara; Spriet, Corentin; Heliot, Laurent

2005-07-15

Physical interactions between transcription factors play important roles in modulating gene expression. Previous in vitro studies have shown a transcriptional synergy between Erg protein, an Ets family member, and Jun/Fos heterodimer, members of the bZip family, which requires direct Erg-Jun protein interactions. Visualization of protein interactions in living cells is a new challenge in biology. For this purpose, we generated fusion proteins of Erg, Fos, and Jun with yellow and cyan fluorescent proteins, YFP and CFP, respectively. After transient expression in HeLa cells, interactions of the resulting fusion proteins were explored by fluorescence resonance energy transfer microscopy (FRET) in fixedmore » and living cells. FRET between YFP-Erg and CFP-Jun was monitored by using photobleaching FRET and fluorescence lifetime imaging microscopy. Both techniques revealed the occurrence of intermolecular FRET between YFP-Erg and CFP-Jun. This is stressed by loss of FRET with an YFP-Erg version carrying a point mutation in its ETS domain. These results provide evidence for the interaction of Erg and Jun proteins in living cells as a critical prerequisite of their transcriptional synergy, but also for the essential role of the Y371 residue, conserved in most Ets proteins, in this interaction.« less

AF4 and AF4N protein complexes: recruitment of P-TEFb kinase, their interactome and potential functions

PubMed Central

Scholz, Bastian; Kowarz, Eric; Rössler, Tanja; Ahmad, Khalil; Steinhilber, Dieter; Marschalek, Rolf

2015-01-01

AF4/AFF1 and AF5/AFF4 are the molecular backbone to assemble “super-elongation complexes” (SECs) that have two main functions: (1) control of transcriptional elongation by recruiting the positive transcription elongation factor b (P-TEFb = CyclinT1/CDK9) that is usually stored in inhibitory 7SK RNPs; (2) binding of different histone methyltransferases, like DOT1L, NSD1 and CARM1. This way, transcribed genes obtain specific histone signatures (e.g. H3K79me2/3, H3K36me2) to generate a transcriptional memory system. Here we addressed several questions: how is P-TEFb recruited into SEC, how is the AF4 interactome composed, and what is the function of the naturally occuring AF4N protein variant which exhibits only the first 360 amino acids of the AF4 full-length protein. Noteworthy, shorter protein variants are a specific feature of all AFF protein family members. Here, we demonstrate that full-length AF4 and AF4N are both catalyzing the transition of P-TEFb from 7SK RNP to their N-terminal domain. We have also mapped the protein-protein interaction network within both complexes. In addition, we have first evidence that the AF4N protein also recruits TFIIH and the tumor suppressor MEN1. This indicate that AF4N may have additional functions in transcriptional initiation and in MEN1-dependend transcriptional processes. PMID:26171280

Human Immunodeficiency Virus-Type 1 LTR DNA contains an intrinsic gene producing antisense RNA and protein products

PubMed Central

Ludwig, Linda B; Ambrus, Julian L; Krawczyk, Kristie A; Sharma, Sanjay; Brooks, Stephen; Hsiao, Chiu-Bin; Schwartz, Stanley A

2006-01-01

Background While viruses have long been shown to capitalize on their limited genomic size by utilizing both strands of DNA or complementary DNA/RNA intermediates to code for viral proteins, it has been assumed that human retroviruses have all their major proteins translated only from the plus or sense strand of RNA, despite their requirement for a dsDNA proviral intermediate. Several studies, however, have suggested the presence of antisense transcription for both HIV-1 and HTLV-1. More recently an antisense transcript responsible for the HTLV-1 bZIP factor (HBZ) protein has been described. In this study we investigated the possibility of an antisense gene contained within the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). Results Inspection of published sequences revealed a potential transcription initiator element (INR) situated downstream of, and in reverse orientation to, the usual HIV-1 promoter and transcription start site. This antisense initiator (HIVaINR) suggested the possibility of an antisense gene responsible for RNA and protein production. We show that antisense transcripts are generated, in vitro and in vivo, originating from the TAR DNA of the HIV-1 LTR. To test the possibility that protein(s) could be translated from this novel HIV-1 antisense RNA, recombinant HIV antisense gene-FLAG vectors were designed. Recombinant protein(s) were produced and isolated utilizing carboxy-terminal FLAG epitope (DYKDDDDK) sequences. In addition, affinity-purified antisera to an internal peptide derived from the HIV antisense protein (HAP) sequences identified HAPs from HIV+ human peripheral blood lymphocytes. Conclusion HIV-1 contains an antisense gene in the U3-R regions of the LTR responsible for both an antisense RNA transcript and proteins. This antisense transcript has tremendous potential for intrinsic RNA regulation because of its overlap with the beginning of all HIV-1 sense RNA transcripts by 25 nucleotides. The novel HAPs are encoded in a region of the LTR that has already been shown to be deleted in some HIV-infected long-term survivors and represent new potential targets for vaccine development. PMID:17090330

Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent Protein Kinase PKR

PubMed Central

Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V.; Lokugamage, Nandadeva; Head, Jennifer A.; Ikegami, Tetsuro

2012-01-01

Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-β mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2α through PKR activation, while retaining the function to inhibit host general transcription including IFN-β gene inhibition. MP-12 NSs but not R173A NSs binds to wt PKR. R173A NSs formed filamentous structure in nucleus in a mosaic pattern, which was distinct from MP-12 NSs filament pattern. Due to early phosphorylation of eIF2α, rMP12-NSsR173A could not efficiently accumulate viral proteins. Our results suggest that NSs-mediated host general transcription suppression occurs independently of PKR degradation, while the PKR degradation is important to inhibit the phosphorylation of eIF2α in infected cells undergoing host general transcription suppression. PMID:23063407

Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent protein kinase PKR.

PubMed

Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V; Lokugamage, Nandadeva; Head, Jennifer A; Ikegami, Tetsuro

2013-01-20

Rift Valley fever virus (RVFV) encodes one major virulence factor, the NSs protein. NSs suppresses host general transcription, including interferon (IFN)-β mRNA synthesis, and promotes degradation of the dsRNA-dependent protein kinase (PKR). We generated a novel RVFV mutant (rMP12-NSsR173A) specifically lacking the function to promote PKR degradation. rMP12-NSsR173A infection induces early phosphorylation of eIF2α through PKR activation, while retaining the function to inhibit host general transcription including IFN-β gene inhibition. MP-12 NSs but not R173A NSs binds to wt PKR. R173A NSs formed filamentous structure in nucleus in a mosaic pattern, which was distinct from MP-12 NSs filament pattern. Due to early phosphorylation of eIF2α, rMP12-NSsR173A could not efficiently accumulate viral proteins. Our results suggest that NSs-mediated host general transcription suppression occurs independently of PKR degradation, while the PKR degradation is important to inhibit the phosphorylation of eIF2α in infected cells undergoing host general transcription suppression. Copyright © 2012 Elsevier Inc. All rights reserved.

Light-Regulated Transcription of Genes Encoding Peridinin Chlorophyll a Proteins and the Major Intrinsic Light-Harvesting Complex Proteins in the Dinoflagellate Amphidinium carterae Hulburt (Dinophycae)1

PubMed Central

ten Lohuis, Michael R.; Miller, David J.

1998-01-01

In the dinoflagellate Amphidinium carterae, photoadaptation involves changes in the transcription of genes encoding both of the major classes of light-harvesting proteins, the peridinin chlorophyll a proteins (PCPs) and the major a/c-containing intrinsic light-harvesting proteins (LHCs). PCP and LHC transcript levels were increased up to 86- and 6-fold higher, respectively, under low-light conditions relative to cells grown at high illumination. These increases in transcript abundance were accompanied by decreases in the extent of methylation of CpG and CpNpG motifs within or near PCP- and LHC-coding regions. Cytosine methylation levels in A. carterae are therefore nonstatic and may vary with environmental conditions in a manner suggestive of involvement in the regulation of gene expression. However, chemically induced undermethylation was insufficient in activating transcription, because treatment with two methylation inhibitors had no effect on PCP mRNA or protein levels. Regulation of gene activity through changes in DNA methylation has traditionally been assumed to be restricted to higher eukaryotes (deuterostomes and green plants); however, the atypically large genomes of dinoflagellates may have generated the requirement for systems of this type in a relatively “primitive” organism. Dinoflagellates may therefore provide a unique perspective on the evolution of eukaryotic DNA-methylation systems. PMID:9576788

A novel GhBEE1-Like gene of cotton causes anther indehiscence in transgenic Arabidopsis under uncontrolled transcription level.

PubMed

Chen, Eryong; Wang, Xiaoqian; Gong, Qian; Butt, Hamama Islam; Chen, Yanli; Zhang, Chaojun; Yang, Zuoren; Wu, Zhixia; Ge, Xiaoyang; Zhang, Xianlong; Li, Fuguang; Zhang, Xueyan

2017-09-05

Male-sterile lines are very important for selective breeding, and anther dehiscence defect is an effective way to generate male-sterile lines. Although several bHLH-family proteins in Arabidopsis have been characterized, little is known about the role of bHLH-family proteins in cotton. Here, we isolated a novel bHLH protein from cotton (Gossypium hirsutum), named GhBEE1-Like. Protein domain analysis showed that GhBEE1-Like contained a basic domain and an HLH domain. Subcellular localization analysis revealed that GhBEE1-Like was a nuclear-localized protein. Expression pattern analysis showed GhBEE1-Like was highly expressed in floral organs, and its expression was induced by the active brassinosteroid (BR) substance 24-epi-BL. GhBEE1-Like overexpression in Arabidopsis resulted in two types of transgenic lines, one with normal anther dehiscence and the other with defective anther dehiscence. Semi-qRT-PCR and qRT-PCR analyses revealed that GhBEE1-Like transcript levels acted as a check-point determining how anther dehiscence proceeds in these transgenic lines; regulated transcript levels result in normal anther dehiscence, whereas uncontrolled transcript levels lead to anther indehiscence. These results suggest that GhBEE1-Like plays an important role via its accumulation in regulating anther dehiscence. Therefore, controlling the level of GhBEE1-Like expression in cotton could be a convenient tool for generating male-sterile lines to use in selective breeding. Copyright © 2017. Published by Elsevier B.V.

A toolbox of immunoprecipitation-grade monoclonal antibodies to human transcription factors.

PubMed

Venkataraman, Anand; Yang, Kun; Irizarry, Jose; Mackiewicz, Mark; Mita, Paolo; Kuang, Zheng; Xue, Lin; Ghosh, Devlina; Liu, Shuang; Ramos, Pedro; Hu, Shaohui; Bayron Kain, Diane; Keegan, Sarah; Saul, Richard; Colantonio, Simona; Zhang, Hongyan; Behn, Florencia Pauli; Song, Guang; Albino, Edisa; Asencio, Lillyann; Ramos, Leonardo; Lugo, Luvir; Morell, Gloriner; Rivera, Javier; Ruiz, Kimberly; Almodovar, Ruth; Nazario, Luis; Murphy, Keven; Vargas, Ivan; Rivera-Pacheco, Zully Ann; Rosa, Christian; Vargas, Moises; McDade, Jessica; Clark, Brian S; Yoo, Sooyeon; Khambadkone, Seva G; de Melo, Jimmy; Stevanovic, Milanka; Jiang, Lizhi; Li, Yana; Yap, Wendy Y; Jones, Brittany; Tandon, Atul; Campbell, Elliot; Montelione, Gaetano T; Anderson, Stephen; Myers, Richard M; Boeke, Jef D; Fenyö, David; Whiteley, Gordon; Bader, Joel S; Pino, Ignacio; Eichinger, Daniel J; Zhu, Heng; Blackshaw, Seth

2018-03-19

A key component of efforts to address the reproducibility crisis in biomedical research is the development of rigorously validated and renewable protein-affinity reagents. As part of the US National Institutes of Health (NIH) Protein Capture Reagents Program (PCRP), we have generated a collection of 1,406 highly validated immunoprecipitation- and/or immunoblotting-grade mouse monoclonal antibodies (mAbs) to 737 human transcription factors, using an integrated production and validation pipeline. We used HuProt human protein microarrays as a primary validation tool to identify mAbs with high specificity for their cognate targets. We further validated PCRP mAbs by means of multiple experimental applications, including immunoprecipitation, immunoblotting, chromatin immunoprecipitation followed by sequencing (ChIP-seq), and immunohistochemistry. We also conducted a meta-analysis that identified critical variables that contribute to the generation of high-quality mAbs. All validation data, protocols, and links to PCRP mAb suppliers are available at http://proteincapture.org.

Serum MX2 Protein as Candidate Biomarker for Early Pregnancy Diagnosis in Buffalo.

PubMed

Buragohain, L; Kumar, R; Nanda, T; Phulia, S K; Mohanty, A K; Kumar, S; Balhara, S; Ghuman, Sps; Singh, I; Balhara, A K

2016-08-01

Interferon-tau (IFN-τ)-induced molecular markers such as ubiquitin-like modifier (ISG15), 2',5'-oligoadenylate synthetase 1 (OAS1) and myxovirus resistance genes (MX1 and MX2) have generated immense attention towards developing diagnostic tools for early diagnosis of pregnancy in bovine. These molecules are expressed at transcriptional level in peripheral nucleated cells. However, their presence in the serum is still a question mark. This study reports sequential changes in expression of MX2 transcript in whole blood and serum MX2 protein level on days 0, 7, 14, 21, 28 and 35 in pregnant (n = 9) buffalo heifers, and on days 0, 7 and 14 in non-inseminated (n = 8) and inseminated non-pregnant (n = 10) control animals. In non-inseminated and inseminated non-pregnant heifers, the differential expression of MX2 transcript and MX2 protein level remained similar between day 7 and 14 post-oestrus. However, in pregnant heifers, on 14th and 28th day post-insemination MX2 transcript was 16.38 ± 1.57 and 28.16 ± 1.91 times upregulated as compared to day 0. Similarly, serum MX2 protein concentration followed analogous trend as MX2 transcript and increased gradually with the progression of pregnancy. Correlation analysis between expression of MX2 transcript and its serum protein level showed a significant positive correlation in pregnant animals, while it was random in other two groups. Therefore, MX2 surge at transcriptional and serum protein level after day 14-28 of pregnancy in buffalo holds potential for its use in early pregnancy detection. © 2016 Blackwell Verlag GmbH.

Mapping the architecture of the HIV-lTat circuit: A decision-making circuit that lacks bistability and exploits stochastic noise

PubMed Central

Razooky, Brandon S.; Weinberger, Leor S.

2014-01-01

Upon infection of a CD4+ T cell, HIV-l appears to ‘choose’ between two alternate fates: active replication or a long-lived dormant statetermed proviral latency. A transcriptional positive-feedback loop generated by the HIV-l Tat protein appears sufficient to mediate this decision. Here, we describea coupled wet-lab and computational approach that uses mathematical modeling and live-cell time-lapse microscopy to map the architecture of the HIV-l Tat transcriptional regulatorycircuit and generate predictive models of HIV-l latency. This approach provided the first characterization of a ‘decision-making’ circuit that lacks bistability andinstead exploits stochastic fluctuations in cellular molecules (i.e. noise) to generate a decision between an on or off transcriptional state. PMID:21167940

High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

PubMed Central

Dortay, Hakan; Akula, Usha Madhuri; Westphal, Christin; Sittig, Marie; Mueller-Roeber, Bernd

2011-01-01

Protein expression in heterologous hosts for functional studies is a cumbersome effort. Here, we report a superior platform for parallel protein expression in vivo and in vitro. The platform combines highly efficient ligation-independent cloning (LIC) with instantaneous detection of expressed proteins through N- or C-terminal fusions to infrared fluorescent protein (IFP). For each open reading frame, only two PCR fragments are generated (with three PCR primers) and inserted by LIC into ten expression vectors suitable for protein expression in microbial hosts, including Escherichia coli, Kluyveromyces lactis, Pichia pastoris, the protozoon Leishmania tarentolae, and an in vitro transcription/translation system. Accumulation of IFP-fusion proteins is detected by infrared imaging of living cells or crude protein extracts directly after SDS-PAGE without additional processing. We successfully employed the LIC-IFP platform for in vivo and in vitro expression of ten plant and fungal proteins, including transcription factors and enzymes. Using the IFP reporter, we additionally established facile methods for the visualisation of protein-protein interactions and the detection of DNA-transcription factor interactions in microtiter and gel-free format. We conclude that IFP represents an excellent reporter for high-throughput protein expression and analysis, which can be easily extended to numerous other expression hosts using the setup reported here. PMID:21541323

Identification of PEG-induced water stress responsive transcripts using co-expression network in Eucalyptus grandis.

PubMed

Ghosh Dasgupta, Modhumita; Dharanishanthi, Veeramuthu

2017-09-05

Ecophysiological studies in Eucalyptus have shown that water is the principal factor limiting stem growth. Effect of water deficit conditions on physiological and biochemical parameters has been extensively reported in Eucalyptus. The present study was conducted to identify major polyethylene glycol induced water stress responsive transcripts in Eucalyptus grandis using gene co-expression network. A customized array representing 3359 water stress responsive genes was designed to document their expression in leaves of E. grandis cuttings subjected to -0.225MPa of PEG treatment. The differentially expressed transcripts were documented and significantly co-expressed transcripts were used for construction of network. The co-expression network was constructed with 915 nodes and 3454 edges with degree ranging from 2 to 45. Ninety four GO categories and 117 functional pathways were identified in the network. MCODE analysis generated 27 modules and module 6 with 479 nodes and 1005 edges was identified as the biologically relevant network. The major water responsive transcripts represented in the module included dehydrin, osmotin, LEA protein, expansin, arabinogalactans, heat shock proteins, major facilitator proteins, ARM repeat proteins, raffinose synthase, tonoplast intrinsic protein and transcription factors like DREB2A, ARF9, AGL24, UNE12, WLIM1 and MYB66, MYB70, MYB 55, MYB 16 and MYB 103. The coordinated analysis of gene expression patterns and coexpression networks developed in this study identified an array of transcripts that may regulate PEG induced water stress responses in E. grandis. Copyright © 2017 Elsevier B.V. All rights reserved.

Alternative polyadenylation of mRNA precursors

PubMed Central

Tian, Bin; Manley, James L.

2017-01-01

Alternative polyadenylation (APA) is an RNA-processing mechanism that generates distinct 3′ termini on mRNAs and other RNA polymerase II transcripts. It is widespread across all eukaryotic species and is recognized as a major mechanism of gene regulation. APA exhibits tissue specificity and is important for cell proliferation and differentiation. In this Review, we discuss the roles of APA in diverse cellular processes, including mRNA metabolism, protein diversification and protein localization, and more generally in gene regulation. We also discuss the molecular mechanisms underlying APA, such as variation in the concentration of core processing factors and RNA-binding proteins, as well as transcription-based regulation. PMID:27677860

Transcription and DNA Damage: Holding Hands or Crossing Swords?

PubMed

D'Alessandro, Giuseppina; d'Adda di Fagagna, Fabrizio

2017-10-27

Transcription has classically been considered a potential threat to genome integrity. Collision between transcription and DNA replication machinery, and retention of DNA:RNA hybrids, may result in genome instability. On the other hand, it has been proposed that active genes repair faster and preferentially via homologous recombination. Moreover, while canonical transcription is inhibited in the proximity of DNA double-strand breaks, a growing body of evidence supports active non-canonical transcription at DNA damage sites. Small non-coding RNAs accumulate at DNA double-strand break sites in mammals and other organisms, and are involved in DNA damage signaling and repair. Furthermore, RNA binding proteins are recruited to DNA damage sites and participate in the DNA damage response. Here, we discuss the impact of transcription on genome stability, the role of RNA binding proteins at DNA damage sites, and the function of small non-coding RNAs generated upon damage in the signaling and repair of DNA lesions. Copyright © 2016 Elsevier Ltd. All rights reserved.

HIV-1 Tat protein promotes formation of more-processive elongation complexes.

PubMed Central

Marciniak, R A; Sharp, P A

1991-01-01

The Tat protein of HIV-1 trans-activates transcription in vitro in a cell-free extract of HeLa nuclei. Quantitative analysis of the efficiency of elongation revealed that a majority of the elongation complexes generated by the HIV-1 promoter were not highly processive and terminated within the first 500 nucleotides. Tat trans-activation of transcription from the HIV-1 promoter resulted from an increase in processive character of the elongation complexes. More specifically, the analysis suggests that there exist two classes of elongation complexes initiating from the HIV promoter: a less-processive form and a more-processive form. Addition of purified Tat protein was found to increase the abundance of the more-processive class of elongation complex. The purine nucleoside analog, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) inhibits transcription in this reaction by decreasing the efficiency of elongation. Surprisingly, stimulation of transcription elongation by Tat was preferentially inhibited by the addition of DRB. Images PMID:1756726

In vitro transcripts of wild-type and fluorescent protein-tagged triticum mosaic virus (family potyviridae) are biologically active in wheat

USDA-ARS?s Scientific Manuscript database

Triticum mosaic virus (TriMV) (genus Poacevirus, family Potyviridae) is a recently described eriophyid mite-transmitted wheat virus. In vitro RNA transcripts generated from full-length cDNA clones of TriMV proved infectious on wheat, and the progeny virus was efficiently transmitted by wheat curl m...

Extensive Use of RNA-Binding Proteins in Drosophila Sensory Neuron Dendrite Morphogenesis

PubMed Central

Olesnicky, Eugenia C.; Killian, Darrell J.; Garcia, Evelyn; Morton, Mary C.; Rathjen, Alan R.; Sola, Ismail E.; Gavis, Elizabeth R.

2013-01-01

The large number of RNA-binding proteins and translation factors encoded in the Drosophila and other metazoan genomes predicts widespread use of post-transcriptional regulation in cellular and developmental processes. Previous studies identified roles for several RNA-binding proteins in dendrite branching morphogenesis of Drosophila larval sensory neurons. To determine the larger contribution of post-transcriptional gene regulation to neuronal morphogenesis, we conducted an RNA interference screen to identify additional Drosophila proteins annotated as either RNA-binding proteins or translation factors that function in producing the complex dendritic trees of larval class IV dendritic arborization neurons. We identified 88 genes encoding such proteins whose knockdown resulted in aberrant dendritic morphology, including alterations in dendritic branch number, branch length, field size, and patterning of the dendritic tree. In particular, splicing and translation initiation factors were associated with distinct and characteristic phenotypes, suggesting that different morphogenetic events are best controlled at specific steps in post-transcriptional messenger RNA metabolism. Many of the factors identified in the screen have been implicated in controlling the subcellular distributions and translation of maternal messenger RNAs; thus, common post-transcriptional regulatory strategies may be used in neurogenesis and in the generation of asymmetry in the female germline and embryo. PMID:24347626

Isolation of genes differentially expressed during development and ripening of Fragaria chiloensis fruit by suppression subtractive hybridization.

PubMed

Pimentel, Paula; Salvatierra, Ariel; Moya-León, María Alejandra; Herrera, Raúl

2010-09-15

Fragaria chiloensis, the native Chilean strawberry, is noted for its good fruit quality characters. However, it is a highly perishable fruit due to its rapid softening. With the aim to screen for genes differentially expressed during development and ripening of strawberry fruit, the subtractive suppressive hybridization (SSH) methodology was employed. Six libraries were generated contrasting transcripts from four different developmental stages. A set of 1807 genes was isolated and characterized. In our EST collection, approximately 90% of partial cDNAs showed significant similarity to proteins with known or unknown function registered in databases. Among them, proteins related to protein fate were identified in a large green fruit library and protein related with cellular transport, cell wall-related proteins, and transcription regulators were identified in a ripe fruit library. Thirteen genes were analyzed by qRT-PCR during development and ripening of the Chilean strawberry fruit. The information generated in this study provides new clues to aid the understanding of the ripening process in F. chiloensis fruit. Copyright 2010 Elsevier GmbH. All rights reserved.

PAR-CLIP data indicate that Nrd1-Nab3-dependent transcription termination regulates expression of hundreds of protein coding genes in yeast

PubMed Central

2014-01-01

Background Nrd1 and Nab3 are essential sequence-specific yeast RNA binding proteins that function as a heterodimer in the processing and degradation of diverse classes of RNAs. These proteins also regulate several mRNA coding genes; however, it remains unclear exactly what percentage of the mRNA component of the transcriptome these proteins control. To address this question, we used the pyCRAC software package developed in our laboratory to analyze CRAC and PAR-CLIP data for Nrd1-Nab3-RNA interactions. Results We generated high-resolution maps of Nrd1-Nab3-RNA interactions, from which we have uncovered hundreds of new Nrd1-Nab3 mRNA targets, representing between 20 and 30% of protein-coding transcripts. Although Nrd1 and Nab3 showed a preference for binding near 5′ ends of relatively short transcripts, they bound transcripts throughout coding sequences and 3′ UTRs. Moreover, our data for Nrd1-Nab3 binding to 3′ UTRs was consistent with a role for these proteins in the termination of transcription. Our data also support a tight integration of Nrd1-Nab3 with the nutrient response pathway. Finally, we provide experimental evidence for some of our predictions, using northern blot and RT-PCR assays. Conclusions Collectively, our data support the notion that Nrd1 and Nab3 function is tightly integrated with the nutrient response and indicate a role for these proteins in the regulation of many mRNA coding genes. Further, we provide evidence to support the hypothesis that Nrd1-Nab3 represents a failsafe termination mechanism in instances of readthrough transcription. PMID:24393166

Transcriptator: An Automated Computational Pipeline to Annotate Assembled Reads and Identify Non Coding RNA.

PubMed

Tripathi, Kumar Parijat; Evangelista, Daniela; Zuccaro, Antonio; Guarracino, Mario Rosario

2015-01-01

RNA-seq is a new tool to measure RNA transcript counts, using high-throughput sequencing at an extraordinary accuracy. It provides quantitative means to explore the transcriptome of an organism of interest. However, interpreting this extremely large data into biological knowledge is a problem, and biologist-friendly tools are lacking. In our lab, we developed Transcriptator, a web application based on a computational Python pipeline with a user-friendly Java interface. This pipeline uses the web services available for BLAST (Basis Local Search Alignment Tool), QuickGO and DAVID (Database for Annotation, Visualization and Integrated Discovery) tools. It offers a report on statistical analysis of functional and Gene Ontology (GO) annotation's enrichment. It helps users to identify enriched biological themes, particularly GO terms, pathways, domains, gene/proteins features and protein-protein interactions related informations. It clusters the transcripts based on functional annotations and generates a tabular report for functional and gene ontology annotations for each submitted transcript to the web server. The implementation of QuickGo web-services in our pipeline enable the users to carry out GO-Slim analysis, whereas the integration of PORTRAIT (Prediction of transcriptomic non coding RNA (ncRNA) by ab initio methods) helps to identify the non coding RNAs and their regulatory role in transcriptome. In summary, Transcriptator is a useful software for both NGS and array data. It helps the users to characterize the de-novo assembled reads, obtained from NGS experiments for non-referenced organisms, while it also performs the functional enrichment analysis of differentially expressed transcripts/genes for both RNA-seq and micro-array experiments. It generates easy to read tables and interactive charts for better understanding of the data. The pipeline is modular in nature, and provides an opportunity to add new plugins in the future. Web application is freely available at: http://www-labgtp.na.icar.cnr.it/Transcriptator.

Single step generation of protein arrays from DNA by cell-free expression and in situ immobilisation (PISA method).

PubMed

He, M; Taussig, M J

2001-08-01

We describe a format for production of protein arrays termed 'protein in situ array' (PISA). A PISA is rapidly generated in one step directly from PCR-generated DNA fragments by cell-free protein expression and in situ immobilisation at a surface. The template for expression is DNA encoding individual proteins or domains, which is produced by PCR using primers designed from information in DNA databases. Coupled transcription and translation is carried out on a surface to which the tagged protein adheres as soon as it is synthesised. Because proteins generated by cell-free synthesis are usually soluble and functional, this method can overcome problems of insolubility or degradation associated with bacterial expression of recombinant proteins. Moreover, the use of PCR-generated DNA enables rapid production of proteins or domains based on genome information alone and will be particularly useful where cloned material is not available. Here we show that human single-chain antibody fragments (three domain, V(H)/K form) and an enzyme (luciferase) can be functionally arrayed by the PISA method.

Reactive oxygen species-generating mitochondrial DNA mutation up-regulates hypoxia-inducible factor-1alpha gene transcription via phosphatidylinositol 3-kinase-Akt/protein kinase C/histone deacetylase pathway.

PubMed

Koshikawa, Nobuko; Hayashi, Jun-Ichi; Nakagawara, Akira; Takenaga, Keizo

2009-11-27

Lewis lung carcinoma-derived high metastatic A11 cells constitutively overexpress hypoxia-inducible factor (HIF)-1alpha mRNA compared with low metastatic P29 cells. Because A11 cells exclusively possess a G13997A mutation in the mitochondrial NADH dehydrogenase subunit 6 (ND6) gene, we addressed here a causal relationship between the ND6 mutation and the activation of HIF-1alpha transcription, and we investigated the potential mechanism. Using trans-mitochondrial cybrids between A11 and P29 cells, we found that the ND6 mutation was directly involved in HIF-1alpha mRNA overexpression. Stimulation of HIF-1alpha transcription by the ND6 mutation was mediated by overproduction of reactive oxygen species (ROS) and subsequent activation of phosphatidylinositol 3-kinase (PI3K)-Akt and protein kinase C (PKC) signaling pathways. The up-regulation of HIF-1alpha transcription was abolished by mithramycin A, an Sp1 inhibitor, but luciferase reporter and chromatin immunoprecipitation assays indicated that Sp1 was necessary but not sufficient for HIF-1alpha mRNA overexpression in A11 cells. On the other hand, trichostatin A, a histone deacetylase (HDAC) inhibitor, markedly suppressed HIF-1alpha transcription in A11 cells. In accordance with this, HDAC activity was high in A11 cells but low in P29 cells and in A11 cells treated with the ROS scavenger ebselene, the PI3K inhibitor LY294002, and the PKC inhibitor Ro31-8220. These results suggest that the ROS-generating ND6 mutation increases HIF-1alpha transcription via the PI3K-Akt/PKC/HDAC pathway, leading to HIF-1alpha protein accumulation in hypoxic tumor cells.

Drosophila Uri, a PP1α binding protein, is essential for viability, maintenance of DNA integrity and normal transcriptional activity

PubMed Central

Kirchner, Jasmin; Vissi, Emese; Gross, Sascha; Szoor, Balazs; Rudenko, Andrey; Alphey, Luke; White-Cooper, Helen

2008-01-01

Background Protein phosphatase 1 (PP1) is involved in diverse cellular processes, and is targeted to substrates via interaction with many different protein binding partners. PP1 catalytic subunits (PP1c) fall into PP1α and PP1β subfamilies based on sequence analysis, however very few PP1c binding proteins have been demonstrated to discriminate between PP1α and PP1β. Results URI (unconventional prefoldin RPB5 interactor) is a conserved molecular chaperone implicated in a variety of cellular processes, including the transcriptional response to nutrient signalling and maintenance of DNA integrity. We show that Drosophila Uri binds PP1α with much higher affinity than PP1β, and that this ability to discriminate between PP1c forms is conserved to humans. Most Uri is cytoplasmic, however we found some protein associated with active RNAPII on chromatin. We generated a uri loss of function allele, and show that uri is essential for viability in Drosophila. uri mutants have transcriptional defects, reduced cell viability and differentiation in the germline, and accumulate DNA damage in their nuclei. Conclusion Uri is the first PP1α specific binding protein to be described in Drosophila. Uri protein plays a role in transcriptional regulation. Activity of uri is required to maintain DNA integrity and cell survival in normal development. PMID:18412953

27-Hydroxycholesterol increases Myc protein stability via suppressing PP2A, SCP1 and FBW7 transcription in MCF-7 breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Li-Ming; Liang, Zi-Rui; Zhou, Ke-Ren

27-hydroxycholesterol (27-HC), the most abundant metabolite of cholesterol, is a risk factor for breast cancer. It can increase the proliferation of breast cancer cells and promote the metastasis of breast tumours in mouse models. Myc is a critical oncoprotein overexpressed in breast cancer. However, whether 27-HC affects Myc expression has not been reported. In the current study, we aimed to investigate the effects of 27-HC on Myc and the underlying mechanisms in MCF-7 breast cancer cells. Our data demonstrated that 27-HC activated Myc via increasing its protein stability. Three key negative modulators of Myc protein stability, PP2A, SCP1 and FBW7,more » were suppressed by 27-HC at the transcriptional level. We performed a data-mining analysis of the chromatin immunoprecipitation with next-generation DNA sequencing (ChIP-Seq) data in the ChIPBase, and discovered that a number of putative transcription factors (TFs), including Myc itself, were involved in the transcriptional regulation of PP2A, SCP1 and FBW7. Our results provide a novel mechanistic insight into the activation of Myc by 27-HC via transcriptional repression of PP2A, SCP1 and FBW7 to increase Myc protein stability in breast cancer cells. - Highlights: • 27-Hydroxycholesterol (27-HC) activates Myc via increasing its protein stability. • 27-HC inhibits PP2A and SCP1 transcription to block pS62-Myc dephosphorylation. • 27-HC suppresses FBW7 transcription to prevent pT58-Myc degradation.« less

A Role for Timely Nuclear Translocation of Clock Repressor Proteins in Setting Circadian Clock Speed

PubMed Central

Lee, Euna

2014-01-01

By means of a circadian clock system, all the living organisms on earth including human beings can anticipate the environmental rhythmic changes such as light/dark and warm/cold periods in a daily as well as in a yearly manner. Anticipating such environmental changes provide organisms with survival benefits via manifesting behavior and physiology at an advantageous time of the day and year. Cell-autonomous circadian oscillators, governed by transcriptional feedback loop composed of positive and negative elements, are organized into a hierarchical system throughout the organisms and generate an oscillatory expression of a clock gene by itself as well as clock controlled genes (ccgs) with a 24 hr periodicity. In the feedback loop, hetero-dimeric transcription factor complex induces the expression of negative regulatory proteins, which in turn represses the activity of transcription factors to inhibit their own transcription. Thus, for robust oscillatory rhythms of the expression of clock genes as well as ccgs, the precise control of subcellular localization and/or timely translocation of core clock protein are crucial. Here, we discuss how sub-cellular localization and nuclear translocation are controlled in a time-specific manner focusing on the negative regulatory clock proteins. PMID:25258565

Transcriptional Activity and Nuclear Localization of Cabut, the Drosophila Ortholog of Vertebrate TGF-β-Inducible Early-Response Gene (TIEG) Proteins

PubMed Central

Belacortu, Yaiza; Weiss, Ron; Kadener, Sebastian; Paricio, Nuria

2012-01-01

Background Cabut (Cbt) is a C2H2-class zinc finger transcription factor involved in embryonic dorsal closure, epithelial regeneration and other developmental processes in Drosophila melanogaster. Cbt orthologs have been identified in other Drosophila species and insects as well as in vertebrates. Indeed, Cbt is the Drosophila ortholog of the group of vertebrate proteins encoded by the TGF-ß-inducible early-response genes (TIEGs), which belong to Sp1-like/Krüppel-like family of transcription factors. Several functional domains involved in transcriptional control and subcellular localization have been identified in the vertebrate TIEGs. However, little is known of whether these domains and functions are also conserved in the Cbt protein. Methodology/Principal Findings To determine the transcriptional regulatory activity of the Drosophila Cbt protein, we performed Gal4-based luciferase assays in S2 cells and showed that Cbt is a transcriptional repressor and able to regulate its own expression. Truncated forms of Cbt were then generated to identify its functional domains. This analysis revealed a sequence similar to the mSin3A-interacting repressor domain found in vertebrate TIEGs, although located in a different part of the Cbt protein. Using β-Galactosidase and eGFP fusion proteins, we also showed that Cbt contains the bipartite nuclear localization signal (NLS) previously identified in TIEG proteins, although it is non-functional in insect cells. Instead, a monopartite NLS, located at the amino terminus of the protein and conserved across insects, is functional in Drosophila S2 and Spodoptera exigua Sec301 cells. Last but not least, genetic interaction and immunohistochemical assays suggested that Cbt nuclear import is mediated by Importin-α2. Conclusions/Significance Our results constitute the first characterization of the molecular mechanisms of Cbt-mediated transcriptional control as well as of Cbt nuclear import, and demonstrate the existence of similarities and differences in both aspects of Cbt function between the insect and the vertebrate TIEG proteins. PMID:22359651

Heat Priming Induces Trans-generational Tolerance to High Temperature Stress in Wheat

PubMed Central

Wang, Xiao; Xin, Caiyun; Cai, Jian; Zhou, Qin; Dai, Tingbo; Cao, Weixing; Jiang, Dong

2016-01-01

Wheat plants are very sensitive to high temperature stress during grain filling. Effects of heat priming applied to the first generation on tolerance of the successive generation to post-anthesis high temperature stress were investigated. Compared with the progeny of non-heat primed plants (NH), the progeny of heat-primed plants (PH) possessed higher grain yield, leaf photosynthesis and activities of antioxidant enzymes and lower cell membrane damage under high temperature stress. In the transcriptome profile, 1430 probes showed obvious difference in expression between PH and NH. These genes were related to signal transduction, transcription, energy, defense, and protein destination and storage, respectively. The gene encoding the lysine-specific histone demethylase 1 (LSD1) which was involved in histone demethylation related to epigenetic modification was up-regulated in the PH compared with NH. The proteome analysis indicated that the proteins involved in photosynthesis, energy production and protein destination and storage were up-regulated in the PH compared with NH. In short, thermos-tolerance was induced through heritable epigenetic alternation and signaling transduction, both processes further triggered prompt modifications of defense related responses in anti-oxidation, transcription, energy production, and protein destination and storage in the progeny of the primed plants under high temperature stress. It was concluded that trans-generation thermo-tolerance was induced by heat priming in the first generation, and this might be an effective measure to cope with severe high-temperature stresses during key growth stages in wheat production. PMID:27148324

Generation and Surface Localization of Intact M Protein in Streptococcus pyogenes Are Dependent on sagA

PubMed Central

Biswas, Indranil; Germon, Pierre; McDade, Kathleen; Scott, June R.

2001-01-01

The M protein is an important surface-located virulence factor of Streptococcus pyogenes, the group A streptococcus (GAS). Expression of M protein is primarily controlled by Mga, a transcriptional activator protein. A recent report suggested that the sag locus, which includes nine genes necessary and sufficient for production of streptolysin S, another GAS virulence factor, is also needed for transcription of emm, encoding the M protein (Z. Li, D. D. Sledjeski, B. Kreikemeyer, A. Podbielski, and M. D. Boyle, J. Bacteriol. 181:6019–6027, 1999). To investigate this in more detail, we constructed an insertion-deletion mutation in sagA, the first gene in the sag locus, in the M6 strain JRS4. The resulting strain, JRS470, produced no detectable streptolysin S and showed a drastic reduction in cell surface-associated M protein, as measured by cell aggregation and Western blot analysis. However, transcription of the emm gene was unaffected by the sagA mutation. Detailed analysis with monoclonal antibodies and an antipeptide antibody showed that the M protein in the sagA mutant strain was truncated so that it lacks the C-repeat region and the C-terminal domain required for anchoring it to the cell surface. This truncated M protein was largely found, as expected, in the culture supernatant. Lack of surface-located M protein made the sagA mutant strain susceptible to phagocytosis. Thus, although sagA does not affect transcription of the M6 protein gene, it is needed for the surface localization of this important virulence factor. PMID:11598078

Systems-wide analysis of manganese deficiency-induced changes in gene activity of Arabidopsis roots

PubMed Central

Rodríguez-Celma, Jorge; Tsai, Yi-Hsiu; Wen, Tuan-Nan; Wu, Yu-Ching; Curie, Catherine; Schmidt, Wolfgang

2016-01-01

Manganese (Mn) is pivotal for plant growth and development, but little information is available regarding the strategies that evolved to improve Mn acquisition and cellular homeostasis of Mn. Using an integrated RNA-based transcriptomic and high-throughput shotgun proteomics approach, we generated a comprehensive inventory of transcripts and proteins that showed altered abundance in response to Mn deficiency in roots of the model plant Arabidopsis. A suite of 22,385 transcripts was consistently detected in three RNA-seq runs; LC-MS/MS-based iTRAQ proteomics allowed the unambiguous determination of 11,606 proteins. While high concordance between mRNA and protein expression (R = 0.87) was observed for transcript/protein pairs in which both gene products accumulated differentially upon Mn deficiency, only approximately 10% of the total alterations in the abundance of proteins could be attributed to transcription, indicating a large impact of protein-level regulation. Differentially expressed genes spanned a wide range of biological functions, including the maturation, translation, and transport of mRNAs, as well as primary and secondary metabolic processes. Metabolic analysis by UPLC-qTOF-MS revealed that the steady-state levels of several major glucosinolates were significantly altered upon Mn deficiency in both roots and leaves, possibly as a compensation for increased pathogen susceptibility under conditions of Mn deficiency. PMID:27804982

The significance of alternative transcripts for Caenorhabditis elegans transcription factor genes, based on expression pattern analysis

PubMed Central

2013-01-01

Background Sequence-specific DNA-binding proteins, with their paramount importance in the regulation of expression of the genetic material, are encoded by approximately 5% of the genes in an animal’s genome. But it is unclear to what extent alternative transcripts from these genes may further increase the complexity of the transcription factor complement. Results Of the 938 potential C. elegans transcription factor genes, 197 were annotated in WormBase as encoding at least two distinct isoforms. Evaluation of prior evidence identified, with different levels of confidence, 50 genes with alternative transcript starts, 23 with alternative transcript ends, 35 with alternative splicing and 34 with alternative transcripts generated by a combination of mechanisms, leaving 55 that were discounted. Expression patterns were determined for transcripts for a sample of 29 transcription factor genes, concentrating on those with alternative transcript starts for which the evidence was strongest. Seamless fosmid recombineering was used to generate reporter gene fusions with minimal modification to assay expression of specific transcripts while maintaining the broad genomic DNA context and alternative transcript production. Alternative transcription factor gene transcripts were typically expressed with identical or substantially overlapping distributions rather than in distinct domains. Conclusions Increasingly sensitive sequencing technologies will reveal rare transcripts but many of these are clearly non-productive. The majority of the transcription factor gene alternative transcripts that are productive may represent tolerable noise rather than encoding functionally distinct isoforms. PMID:23586691

ReTrOS: a MATLAB toolbox for reconstructing transcriptional activity from gene and protein expression data.

PubMed

Minas, Giorgos; Momiji, Hiroshi; Jenkins, Dafyd J; Costa, Maria J; Rand, David A; Finkenstädt, Bärbel

2017-06-26

Given the development of high-throughput experimental techniques, an increasing number of whole genome transcription profiling time series data sets, with good temporal resolution, are becoming available to researchers. The ReTrOS toolbox (Reconstructing Transcription Open Software) provides MATLAB-based implementations of two related methods, namely ReTrOS-Smooth and ReTrOS-Switch, for reconstructing the temporal transcriptional activity profile of a gene from given mRNA expression time series or protein reporter time series. The methods are based on fitting a differential equation model incorporating the processes of transcription, translation and degradation. The toolbox provides a framework for model fitting along with statistical analyses of the model with a graphical interface and model visualisation. We highlight several applications of the toolbox, including the reconstruction of the temporal cascade of transcriptional activity inferred from mRNA expression data and protein reporter data in the core circadian clock in Arabidopsis thaliana, and how such reconstructed transcription profiles can be used to study the effects of different cell lines and conditions. The ReTrOS toolbox allows users to analyse gene and/or protein expression time series where, with appropriate formulation of prior information about a minimum of kinetic parameters, in particular rates of degradation, users are able to infer timings of changes in transcriptional activity. Data from any organism and obtained from a range of technologies can be used as input due to the flexible and generic nature of the model and implementation. The output from this software provides a useful analysis of time series data and can be incorporated into further modelling approaches or in hypothesis generation.

Two-stage control of an oxidative stress regulon: the Escherichia coli SoxR protein triggers redox-inducible expression of the soxS regulatory gene.

PubMed Central

Nunoshiba, T; Hidalgo, E; Amábile Cuevas, C F; Demple, B

1992-01-01

Escherichia coli responds to the redox stress imposed by superoxide-generating agents such as paraquat by activating the synthesis of as many as 80 polypeptides. Expression of a key group of these inducible proteins is controlled at the transcriptional level by the soxRS locus (the soxRS regulon). A two-stage control system was hypothesized for soxRS, in which an intracellular redox signal would trigger the SoxR protein as a transcriptional activator of the soxS gene and the resulting increased levels of SoxS protein would activate transcription of the various soxRS regulon genes (B. Demple and C.F. Amábile Cuevas, Cell 67:837-839, 1990). We have constructed operon fusions of the E. coli lac genes to the soxS promoter to monitor soxS transcription. Expression from the soxS promoter is strongly inducible by paraquat in a manner strictly dependent on a functional soxR gene. Several other superoxide-generating agents also trigger soxR(+)-dependent soxS expression, and the inductions by paraquat and phenazine methosulfate were dependent on the presence of oxygen. Numerous other oxidative stress agents (H2O2, gamma rays, heat shock, etc.) failed to induce soxS, while aerobic growth of superoxide dismutase-deficient bacteria triggered soxR-dependent soxS expression. These results indicate a specific redox signal for soxS induction. A direct role for SoxR protein in the activation of the soxS gene is indicated by band-shift and DNase I footprinting experiments that demonstrate specific binding of the SoxR protein in cell extracts to the soxS promoter. The mode of SoxR binding to DNA appears to be similar to that of its homolog MerR in that the SoxR footprint spans the -10 to -35 region of the soxS promoter. Images PMID:1400156

The artificial gene Jazz, a transcriptional regulator of utrophin, corrects the dystrophic pathology in mdx mice.

PubMed

Di Certo, Maria Grazia; Corbi, Nicoletta; Strimpakos, Georgios; Onori, Annalisa; Luvisetto, Siro; Severini, Cinzia; Guglielmotti, Angelo; Batassa, Enrico Maria; Pisani, Cinzia; Floridi, Aristide; Benassi, Barbara; Fanciulli, Maurizio; Magrelli, Armando; Mattei, Elisabetta; Passananti, Claudio

2010-03-01

The absence of the cytoskeletal protein dystrophin results in Duchenne muscular dystrophy (DMD). The utrophin protein is the best candidate for dystrophin replacement in DMD patients. To obtain therapeutic levels of utrophin expression in dystrophic muscle, we developed an alternative strategy based on the use of artificial zinc finger transcription factors (ZF ATFs). The ZF ATF 'Jazz' was recently engineered and tested in vivo by generating a transgenic mouse specifically expressing Jazz at the muscular level. To validate the ZF ATF technology for DMD treatment we generated a second mouse model by crossing Jazz-transgenic mice with dystrophin-deficient mdx mice. Here, we show that the artificial Jazz protein restores sarcolemmal integrity and prevents the development of the dystrophic disease in mdx mice. This exclusive animal model establishes the notion that utrophin-based therapy for DMD can be efficiently developed using ZF ATF technology and candidates Jazz as a novel therapeutic molecule for DMD therapy.

Transcription factor-based modulation of neural stem cell differentiation using direct protein transduction

PubMed Central

Stock, Kristin; Nolden, Lars; Edenhofer, Frank; Quandel, Tamara

2010-01-01

In contrast to conventional gene transfer strategies, the direct introduction of recombinant proteins into cells bypasses the risk of insertional mutagenesis and offers an alternative to genetic intervention. Here, we explore whether protein transduction of the gliogenic transcription factor Nkx2.2 can be used to promote oligodendroglial differentiation of mouse embryonic stem cell (ESC)-derived neural stem cells (NSC). To that end, a recombinant cell-permeant form of Nkx2.2 protein was generated. Exposure of ESC-derived NSC to the recombinant protein and initiation of differentiation resulted in a two-fold increase in the number of oligodendrocytes. Furthermore, Nkx2.2-transduced cells exhibited a more mature oligodendroglial phenotype. Comparative viral gene transfer studies showed that the biological effect of Nkx2.2 protein transduction is comparable to that obtained by lentiviral transduction. The results of this proof-of-concept study depict direct intracellular delivery of transcription factors as alternative modality to control lineage differentiation in NSC cultures without genetic modification. Electronic supplementary material The online version of this article (doi:10.1007/s00018-010-0347-1) contains supplementary material, which is available to authorized users. PMID:20352468

Efficient generation of transgenic reporter strains and analysis of expression patterns in Caenorhabditis elegans using Library MosSCI

PubMed Central

Kaymak, Ebru; Farley, Brian M.; Hay, Samantha A.; Li, Chihua; Ho, Samantha; Hartman, Daniel J.; Ryder, Sean P.

2016-01-01

Background In C. elegans, germline development and early embryogenesis rely on post-transcriptional regulation of maternally transcribed mRNAs. In many cases, the 3′UTR is sufficient to govern the expression patterns of these transcripts. Several RNA-binding proteins are required to regulate maternal mRNAs through the 3′UTR. Despite intensive efforts to map RNA-binding protein-mRNA interactions in vivo, the biological impact of most binding events remains unknown. Reporter studies using single copy integrated transgenes are essential to evaluate the functional consequences of interactions between RNA-binding proteins and their associated mRNAs. Results In this report, we present an efficient method of generating reporter strains with improved throughput by using a library variant of MosSCI transgenesis. Furthermore, using RNA interference, we identify the suite of RBPs that control the expression pattern of five different maternal mRNAs. Conclusions The results provide a generalizable and efficient strategy to assess the functional relevance of protein-RNA interactions in vivo, and reveal new regulatory connections between key RNA-binding proteins and their maternal mRNA targets. PMID:27294288

A Change in SHATTERPROOF Protein Lies at the Origin of a Fruit Morphological Novelty and a New Strategy for Seed Dispersal in Medicago Genus1[C][W

PubMed Central

Fourquin, Chloé; del Cerro, Carolina; Victoria, Filipe C.; Vialette-Guiraud, Aurélie; de Oliveira, Antonio C.; Ferrándiz, Cristina

2013-01-01

Angiosperms are the most diverse and numerous group of plants, and it is generally accepted that this evolutionary success owes in part to the diversity found in fruits, key for protecting the developing seeds and ensuring seed dispersal. Although studies on the molecular basis of morphological innovations are few, they all illustrate the central role played by transcription factors acting as developmental regulators. Here, we show that a small change in the protein sequence of a MADS-box transcription factor correlates with the origin of a highly modified fruit morphology and the change in seed dispersal strategies that occurred in Medicago, a genus belonging to the large legume family. This protein sequence modification alters the functional properties of the protein, affecting the affinities for other protein partners involved in high-order complexes. Our work illustrates that variation in coding regions can generate evolutionary novelties not based on gene duplication/subfunctionalization but by interactions in complex networks, contributing also to the current debate on the relative importance of changes in regulatory or coding regions of master regulators in generating morphological novelties. PMID:23640757

Basic Principles and Emerging Concepts in the Redox Control of Transcription Factors

PubMed Central

Flohé, Leopold

2011-01-01

Abstract Convincing concepts of redox control of gene transcription have been worked out for prokaryotes and lower eukaryotes, whereas the knowledge on complex mammalian systems still resembles a patchwork of poorly connected findings. The article, therefore, reviews principles of redox regulation with special emphasis on chemical feasibility, kinetic requirements, specificity, and physiological context, taking well investigated mammalian transcription factor systems, nuclear transcription factor of bone marrow-derived lymphocytes (NF-κB), and kelch-like ECH-associated protein-1 (Keap1)/Nrf2, as paradigms. Major conclusions are that (i) direct signaling by free radicals is restricted to O2•− and •NO and can be excluded for fast reacting radicals such as •OH, •OR, or Cl•; (ii) oxidant signals are H2O2, enzymatically generated lipid hydroperoxides, and peroxynitrite; (iii) free radical damage is sensed via generation of Michael acceptors; (iv) protein thiol oxidation/alkylation is the prominent mechanism to modulate function; (v) redox sensors must be thiol peroxidases by themselves or proteins with similarly reactive cysteine or selenocysteine (Sec) residues to kinetically compete with glutathione peroxidase (GPx)- and peroxiredoxin (Prx)-type peroxidases or glutathione-S-transferases, respectively, a postulate that still has to be verified for putative mammalian sensors. S-transferases and Prxs are considered for system complementation. The impact of NF-κB and Nrf2 on hormesis, management of inflammatory diseases, and cancer prevention is critically discussed. Antioxid. Redox Signal. 15, 2335–2381. PMID:21194351

Transcriptomic analysis reveals numerous diverse protein kinases and transcription factors involved in desiccation tolerance in the resurrection plant Myrothamnus flabellifolia

PubMed Central

Ma, Chao; Wang, Hong; Macnish, Andrew J; Estrada-Melo, Alejandro C; Lin, Jing; Chang, Youhong; Reid, Michael S; Jiang, Cai-Zhong

2015-01-01

The woody resurrection plant Myrothamnus flabellifolia has remarkable tolerance to desiccation. Pyro-sequencing technology permitted us to analyze the transcriptome of M. flabellifolia during both dehydration and rehydration. We identified a total of 8287 and 8542 differentially transcribed genes during dehydration and rehydration treatments respectively. Approximately 295 transcription factors (TFs) and 484 protein kinases (PKs) were up- or down-regulated in response to desiccation stress. Among these, the transcript levels of 53 TFs and 91 PKs increased rapidly and peaked early during dehydration. These regulators transduce signal cascades of molecular pathways, including the up-regulation of ABA-dependent and independent drought stress pathways and the activation of protective mechanisms for coping with oxidative damage. Antioxidant systems are up-regulated, and the photosynthetic system is modified to reduce ROS generation. Secondary metabolism may participate in the desiccation tolerance of M. flabellifolia as indicated by increases in transcript abundance of genes involved in isopentenyl diphosphate biosynthesis. Up-regulation of genes encoding late embryogenesis abundant proteins and sucrose phosphate synthase is also associated with increased tolerance to desiccation. During rehydration, the transcriptome is also enriched in transcripts of genes encoding TFs and PKs, as well as genes involved in photosynthesis, and protein synthesis. The data reported here contribute comprehensive insights into the molecular mechanisms of desiccation tolerance in M. flabellifolia. PMID:26504577

Regulation of enzyme activities in carnivorous pitcher plants of the genus Nepenthes.

PubMed

Saganová, Michaela; Bokor, Boris; Stolárik, Tibor; Pavlovič, Andrej

2018-05-16

Nepenthes regulates enzyme activities by sensing stimuli from the insect prey. Protein is the best inductor mimicking the presence of an insect prey. Carnivorous plants of the genus Nepenthes have evolved passive pitcher traps for prey capture. In this study, we investigated the ability of chemical signals from a prey (chitin, protein, and ammonium) to induce transcription and synthesis of digestive enzymes in Nepenthes × Mixta. We used real-time PCR and specific antibodies generated against the aspartic proteases nepenthesins, and type III and type IV chitinases to investigate the induction of digestive enzyme synthesis in response to different chemical stimuli from the prey. Transcription of nepenthesins was strongly induced by ammonium, protein and live prey; chitin induced transcription only very slightly. This is in accordance with the amount of released enzyme and proteolytic activity in the digestive fluid. Although transcription of type III chitinase was induced by all investigated stimuli, a significant accumulation of the enzyme in the digestive fluid was found mainly after protein and live prey addition. Protein and live prey were also the best inducers for accumulation of type IV chitinase in the digestive fluid. Although ammonium strongly induced transcription of all investigated genes probably through membrane depolarization, strong acidification of the digestive fluid affected stability and abundance of both chitinases in the digestive fluid. The study showed that the proteins are universal inductors of enzyme activities in carnivorous pitcher plants best mimicking the presence of insect prey. This is not surprising, because proteins are a much valuable source of nitrogen, superior to chitin. Extensive vesicular activity was observed in prey-activated glands.

Mechanistic Insight into the Host Transcription Inhibition Function of Rift Valley Fever Virus NSs and Its Importance in Virulence

PubMed Central

Terasaki, Kaori; Ramirez, Sydney I.; Makino, Shinji

2016-01-01

Rift Valley fever virus (RVFV), a member of the genus Phlebovirus within the family Bunyaviridae, causes periodic outbreaks in livestocks and humans in countries of the African continent and Middle East. RVFV NSs protein, a nonstructural protein, is a major virulence factor that exhibits several important biological properties. These include suppression of general transcription, inhibition of IFN-β promoter induction and degradation of double-stranded RNA-dependent protein kinase R. Although each of these biological functions of NSs are considered important for countering the antiviral response in the host, the individual contributions of these functions towards RVFV virulence remains unclear. To examine this, we generated two RVFV MP-12 strain-derived mutant viruses. Each carried mutations in NSs that specifically targeted its general transcription inhibition function without affecting its ability to degrade PKR and inhibit IFN-β promoter induction, through its interaction with Sin3-associated protein 30, a part of the repressor complex at the IFN-β promoter. Using these mutant viruses, we have dissected the transcription inhibition function of NSs and examined its importance in RVFV virulence. Both NSs mutant viruses exhibited a differentially impaired ability to inhibit host transcription when compared with MP-12. It has been reported that NSs suppresses general transcription by interfering with the formation of the transcription factor IIH complex, through the degradation of the p62 subunit and sequestration of the p44 subunit. Our study results lead us to suggest that the ability of NSs to induce p62 degradation is the major contributor to its general transcription inhibition property, whereas its interaction with p44 may not play a significant role in this function. Importantly, RVFV MP-12-NSs mutant viruses with an impaired general transcription inhibition function showed a reduced cytotoxicity in cell culture and attenuated virulence in young mice, compared with its parental virus MP-12, highlighting the contribution of NSs-mediated general transcription inhibition towards RVFV virulence. PMID:27711108

Mechanistic Insight into the Host Transcription Inhibition Function of Rift Valley Fever Virus NSs and Its Importance in Virulence.

PubMed

Terasaki, Kaori; Ramirez, Sydney I; Makino, Shinji

2016-10-01

Rift Valley fever virus (RVFV), a member of the genus Phlebovirus within the family Bunyaviridae, causes periodic outbreaks in livestocks and humans in countries of the African continent and Middle East. RVFV NSs protein, a nonstructural protein, is a major virulence factor that exhibits several important biological properties. These include suppression of general transcription, inhibition of IFN-β promoter induction and degradation of double-stranded RNA-dependent protein kinase R. Although each of these biological functions of NSs are considered important for countering the antiviral response in the host, the individual contributions of these functions towards RVFV virulence remains unclear. To examine this, we generated two RVFV MP-12 strain-derived mutant viruses. Each carried mutations in NSs that specifically targeted its general transcription inhibition function without affecting its ability to degrade PKR and inhibit IFN-β promoter induction, through its interaction with Sin3-associated protein 30, a part of the repressor complex at the IFN-β promoter. Using these mutant viruses, we have dissected the transcription inhibition function of NSs and examined its importance in RVFV virulence. Both NSs mutant viruses exhibited a differentially impaired ability to inhibit host transcription when compared with MP-12. It has been reported that NSs suppresses general transcription by interfering with the formation of the transcription factor IIH complex, through the degradation of the p62 subunit and sequestration of the p44 subunit. Our study results lead us to suggest that the ability of NSs to induce p62 degradation is the major contributor to its general transcription inhibition property, whereas its interaction with p44 may not play a significant role in this function. Importantly, RVFV MP-12-NSs mutant viruses with an impaired general transcription inhibition function showed a reduced cytotoxicity in cell culture and attenuated virulence in young mice, compared with its parental virus MP-12, highlighting the contribution of NSs-mediated general transcription inhibition towards RVFV virulence.

De novo assembly and characterization of the Trichuris trichiura adult worm transcriptome using Ion Torrent sequencing.

PubMed

Santos, Leonardo N; Silva, Eduardo S; Santos, André S; De Sá, Pablo H; Ramos, Rommel T; Silva, Artur; Cooper, Philip J; Barreto, Maurício L; Loureiro, Sebastião; Pinheiro, Carina S; Alcantara-Neves, Neuza M; Pacheco, Luis G C

2016-07-01

Infection with helminthic parasites, including the soil-transmitted helminth Trichuris trichiura (human whipworm), has been shown to modulate host immune responses and, consequently, to have an impact on the development and manifestation of chronic human inflammatory diseases. De novo derivation of helminth proteomes from sequencing of transcriptomes will provide valuable data to aid identification of parasite proteins that could be evaluated as potential immunotherapeutic molecules in near future. Herein, we characterized the transcriptome of the adult stage of the human whipworm T. trichiura, using next-generation sequencing technology and a de novo assembly strategy. Nearly 17.6 million high-quality clean reads were assembled into 6414 contiguous sequences, with an N50 of 1606bp. In total, 5673 protein-encoding sequences were confidentially identified in the T. trichiura adult worm transcriptome; of these, 1013 sequences represent potential newly discovered proteins for the species, most of which presenting orthologs already annotated in the related species T. suis. A number of transcripts representing probable novel non-coding transcripts for the species T. trichiura were also identified. Among the most abundant transcripts, we found sequences that code for proteins involved in lipid transport, such as vitellogenins, and several chitin-binding proteins. Through a cross-species expression analysis of gene orthologs shared by T. trichiura and the closely related parasites T. suis and T. muris it was possible to find twenty-six protein-encoding genes that are consistently highly expressed in the adult stages of the three helminth species. Additionally, twenty transcripts could be identified that code for proteins previously detected by mass spectrometry analysis of protein fractions of the whipworm somatic extract that present immunomodulatory activities. Five of these transcripts were amongst the most highly expressed protein-encoding sequences in the T. trichiura adult worm. Besides, orthologs of proteins demonstrated to have potent immunomodulatory properties in related parasitic helminths were also predicted from the T. trichiura de novo assembled transcriptome. Copyright © 2016. Published by Elsevier B.V.

Genome-Wide Spectra of Transcription Insertions and Deletions Reveal That Slippage Depends on RNA:DNA Hybrid Complementarity.

PubMed

Traverse, Charles C; Ochman, Howard

2017-08-29

Advances in sequencing technologies have enabled direct quantification of genome-wide errors that occur during RNA transcription. These errors occur at rates that are orders of magnitude higher than rates during DNA replication, but due to technical difficulties such measurements have been limited to single-base substitutions and have not yet quantified the scope of transcription insertions and deletions. Previous reporter gene assay findings suggested that transcription indels are produced exclusively by elongation complex slippage at homopolymeric runs, so we enumerated indels across the protein-coding transcriptomes of Escherichia coli and Buchnera aphidicola , which differ widely in their genomic base compositions and incidence of repeat regions. As anticipated from prior assays, transcription insertions prevailed in homopolymeric runs of A and T; however, transcription deletions arose in much more complex sequences and were rarely associated with homopolymeric runs. By reconstructing the relocated positions of the elongation complex as inferred from the sequences inserted or deleted during transcription, we show that continuation of transcription after slippage hinges on the degree of nucleotide complementarity within the RNA:DNA hybrid at the new DNA template location. IMPORTANCE The high level of mistakes generated during transcription can result in the accumulation of malfunctioning and misfolded proteins which can alter global gene regulation and in the expenditure of energy to degrade these nonfunctional proteins. The transcriptome-wide occurrence of base substitutions has been elucidated in bacteria, but information on transcription insertions and deletions-errors that potentially have more dire effects on protein function-is limited to reporter gene constructs. Here, we capture the transcriptome-wide spectrum of insertions and deletions in Escherichia coli and Buchnera aphidicola and show that they occur at rates approaching those of base substitutions. Knowledge of the full extent of sequences subject to transcription indels supports a new model of bacterial transcription slippage, one that relies on the number of complementary bases between the transcript and the DNA template to which it slipped. Copyright © 2017 Traverse and Ochman.

The venom gland transcriptome of the Desert Massasauga Rattlesnake (Sistrurus catenatus edwardsii): towards an understanding of venom composition among advanced snakes (Superfamily Colubroidea)

PubMed Central

Pahari, Susanta; Mackessy, Stephen P; Kini, R Manjunatha

2007-01-01

Background Snake venoms are complex mixtures of pharmacologically active proteins and peptides which belong to a small number of superfamilies. Global cataloguing of the venom transcriptome facilitates the identification of new families of toxins as well as helps in understanding the evolution of venom proteomes. Results We have constructed a cDNA library of the venom gland of a threatened rattlesnake (a pitviper), Sistrurus catenatus edwardsii (Desert Massasauga), and sequenced 576 ESTs. Our results demonstrate a high abundance of serine proteinase and metalloproteinase transcripts, indicating that the disruption of hemostasis is a principle mechanism of action of the venom. In addition to the transcripts encoding common venom proteins, we detected two varieties of low abundance unique transcripts in the library; these encode for three-finger toxins and a novel toxin possibly generated from the fusion of two genes. We also observed polyadenylated ribosomal RNAs in the venom gland library, an interesting preliminary obsevation of this unusual phenomenon in a reptilian system. Conclusion The three-finger toxins are characteristic of most elapid venoms but are rare in viperid venoms. We detected several ESTs encoding this group of toxins in this study. We also observed the presence of a transcript encoding a fused protein of two well-characterized toxins (Kunitz/BPTI and Waprins), and this is the first report of this kind of fusion in a snake toxin transcriptome. We propose that these new venom proteins may have ancillary functions for envenomation. The presence of a fused toxin indicates that in addition to gene duplication and accelerated evolution, exon shuffling or transcriptional splicing may also contribute to generating the diversity of toxins and toxin isoforms observed among snake venoms. The detection of low abundance toxins, as observed in this and other studies, indicates a greater compositional similarity of venoms (though potency will differ) among advanced snakes than has been previously recognized. PMID:18096037

Transcriptomic Analysis of Grapevine (cv. Summer Black) Leaf, Using the Illumina Platform

PubMed Central

Pervaiz, Tariq; Haifeng, Jia; Salman Haider, Muhammad; Cheng, Zhang; Cui, Mengjie; Wang, Mengqi; Cui, Liwen; Wang, Xicheng; Fang, Jinggui

2016-01-01

Proceeding to illumina sequencing, determining RNA integrity numbers for poly RNA were separated from each of the four developmental stages of cv. Summer Black leaves by using Illumina HiSeq™ 2000. The sums of 272,941,656 reads were generated from vitis vinifera leaf at four different developmental stages, with more than 27 billion nucleotides of the sequence data. At each growth stage, RNA samples were indexed through unique nucleic acid identifiers and sequenced. KEGG annotation results depicted that the highest number of transcripts in 2,963 (2Avs4A) followed by 1Avs4A (2,920), and 3Avs4A (2,294) out of 15,614 (71%) transcripts were recorded. In comparison, a total of 1,532 transcripts were annotated in GOs, including Cellular component, with the highest number in “Cell part” 251 out of 353 transcripts (71.1%), followed by intracellular organelle 163 out of 353 transcripts (46.2%), while in molecular function and metabolic process 375 out of 525 (71.4%) transcripts, multicellular organism process 40 out of 525 (7.6%) transcripts in biological process were most common in 1Avs2A. While in case of 1Avs3A, cell part 476 out of 662 transcripts (71.9%), and membrane-bounded organelle 263 out of 662 transcripts (39.7%) were recorded in Cellular component. In the grapevine transcriptome, during the initial stages of leaf development 1Avs2A showed single transcript was down-regulated and none of them were up-regulated. While in comparison of 1A to 3A showed one up-regulated (photosystem II reaction center protein C) and one down regulated (conserved gene of unknown function) transcripts, during the hormone regulating pathway namely SAUR-like auxin-responsive protein family having 2 up-regulated and 7 down-regulated transcripts, phytochrome-associated protein showed 1 up-regulated and 9 down-regulated transcripts, whereas genes associated with the Leucine-rich repeat protein kinase family protein showed 7 up-regulated and 1 down-regulated transcript, meanwhile Auxin Resistant 2 has single up-regulated transcript in second developmental stage, although 3 were down-regulated at lateral growth stages (3A and 4A). In the present study, 489 secondary metabolic pathways related genes were identified during leaf growth, which mainly includes alkaloid (40), anthocyanins (21), Diterpenoid (144), Monoterpenoid (90) and Flavonoids (93). Quantitative real-time PCR was applied to validate 10 differentially expressed transcripts patterns from flower, leaf and fruit metabolic pathways at different growth stages. PMID:26824474

Tight Junction–Associated Signaling Pathways Modulate Cell Proliferation in Uveal Melanoma

PubMed Central

Jayagopal, Ashwath; Yang, Jin-Long; Haselton, Frederick R.; Chang, Min S.

2011-01-01

Purpose. To investigate the role of tight junction (TJ)–associated signaling pathways in the proliferation of uveal melanoma. Methods. Human uveal melanoma cell lines overexpressing the TJ molecule blood vessel epicardial substance (Bves) were generated. The effects of Bves overexpression on TJ protein expression, cell proliferation, and cell cycle distribution were quantified. In addition, localization and transcription activity of the TJ-associated protein ZO-1–associated nucleic acid binding protein (ZONAB) were evaluated using immunofluorescence and bioluminescence reporter assays to study the involvement of Bves signaling in cell proliferation-associated pathways. Results. Bves overexpression in uveal melanoma cell lines resulted in increased expression of the TJ proteins occludin and ZO-1, reduced cell proliferation, and increased sequestration of ZONAB at TJs and reduced ZONAB transcriptional activity. Conclusions. TJ proteins are present in uveal melanoma, and TJ-associated signaling pathways modulate cell signaling pathways relevant to proliferation in uveal melanoma. PMID:20861479

Amino Acids of Epstein-Barr Virus Nuclear Antigen 3A Essential for Repression of Jκ-Mediated Transcription and Their Evolutionary Conservation

PubMed Central

Dalbiès-Tran, Rozenn; Stigger-Rosser, Evelyn; Dotson, Travis; Sample, Clare E.

2001-01-01

Epstein-Barr virus (EBV) nuclear antigen 3A (EBNA-3A) is essential for virus-mediated immortalization of B lymphocytes in vitro and is believed to regulate transcription of cellular and/or viral genes. One known mechanism of regulation is through its interaction with the cellular transcription factor Jκ. This interaction downregulates transcription mediated by EBNA-2 and Jκ. To identify the amino acids that play a role in this interaction, we have generated mutant EBNA-3A proteins. A mutant EBNA-3A protein in which alanine residues were substituted for amino acids 199, 200, and 202 no longer downregulated transcription. Surprisingly, this mutant protein remained able to coimmunoprecipitate with Jκ. Using a reporter gene assay based on the recruitment of Jκ by various regions spanning EBNA-3A, we have shown that this mutation abolished binding of Jκ to the N-proximal region (amino acids 125 to 222) and that no other region of EBNA-3A alone was sufficient to mediate an association with Jκ. To determine the biological significance of the interaction of EBNA-3A with Jκ, we have studied its conservation in the simian lymphocryptovirus herpesvirus papio (HVP) by cloning HVP-3A, the homolog of EBNA-3A encoded by this virus. This 903-amino-acid protein exhibited 37% identity with its EBV counterpart, mainly within the amino-terminal half. HVP-3A also interacted with Jκ through a region located between amino acids 127 and 223 and also repressed transcription mediated through EBNA-2 and Jκ. The evolutionary conservation of this function, in proteins that have otherwise significantly diverged, argues strongly for an important biological role in virus-mediated immortalization of B lymphocytes. PMID:11119577

Direct modulation of T-box riboswitch-controlled transcription by protein synthesis inhibitors

PubMed Central

Stamatopoulou, Vassiliki; Apostolidi, Maria; Li, Shuang; Lamprinou, Katerina; Papakyriakou, Athanasios

2017-01-01

Abstract Recently, it was discovered that exposure to mainstream antibiotics activate numerous bacterial riboregulators that control antibiotic resistance genes including metabolite-binding riboswitches and other transcription attenuators. However, the effects of commonly used antibiotics, many of which exhibit RNA-binding properties, on the widespread T-box riboswitches, remain unknown. In Staphylococcus aureus, a species-specific glyS T-box controls the supply of glycine for both ribosomal translation and cell wall synthesis, making it a promising target for next-generation antimicrobials. Here, we report that specific protein synthesis inhibitors could either significantly increase T-box-mediated transcription antitermination, while other compounds could suppress it, both in vitro and in vivo. In-line probing of the full-length T-box combined with molecular modelling and docking analyses suggest that the antibiotics that promote transcription antitermination stabilize the T-box:tRNA complex through binding specific positions on stem I and the Staphylococcal-specific stem Sa. By contrast, the antibiotics that attenuate T-box transcription bind to other positions on stem I and do not interact with stem Sa. Taken together, our results reveal that the transcription of essential genes controlled by T-box riboswitches can be directly modulated by commonly used protein synthesis inhibitors. These findings accentuate the regulatory complexities of bacterial response to antimicrobials that involve multiple riboregulators. PMID:28973457

Direct modulation of T-box riboswitch-controlled transcription by protein synthesis inhibitors.

PubMed

Stamatopoulou, Vassiliki; Apostolidi, Maria; Li, Shuang; Lamprinou, Katerina; Papakyriakou, Athanasios; Zhang, Jinwei; Stathopoulos, Constantinos

2017-09-29

Recently, it was discovered that exposure to mainstream antibiotics activate numerous bacterial riboregulators that control antibiotic resistance genes including metabolite-binding riboswitches and other transcription attenuators. However, the effects of commonly used antibiotics, many of which exhibit RNA-binding properties, on the widespread T-box riboswitches, remain unknown. In Staphylococcus aureus, a species-specific glyS T-box controls the supply of glycine for both ribosomal translation and cell wall synthesis, making it a promising target for next-generation antimicrobials. Here, we report that specific protein synthesis inhibitors could either significantly increase T-box-mediated transcription antitermination, while other compounds could suppress it, both in vitro and in vivo. In-line probing of the full-length T-box combined with molecular modelling and docking analyses suggest that the antibiotics that promote transcription antitermination stabilize the T-box:tRNA complex through binding specific positions on stem I and the Staphylococcal-specific stem Sa. By contrast, the antibiotics that attenuate T-box transcription bind to other positions on stem I and do not interact with stem Sa. Taken together, our results reveal that the transcription of essential genes controlled by T-box riboswitches can be directly modulated by commonly used protein synthesis inhibitors. These findings accentuate the regulatory complexities of bacterial response to antimicrobials that involve multiple riboregulators. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Global Analysis of Transcription Factor-Binding Sites in Yeast Using ChIP-Seq

PubMed Central

Lefrançois, Philippe; Gallagher, Jennifer E. G.; Snyder, Michael

2016-01-01

Transcription factors influence gene expression through their ability to bind DNA at specific regulatory elements. Specific DNA-protein interactions can be isolated through the chromatin immunoprecipitation (ChIP) procedure, in which DNA fragments bound by the protein of interest are recovered. ChIP is followed by high-throughput DNA sequencing (Seq) to determine the genomic provenance of ChIP DNA fragments and their relative abundance in the sample. This chapter describes a ChIP-Seq strategy adapted for budding yeast to enable the genome-wide characterization of binding sites of transcription factors (TFs) and other DNA-binding proteins in an efficient and cost-effective way. Yeast strains with epitope-tagged TFs are most commonly used for ChIP-Seq, along with their matching untagged control strains. The initial step of ChIP involves the cross-linking of DNA and proteins. Next, yeast cells are lysed and sonicated to shear chromatin into smaller fragments. An antibody against an epitope-tagged TF is used to pull down chromatin complexes containing DNA and the TF of interest. DNA is then purified and proteins degraded. Specific barcoded adapters for multiplex DNA sequencing are ligated to ChIP DNA. Short DNA sequence reads (28–36 base pairs) are parsed according to the barcode and aligned against the yeast reference genome, thus generating a nucleotide-resolution map of transcription factor-binding sites and their occupancy. PMID:25213249

A Long Terminal Repeat-Containing Retrotransposon of Schizosaccharomyces pombe Expresses a Gag-Like Protein That Assembles into Virus-Like Particles Which Mediate Reverse Transcription

PubMed Central

Teysset, Laure; Dang, Van-Dinh; Kim, Min Kyung; Levin, Henry L.

2003-01-01

The Tf1 element of Schizosaccharomyces pombe is a long terminal repeat-containing retrotransposon that encodes functional protease, reverse transcriptase, and integrase proteins. Although these proteins are known to be necessary for protein processing, reverse transcription, and integration, respectively, the function of the protein thought to be Gag has not been determined. We present here the first electron microscopy of Tf1 particles. We tested whether the putative Gag of Tf1 was required for particle formation, packaging of RNA, and reverse transcription. We generated deletions of 10 amino acids in each of the four hydrophilic domains of the protein and found that all four mutations reduced transposition activity. The N-terminal deletion removed a nuclear localization signal and inhibited nuclear import of the transposon. The two mutations in the center of Gag destabilized the protein and resulted in no virus-like particles. The C-terminal deletion caused a defect in RNA packaging and, as a result, low levels of cDNA. The electron microscopy of cells expressing a truncated Tf1 showed that Gag alone was sufficient for the formation of virus-like particles. Taken together, these results indicate that Tf1 encodes a Gag protein that is a functional equivalent of the Gag proteins of retroviruses. PMID:12692246

Nuclear cereblon modulates transcriptional activity of Ikaros and regulates its downstream target, enkephalin, in human neuroblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wada, Takeyoshi; Asahi, Toru; Research Organization for Nano & Life Innovation, Waseda University #03C309, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480

2016-08-26

The gene coding cereblon (CRBN) was originally identified in genetic linkage analysis of mild autosomal recessive nonsyndromic intellectual disability. CRBN has broad localization in both the cytoplasm and nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. First, we generated a series of CRBN deletion mutants and determined the regions responsible for the nuclear localization. Only CRBN protein lacking the N-terminal region was localized outside of the nucleus, suggesting that the N-terminal region is important for its nuclear localization. CRBN was also identified as amore » thalidomide-binding protein and component of the cullin-4-containing E3 ubiquitin ligase complex. Thalidomide has been reported to be involved in the regulation of the transcription factor Ikaros by CRBN-mediated degradation. To investigate the nuclear functions of CRBN, we performed co-immunoprecipitation experiments and evaluated the binding of CRBN to Ikaros. As a result, we found that CRBN was associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. In luciferase reporter gene experiments, CRBN modulated transcriptional activity of Ikaros. Furthermore, we found that CRBN modulated Ikaros-mediated transcriptional repression of the proenkephalin gene by binding to its promoter region. These results suggest that CRBN binds to Ikaros via its N-terminal region and regulates transcriptional activities of Ikaros and its downstream target, enkephalin. - Highlights: • We found that CRBN is a nucleocytoplasmic shutting protein and identified the key domain for nucleocytoplasmic shuttling. • CRBN associates with the transcription factor Ikaros via the N-terminal domain. • CRBN modulates Ikaros-mediated transcriptional regulation and its downstream target, enkephalin.« less

Nutrient sensing by the mitochondrial transcription machinery dictates oxidative phosphorylation

PubMed Central

Liu, Lijun; Nam, Minwoo; Fan, Wei; Akie, Thomas E.; Hoaglin, David C.; Gao, Guangping; Keaney, John F.; Cooper, Marcus P.

2014-01-01

Sirtuin 3 (SIRT3), an important regulator of energy metabolism and lipid oxidation, is induced in fasted liver mitochondria and implicated in metabolic syndrome. In fasted liver, SIRT3-mediated increases in substrate flux depend on oxidative phosphorylation (OXPHOS), but precisely how OXPHOS meets the challenge of increased substrate oxidation in fasted liver remains unclear. Here, we show that liver mitochondria in fasting mice adapt to the demand of increased substrate oxidation by increasing their OXPHOS efficiency. In response to cAMP signaling, SIRT3 deacetylated and activated leucine-rich protein 130 (LRP130; official symbol, LRPPRC), promoting a mitochondrial transcriptional program that enhanced hepatic OXPHOS. Using mass spectrometry, we identified SIRT3-regulated lysine residues in LRP130 that generated a lysine-to-arginine (KR) mutant of LRP130 that mimics deacetylated protein. Compared with wild-type LRP130 protein, expression of the KR mutant increased mitochondrial transcription and OXPHOS in vitro. Indeed, even when SIRT3 activity was abolished, activation of mitochondrial transcription and OXPHOS by the KR mutant remained robust, further highlighting the contribution of LRP130 deacetylation to increased OXPHOS in fasted liver. These data establish a link between nutrient sensing and mitochondrial transcription that regulates OXPHOS in fasted liver and may explain how fasted liver adapts to increased substrate oxidation. PMID:24430182

The Nutrient-Sensing Hexosamine Biosynthetic Pathway as the Hub of Cancer Metabolic Rewiring.

PubMed

Chiaradonna, Ferdinando; Ricciardiello, Francesca; Palorini, Roberta

2018-06-02

Alterations in glucose and glutamine utilizing pathways and in fatty acid metabolism are currently considered the most significant and prevalent metabolic changes observed in almost all types of tumors. Glucose, glutamine and fatty acids are the substrates for the hexosamine biosynthetic pathway (HBP). This metabolic pathway generates the "sensing molecule" UDP- N -Acetylglucosamine (UDP-Glc N Ac). UDP-Glc N Ac is the substrate for the enzymes involved in protein N - and O -glycosylation, two important post-translational modifications (PTMs) identified in several proteins localized in the extracellular space, on the cell membrane and in the cytoplasm, nucleus and mitochondria. Since protein glycosylation controls several key aspects of cell physiology, aberrant protein glycosylation has been associated with different human diseases, including cancer. Here we review recent evidence indicating the tight association between the HBP flux and cell metabolism, with particular emphasis on the post-transcriptional and transcriptional mechanisms regulated by the HBP that may cause the metabolic rewiring observed in cancer. We describe the implications of both protein O - and N -glycosylation in cancer cell metabolism and bioenergetics; focusing our attention on the effect of these PTMs on nutrient transport and on the transcriptional regulation and function of cancer-specific metabolic pathways.

De novo Assembly of Leaf Transcriptome in the Medicinal Plant Andrographis paniculata

PubMed Central

Cherukupalli, Neeraja; Divate, Mayur; Mittapelli, Suresh R.; Khareedu, Venkateswara R.; Vudem, Dashavantha R.

2016-01-01

Andrographis paniculata is an important medicinal plant containing various bioactive terpenoids and flavonoids. Despite its importance in herbal medicine, no ready-to-use transcript sequence information of this plant is made available in the public data base, this study mainly deals with the sequencing of RNA from A. paniculata leaf using Illumina HiSeq™ 2000 platform followed by the de novo transcriptome assembly. A total of 189.22 million high quality paired reads were generated and 1,70,724 transcripts were predicted in the primary assembly. Secondary assembly generated a transcriptome size of ~88 Mb with 83,800 clustered transcripts. Based on the similarity searches against plant non-redundant protein database, gene ontology, and eukaryotic orthologous groups, 49,363 transcripts were annotated constituting upto 58.91% of the identified unigenes. Annotation of transcripts—using kyoto encyclopedia of genes and genomes database—revealed 5606 transcripts plausibly involved in 140 pathways including biosynthesis of terpenoids and other secondary metabolites. Transcription factor analysis showed 6767 unique transcripts belonging to 97 different transcription factor families. A total number of 124 CYP450 transcripts belonging to seven divergent clans have been identified. Transcriptome revealed 146 different transcripts coding for enzymes involved in the biosynthesis of terpenoids of which 35 contained terpene synthase motifs. This study also revealed 32,341 simple sequence repeats (SSRs) in 23,168 transcripts. Assembled sequences of transcriptome of A. paniculata generated in this study are made available, for the first time, in the TSA database, which provides useful information for functional and comparative genomic analysis besides identification of key enzymes involved in the various pathways of secondary metabolism. PMID:27582746

Control of regulatory T cell and Th17 cell differentiation by inhibitory helix-loop-helix protein Id3

PubMed Central

Maruyama, Takashi; Li, Jun; Vaque, Jose P.; Konkel, Joanne E.; Wang, Weifeng; Zhang, Baojun; Zhang, Pin; Zamarron, Brian; Yu, Dongyang; Wu, Yuntao; Zhuang, Yuan; Gutkind, J. Silvio; Chen, WanJun

2010-01-01

The molecular mechanisms directing Foxp3 gene transcription in CD4+ T cells remain ill defined. We show that deletion of the inhibitory helix-loop-helix (HLH) protein Id3 results in defective Foxp3+ Treg cell generation. We identified two transforming grothw factor-β1 (TGF-β1)-dependent mechanisms that are vital for activation of Foxp3 gene transcription, and are defective in Id3−/− CD4+ T cells. Enhanced binding of the HLH protein E2A to the Foxp3 promoter promoted Foxp3 gene transcription. Id3 was required to relieve inhibition by GATA-3 at the Foxp3 promoter. Further, Id3−/− T cells increased differentiation of Th17 cells in vitro and in a mouse asthma model. A network of factors therefore act in a TGF-β-dependent manner to control Foxp3 expression and inhibit Th17 cell development. PMID:21131965

Rapid discovery of protein interactions by cell-free protein technologies.

PubMed

He, M; Taussig, M J

2007-11-01

Cell-free transcription and translation provides an open, controllable environment for production of correctly folded, soluble proteins and allows the rapid generation of proteins from DNA without the need for cloning. Thus it is becoming an increasingly attractive alternative to conventional in vivo expression systems, especially when parallel expression of multiple proteins is required. Through novel design and exploitation, powerful cell-free technologies of ribosome display and protein in situ arrays have been developed for in vitro production and isolation of protein-binding molecules from large libraries. These technologies can be combined for rapid detection of protein interactions.

Insights into the nature of DNA binding of AbrB-like transcription factors

PubMed Central

Sullivan, Daniel M.; Bobay, Benjamin G.; Kojetin, Douglas J.; Thompson, Richele J.; Rance, Mark; Strauch, Mark A.; Cavanagh, John

2008-01-01

Summary Understanding the DNA recognition and binding by the AbrB-like family of transcriptional regulators is of significant interest since these proteins enable bacteria to elicit the appropriate response to diverse environmental stimuli. Although these ‘transition-state regulator’ proteins have been well characterized at the genetic level, the general and specific mechanisms of DNA binding remain elusive. We present RDC-refined NMR solution structures and dynamic properties of the DNA-binding domains of three Bacillus subtilis transition-state regulators AbrB, Abh, and SpoVT. We combined previously investigated DNase I footprinting, DNA methylation, gel shift assays, mutagenic and NMR studies to generate a structural model of the complex between AbrBN55 and its cognate promoter, abrB8. These investigations have enabled us to generate the first model for the specific nature of the transition-state regulator-DNA interaction. PMID:19000822

LncRNApred: Classification of Long Non-Coding RNAs and Protein-Coding Transcripts by the Ensemble Algorithm with a New Hybrid Feature.

PubMed

Pian, Cong; Zhang, Guangle; Chen, Zhi; Chen, Yuanyuan; Zhang, Jin; Yang, Tao; Zhang, Liangyun

2016-01-01

As a novel class of noncoding RNAs, long noncoding RNAs (lncRNAs) have been verified to be associated with various diseases. As large scale transcripts are generated every year, it is significant to accurately and quickly identify lncRNAs from thousands of assembled transcripts. To accurately discover new lncRNAs, we develop a classification tool of random forest (RF) named LncRNApred based on a new hybrid feature. This hybrid feature set includes three new proposed features, which are MaxORF, RMaxORF and SNR. LncRNApred is effective for classifying lncRNAs and protein coding transcripts accurately and quickly. Moreover,our RF model only requests the training using data on human coding and non-coding transcripts. Other species can also be predicted by using LncRNApred. The result shows that our method is more effective compared with the Coding Potential Calculate (CPC). The web server of LncRNApred is available for free at http://mm20132014.wicp.net:57203/LncRNApred/home.jsp.

Engineering synthetic TALE and CRISPR/Cas9 transcription factors for regulating gene expression.

PubMed

Kabadi, Ami M; Gersbach, Charles A

2014-09-01

Engineered DNA-binding proteins that can be targeted to specific sites in the genome to manipulate gene expression have enabled many advances in biomedical research. This includes generating tools to study fundamental aspects of gene regulation and the development of a new class of gene therapies that alter the expression of endogenous genes. Designed transcription factors have entered clinical trials for the treatment of human diseases and others are in preclinical development. High-throughput and user-friendly platforms for designing synthetic DNA-binding proteins present innovative methods for deciphering cell biology and designing custom synthetic gene circuits. We review two platforms for designing synthetic transcription factors for manipulating gene expression: Transcription activator-like effectors (TALEs) and the RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. We present an overview of each technology and a guide for designing and assembling custom TALE- and CRISPR/Cas9-based transcription factors. We also discuss characteristics of each platform that are best suited for different applications. Copyright © 2014 Elsevier Inc. All rights reserved.

Post-transcriptional bursting in genes regulated by small RNA molecules

NASA Astrophysics Data System (ADS)

Rodrigo, Guillermo

2018-03-01

Gene expression programs in living cells are highly dynamic due to spatiotemporal molecular signaling and inherent biochemical stochasticity. Here we study a mechanism based on molecule-to-molecule variability at the RNA level for the generation of bursts of protein production, which can lead to heterogeneity in a cell population. We develop a mathematical framework to show numerically and analytically that genes regulated post transcriptionally by small RNA molecules can exhibit such bursts due to different states of translation activity (on or off), mostly revealed in a regime of few molecules. We exploit this framework to compare transcriptional and post-transcriptional bursting and also to illustrate how to tune the resulting protein distribution with additional post-transcriptional regulations. Moreover, because RNA-RNA interactions are predictable with an energy model, we define the kinetic constants of on-off switching as functions of the two characteristic free-energy differences of the system, activation and formation, with a nonequilibrium scheme. Overall, post-transcriptional bursting represents a distinctive principle linking gene regulation to gene expression noise, which highlights the importance of the RNA layer beyond the simple information transfer paradigm and significantly contributes to the understanding of the intracellular processes from a first-principles perspective.

Molecular Mechanisms Regulating Muscle Fiber Composition Under Microgravity

NASA Technical Reports Server (NTRS)

Rosenthal, Nadia A.

1999-01-01

The overall goal of this project is to reveal the molecular mechanisms underlying the selective and debilitating atrophy of specific skeletal muscle fiber types that accompanies sustained conditions of microgravity. Since little is currently known about the regulation of fiber-specific gene expression programs in mammalian muscle, elucidation of the basic mechanisms of fiber diversification is a necessary prerequisite to the generation of therapeutic strategies for attenuation of muscle atrophy on earth or in space. Vertebrate skeletal muscle development involves the fusion of undifferentiated mononucleated myoblasts to form multinucleated myofibers, with a concomitant activation of muscle-specific genes encoding proteins that form the force-generating contractile apparatus. The regulatory circuitry controlling skeletal muscle gene expression has been well studied in a number of vertebrate animal systems. The goal of this project has been to achieve a similar level of understanding of the mechanisms underlying the further specification of muscles into different fiber types, and the role played by innervation and physical activity in the maintenance and adaptation of different fiber phenotypes into adulthood. Our recent research on the genetic basis of fiber specificity has focused on the emergence of mature fiber types and have implicated a group of transcriptional regulatory proteins, known as E proteins, in the control of fiber specificity. The restriction of E proteins to selected muscle fiber types is an attractive hypothetical mechanism for the generation of muscle fiber-specific patterns of gene expression. To date our results support a model wherein different E proteins are selectively expressed in muscle cells to determine fiber-restricted gene expression. These studies are a first step to define the molecular mechanisms responsible for the shifts in fiber type under conditions of microgravity, and to determine the potential importance of E proteins as upstream targets for the effects of weightlessness. In the past year we have determined that the expression of E Proteins is restricted to specific fiber types by post-transcriptional mechanisms. By far, the most prevalent mechanism of cellular control for achieving post-transcriptional regulation of gene expression is selective proteolysis -through the ubiquitin -proteasome pathway. Steady-state levels of HEB message are similar in all fast and slow skeletal muscle fiber types, yet the protein is restricted to Type IIX fibers. HEB appears to be a nodal point for regulating fiber-specific transcription, as expression of the transcription factor is regulated at the post-transcriptional level. It is not clear at present whether the regulation is at the level of protein synthesis or degradation. We are now poised to evaluate the biological role of ubiquitination in fiber specific-gene expression by controlling the post-transcriptional expression of E Proteins. The use of metabolic labelling and pharmacological inhibitors of the ubiquitin pathway will be used to identify the mode of regulation of the Type IIX expression pattern. The potential role of specific kinases in effecting the restriction of HEB expression will be examined by using both inhibitors and activators. The results of these studies will provide the necessary information to evaluate the biological role of E proteins in controlling fiber type transitions, and in potentially attenuating the atrophic effects of microgravity conditions. We have also recently shown that ectopic expression of the HEB protein transactivates the Type IIX-specific skeletal a-actin reporter. The 218 bp skeletal a-actin promoter drives transgene expression solely in mature Type IIX fibers. A mouse also carrying the transgene MLCI/HEB (which ectopically expresses the E Protein HEB in Type IIB fibers) forces expression of the skeletal a-actin reporter gene in Type IIB fibers. We can now dissect the composition of this fiber-specific cis-element. The skeletal a-actin promoter is quite compact and has been extensively characterized in vitro for activity and binding factors. The single E box may act as a binding target of myogenic factor/HEB heterodimer to allow for IIX expression. The HEB transcription factor may recognize either the precise flanking sequences of the E Box, or perhaps interacting with other proteins bound nearby, and activating expression in Type IIX fibers. This E box will be both ablated, and alternatively, as ablation may well destroy any muscle-specific transcriptional activity, flanking sequences substituted with those surrounding the E box (El) of the myogenin promoter. Modification of fiber-specific transgene expression will be tested in transgenic mice. The results of these studies will provide basic information on the regulatory circuitry underlying fiber specificity, and will form the basis for building appropriate transgenic regulatory cassettes to effect fiber transitions in subsequent experimental manipulations on unweighted muscles.

Engineering allostery.

PubMed

Raman, Srivatsan; Taylor, Noah; Genuth, Naomi; Fields, Stanley; Church, George M

2014-12-01

Allosteric proteins have great potential in synthetic biology, but our limited understanding of the molecular underpinnings of allostery has hindered the development of designer molecules, including transcription factors with new DNA-binding or ligand-binding specificities that respond appropriately to inducers. Such allosteric proteins could function as novel switches in complex circuits, metabolite sensors, or as orthogonal regulators for independent, inducible control of multiple genes. Advances in DNA synthesis and next-generation sequencing technologies have enabled the assessment of millions of mutants in a single experiment, providing new opportunities to study allostery. Using the classic LacI protein as an example, we describe a genetic selection system using a bidirectional reporter to capture mutants in both allosteric states, allowing the positions most crucial for allostery to be identified. This approach is not limited to bacterial transcription factors, and could reveal new mechanistic insights and facilitate engineering of other major classes of allosteric proteins such as nuclear receptors, two-component systems, G protein-coupled receptors, and protein kinases. Copyright © 2014 Elsevier Ltd. All rights reserved.

Engineering allostery

DOE PAGES

Raman, Srivatsan; Taylor, Noah; Genuth, Naomi; ...

2014-10-08

Allosteric proteins have great potential in synthetic biology, but our limited understanding of the molecular underpinnings of allostery has hindered the development of designer molecules, including transcription factors with new DNA-binding or ligand-binding specificities that respond appropriately to inducers. Such allosteric proteins could function as novel switches in complex circuits, metabolite sensors, or as orthogonal regulators for independent, inducible control of multiple genes. Advances in DNA synthesis and next-generation sequencing technologies have enabled the assessment of millions of mutants in a single experiment, providing new opportunities to study allostery. Using the classic LacI protein as an example, in this papermore » we describe a genetic selection system using a bidirectional reporter to capture mutants in both allosteric states, allowing the positions most crucial for allostery to be identified. Finally, this approach is not limited to bacterial transcription factors, and could reveal new mechanistic insights and facilitate engineering of other major classes of allosteric proteins such as nuclear receptors, two-component systems, G protein-coupled receptors, and protein kinases.« less

Insights into rubber biosynthesis from transcriptome analysis of Hevea brasiliensis latex.

PubMed

Chow, Keng-See; Wan, Kiew-Lian; Isa, Mohd Noor Mat; Bahari, Azlina; Tan, Siang-Hee; Harikrishna, K; Yeang, Hoong-Yeet

2007-01-01

Hevea brasiliensis is the most widely cultivated species for commercial production of natural rubber (cis-polyisoprene). In this study, 10,040 expressed sequence tags (ESTs) were generated from the latex of the rubber tree, which represents the cytoplasmic content of a single cell type, in order to analyse the latex transcription profile with emphasis on rubber biosynthesis-related genes. A total of 3,441 unique transcripts (UTs) were obtained after quality editing and assembly of EST sequences. Functional classification of UTs according to the Gene Ontology convention showed that 73.8% were related to genes of unknown function. Among highly expressed ESTs, a significant proportion encoded proteins related to rubber biosynthesis and stress or defence responses. Sequences encoding rubber particle membrane proteins (RPMPs) belonging to three protein families accounted for 12% of the ESTs. Characterization of these ESTs revealed nine RPMP variants (7.9-27 kDa) including the 14 kDa REF (rubber elongation factor) and 22 kDa SRPP (small rubber particle protein). The expression of multiple RPMP isoforms in latex was shown using antibodies against REF and SRPP. Both EST and quantitative reverse transcription-PCR (QRT-PCR) analyses demonstrated REF and SRPP to be the most abundant transcripts in latex. Besides rubber biosynthesis, comparative sequence analysis showed that the RPMPs are highly similar to sequences in the plant kingdom having stress-related functions. Implications of the RPMP function in cis-polyisoprene biosynthesis in the context of transcript abundance and differential gene expression are discussed.

RNA-seq Transcriptional Profiling of an Arbuscular Mycorrhiza Provides Insights into Regulated and Coordinated Gene Expression in Lotus japonicus and Rhizophagus irregularis.

PubMed

Handa, Yoshihiro; Nishide, Hiroyo; Takeda, Naoya; Suzuki, Yutaka; Kawaguchi, Masayoshi; Saito, Katsuharu

2015-08-01

Gene expression during arbuscular mycorrhizal development is highly orchestrated in both plants and arbuscular mycorrhizal fungi. To elucidate the gene expression profiles of the symbiotic association, we performed a digital gene expression analysis of Lotus japonicus and Rhizophagus irregularis using a HiSeq 2000 next-generation sequencer with a Cufflinks assembly and de novo transcriptome assembly. There were 3,641 genes differentially expressed during arbuscular mycorrhizal development in L. japonicus, approximately 80% of which were up-regulated. The up-regulated genes included secreted proteins, transporters, proteins involved in lipid and amino acid metabolism, ribosomes and histones. We also detected many genes that were differentially expressed in small-secreted peptides and transcription factors, which may be involved in signal transduction or transcription regulation during symbiosis. Co-regulated genes between arbuscular mycorrhizal and root nodule symbiosis were not particularly abundant, but transcripts encoding for membrane traffic-related proteins, transporters and iron transport-related proteins were found to be highly co-up-regulated. In transcripts of arbuscular mycorrhizal fungi, expansion of cytochrome P450 was observed, which may contribute to various metabolic pathways required to accommodate roots and soil. The comprehensive gene expression data of both plants and arbuscular mycorrhizal fungi provide a powerful platform for investigating the functional and molecular mechanisms underlying arbuscular mycorrhizal symbiosis. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

The mycobacterial antibiotic resistance determinant WhiB7 acts as a transcriptional activator by binding the primary sigma factor SigA (RpoV)

PubMed Central

Burian, Ján; Yim, Grace; Hsing, Michael; Axerio-Cilies, Peter; Cherkasov, Artem; Spiegelman, George B.; Thompson, Charles J.

2013-01-01

Tuberculosis therapeutic options are limited by the high intrinsic antibiotic resistance of Mycobacterium tuberculosis. The putative transcriptional regulator WhiB7 is crucial for the activation of systems that provide resistance to diverse antibiotic classes. Here, we used in vitro run-off, two-hybrid assays, as well as mutagenic, complementation and protein pull-down experiments, to characterize WhiB7 as an auto-regulatory, redox-sensitive transcriptional activator in Mycobacterium smegmatis. We provide the first direct biochemical proof that a WhiB protein promotes transcription and also demonstrate that this activity is sensitive to oxidation (diamide). Its partner protein for transcriptional activation was identified as SigA, the primary sigma factor subunit of RNA polymerase. Residues required for the interaction mapped to region 4 of SigA (including R515H) or adjacent domains of WhiB7 (including E63D). WhiB7’s ability to provide a specific spectrum of antibiotic-resistance was dependent on these residues as well as its C-terminal AT-hook module that binds to an AT-rich motif immediately upstream of the −35 hexamer recognized by SigA. These experimentally established constrains, combined with protein structure predictions, were used to generate a working model of the WhiB7–SigA-promoter complex. Inhibitors preventing WhiB7 interactions could allow the use of previously ineffective antibiotics for treatment of mycobacterial diseases. PMID:23990327

The mycobacterial antibiotic resistance determinant WhiB7 acts as a transcriptional activator by binding the primary sigma factor SigA (RpoV).

PubMed

Burian, Ján; Yim, Grace; Hsing, Michael; Axerio-Cilies, Peter; Cherkasov, Artem; Spiegelman, George B; Thompson, Charles J

2013-12-01

Tuberculosis therapeutic options are limited by the high intrinsic antibiotic resistance of Mycobacterium tuberculosis. The putative transcriptional regulator WhiB7 is crucial for the activation of systems that provide resistance to diverse antibiotic classes. Here, we used in vitro run-off, two-hybrid assays, as well as mutagenic, complementation and protein pull-down experiments, to characterize WhiB7 as an auto-regulatory, redox-sensitive transcriptional activator in Mycobacterium smegmatis. We provide the first direct biochemical proof that a WhiB protein promotes transcription and also demonstrate that this activity is sensitive to oxidation (diamide). Its partner protein for transcriptional activation was identified as SigA, the primary sigma factor subunit of RNA polymerase. Residues required for the interaction mapped to region 4 of SigA (including R515H) or adjacent domains of WhiB7 (including E63D). WhiB7's ability to provide a specific spectrum of antibiotic-resistance was dependent on these residues as well as its C-terminal AT-hook module that binds to an AT-rich motif immediately upstream of the -35 hexamer recognized by SigA. These experimentally established constrains, combined with protein structure predictions, were used to generate a working model of the WhiB7-SigA-promoter complex. Inhibitors preventing WhiB7 interactions could allow the use of previously ineffective antibiotics for treatment of mycobacterial diseases.

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome.

PubMed

Camargo, A A; Samaia, H P; Dias-Neto, E; Simão, D F; Migotto, I A; Briones, M R; Costa, F F; Nagai, M A; Verjovski-Almeida, S; Zago, M A; Andrade, L E; Carrer, H; El-Dorry, H F; Espreafico, E M; Habr-Gama, A; Giannella-Neto, D; Goldman, G H; Gruber, A; Hackel, C; Kimura, E T; Maciel, R M; Marie, S K; Martins, E A; Nobrega, M P; Paco-Larson, M L; Pardini, M I; Pereira, G G; Pesquero, J B; Rodrigues, V; Rogatto, S R; da Silva, I D; Sogayar, M C; Sonati, M F; Tajara, E H; Valentini, S R; Alberto, F L; Amaral, M E; Aneas, I; Arnaldi, L A; de Assis, A M; Bengtson, M H; Bergamo, N A; Bombonato, V; de Camargo, M E; Canevari, R A; Carraro, D M; Cerutti, J M; Correa, M L; Correa, R F; Costa, M C; Curcio, C; Hokama, P O; Ferreira, A J; Furuzawa, G K; Gushiken, T; Ho, P L; Kimura, E; Krieger, J E; Leite, L C; Majumder, P; Marins, M; Marques, E R; Melo, A S; Melo, M B; Mestriner, C A; Miracca, E C; Miranda, D C; Nascimento, A L; Nobrega, F G; Ojopi, E P; Pandolfi, J R; Pessoa, L G; Prevedel, A C; Rahal, P; Rainho, C A; Reis, E M; Ribeiro, M L; da Ros, N; de Sa, R G; Sales, M M; Sant'anna, S C; dos Santos, M L; da Silva, A M; da Silva, N P; Silva, W A; da Silveira, R A; Sousa, J F; Stecconi, D; Tsukumo, F; Valente, V; Soares, F; Moreira, E S; Nunes, D N; Correa, R G; Zalcberg, H; Carvalho, A F; Reis, L F; Brentani, R R; Simpson, A J; de Souza, S J; Melo, M

2001-10-09

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription-PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

Mechanical control of cyclic AMP signalling and gene transcription through integrins

NASA Technical Reports Server (NTRS)

Meyer, C. J.; Alenghat, F. J.; Rim, P.; Fong, J. H.; Fabry, B.; Ingber, D. E.

2000-01-01

This study was carried out to discriminate between two alternative hypotheses as to how cells sense mechanical forces and transduce them into changes in gene transcription. Do cells sense mechanical signals through generalized membrane distortion or through specific transmembrane receptors, such as integrins? Here we show that mechanical stresses applied to the cell surface alter the cyclic AMP signalling cascade and downstream gene transcription by modulating local release of signals generated by activated integrin receptors in a G-protein-dependent manner, whereas distortion of integrins in the absence of receptor occupancy has no effect.

Nitrous oxide production and consumption: regulation of gene expression by gas-sensitive transcription factors

PubMed Central

Spiro, Stephen

2012-01-01

Several biochemical mechanisms contribute to the biological generation of nitrous oxide (N2O). N2O generating enzymes include the respiratory nitric oxide (NO) reductase, an enzyme from the flavo-diiron family, and flavohaemoglobin. On the other hand, there is only one enzyme that is known to use N2O as a substrate, which is the respiratory N2O reductase typically found in bacteria capable of denitrification (the respiratory reduction of nitrate and nitrite to dinitrogen). This article will briefly review the properties of the enzymes that make and consume N2O, together with the accessory proteins that have roles in the assembly and maturation of those enzymes. The expression of the genes encoding the enzymes that produce and consume N2O is regulated by environmental signals (typically oxygen and NO) acting through regulatory proteins, which, either directly or indirectly, control the frequency of transcription initiation. The roles and mechanisms of these proteins, and the structures of the regulatory networks in which they participate will also be reviewed. PMID:22451107

Identification of immunity-related genes in the larvae of Protaetia brevitarsis seulensis (Coleoptera: Cetoniidae) by a next-generation sequencing-based transcriptome analysis.

PubMed

Bang, Kyeongrin; Hwang, Sejung; Lee, Jiae; Cho, Saeyoull

2015-01-01

To identify immune-related genes in the larvae of white-spotted flower chafers, next-generation sequencing was conducted with an Illumina HiSeq2000, resulting in 100 million cDNA reads with sequence information from over 10 billion base pairs (bp) and >50× transcriptome coverage. A subset of 77,336 contigs was created, and ∼35,532 sequences matched entries against the NCBI nonredundant database (cutoff, e < 10(-5)). Statistical analysis was performed on the 35,532 contigs. For profiling of the immune response, samples were analyzed by aligning 42 base sequence tags to the de novo reference assembly, comparing levels in immunized larvae to control levels of expression. Of the differentially expressed genes, 3,440 transcripts were upregulated and 3,590 transcripts were downregulated. Many of these genes were confirmed as immune-related genes such as pattern recognition proteins, immune-related signal transduction proteins, antimicrobial peptides, and cellular response proteins, by comparison to published data. © The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.

Nitrous oxide production and consumption: regulation of gene expression by gas-sensitive transcription factors.

PubMed

Spiro, Stephen

2012-05-05

Several biochemical mechanisms contribute to the biological generation of nitrous oxide (N(2)O). N(2)O generating enzymes include the respiratory nitric oxide (NO) reductase, an enzyme from the flavo-diiron family, and flavohaemoglobin. On the other hand, there is only one enzyme that is known to use N(2)O as a substrate, which is the respiratory N(2)O reductase typically found in bacteria capable of denitrification (the respiratory reduction of nitrate and nitrite to dinitrogen). This article will briefly review the properties of the enzymes that make and consume N(2)O, together with the accessory proteins that have roles in the assembly and maturation of those enzymes. The expression of the genes encoding the enzymes that produce and consume N(2)O is regulated by environmental signals (typically oxygen and NO) acting through regulatory proteins, which, either directly or indirectly, control the frequency of transcription initiation. The roles and mechanisms of these proteins, and the structures of the regulatory networks in which they participate will also be reviewed.

Generation of oscillating gene regulatory network motifs

NASA Astrophysics Data System (ADS)

van Dorp, M.; Lannoo, B.; Carlon, E.

2013-07-01

Using an improved version of an evolutionary algorithm originally proposed by François and Hakim [Proc. Natl. Acad. Sci. USAPNASA60027-842410.1073/pnas.0304532101 101, 580 (2004)], we generated small gene regulatory networks in which the concentration of a target protein oscillates in time. These networks may serve as candidates for oscillatory modules to be found in larger regulatory networks and protein interaction networks. The algorithm was run for 105 times to produce a large set of oscillating modules, which were systematically classified and analyzed. The robustness of the oscillations against variations of the kinetic rates was also determined, to filter out the least robust cases. Furthermore, we show that the set of evolved networks can serve as a database of models whose behavior can be compared to experimentally observed oscillations. The algorithm found three smallest (core) oscillators in which nonlinearities and number of components are minimal. Two of those are two-gene modules: the mixed feedback loop, already discussed in the literature, and an autorepressed gene coupled with a heterodimer. The third one is a single gene module which is competitively regulated by a monomer and a dimer. The evolutionary algorithm also generated larger oscillating networks, which are in part extensions of the three core modules and in part genuinely new modules. The latter includes oscillators which do not rely on feedback induced by transcription factors, but are purely of post-transcriptional type. Analysis of post-transcriptional mechanisms of oscillation may provide useful information for circadian clock research, as recent experiments showed that circadian rhythms are maintained even in the absence of transcription.

Recombinant probes for visualizing endogenous synaptic proteins in living neurons

PubMed Central

Gross, Garrett G.; Junge, Jason A.; Mora, Rudy J.; Kwon, Hyung-Bae; Olson, C. Anders; Takahashi, Terry T.; Liman, Emily R.; Ellis-Davies, Graham C.R.; McGee, Aaron W.; Sabatini, Bernardo L.; Roberts, Richard W.; Arnold, Don B.

2013-01-01

Summary The ability to visualize endogenous proteins in living neurons provides a powerful means to interrogate neuronal structure and function. Here we generate recombinant antibody-like proteins, termed FingRs (Fibronectin intrabodies generated with mRNA display), that bind endogenous neuronal proteins PSD-95 and Gephyrin with high affinity and which, when fused to GFP, allow excitatory and inhibitory synapses to be visualized in living neurons. Design of the FingR incorporates a novel transcriptional regulation system that ties FingR expression to the level of the target and reduces background fluorescence. In dissociated neurons and brain slices FingRs generated against PSD-95 and Gephyrin did not affect the expression patterns of their endogenous target proteins or the number or strength of synapses. Together, our data indicate that PSD-95 and Gephyrin FingRs can report the localization and amount of endogenous synaptic proteins in living neurons and thus may be used to study changes in synaptic strength in vivo. PMID:23791193

Imaging mRNA In Vivo, from Birth to Death.

PubMed

Tutucci, Evelina; Livingston, Nathan M; Singer, Robert H; Wu, Bin

2018-05-20

RNA is the fundamental information transfer system in the cell. The ability to follow single messenger RNAs (mRNAs) from transcription to degradation with fluorescent probes gives quantitative information about how the information is transferred from DNA to proteins. This review focuses on the latest technological developments in the field of single-mRNA detection and their usage to study gene expression in both fixed and live cells. By describing the application of these imaging tools, we follow the journey of mRNA from transcription to decay in single cells, with single-molecule resolution. We review current theoretical models for describing transcription and translation that were generated by single-molecule and single-cell studies. These methods provide a basis to study how single-molecule interactions generate phenotypes, fundamentally changing our understating of gene expression regulation.

Accurate RNA consensus sequencing for high-fidelity detection of transcriptional mutagenesis-induced epimutations.

PubMed

Reid-Bayliss, Kate S; Loeb, Lawrence A

2017-08-29

Transcriptional mutagenesis (TM) due to misincorporation during RNA transcription can result in mutant RNAs, or epimutations, that generate proteins with altered properties. TM has long been hypothesized to play a role in aging, cancer, and viral and bacterial evolution. However, inadequate methodologies have limited progress in elucidating a causal association. We present a high-throughput, highly accurate RNA sequencing method to measure epimutations with single-molecule sensitivity. Accurate RNA consensus sequencing (ARC-seq) uniquely combines RNA barcoding and generation of multiple cDNA copies per RNA molecule to eliminate errors introduced during cDNA synthesis, PCR, and sequencing. The stringency of ARC-seq can be scaled to accommodate the quality of input RNAs. We apply ARC-seq to directly assess transcriptome-wide epimutations resulting from RNA polymerase mutants and oxidative stress.

Voltage-Gated Na+ Channel Isoforms and Their mRNA Expression Levels and Protein Abundance in Three Electric Organs and the Skeletal Muscle of the Electric Eel Electrophorus electricus

PubMed Central

Hiong, Kum C.; Boo, Mel V.; Wong, Wai P.; Chew, Shit F.

2016-01-01

This study aimed to obtain the coding cDNA sequences of voltage-gated Na+ channel (scn) α-subunit (scna) and β-subunit (scnb) isoforms from, and to quantify their transcript levels in, the main electric organ (EO), Hunter’s EO, Sach’s EO and the skeletal muscle (SM) of the electric eel, Electrophorus electricus, which can generate both high and low voltage electric organ discharges (EODs). The full coding sequences of two scna (scn4aa and scn4ab) and three scnb (scn1b, scn2b and scn4b) were identified for the first time (except scn4aa) in E. electricus. In adult fish, the scn4aa transcript level was the highest in the main EO and the lowest in the Sach’s EO, indicating that it might play an important role in generating high voltage EODs. For scn4ab/Scn4ab, the transcript and protein levels were unexpectedly high in the EOs, with expression levels in the main EO and the Hunter’s EO comparable to those of scn4aa. As the key domains affecting the properties of the channel were mostly conserved between Scn4aa and Scn4ab, Scn4ab might play a role in electrogenesis. Concerning scnb, the transcript level of scn4b was much higher than those of scn1b and scn2b in the EOs and the SM. While the transcript level of scn4b was the highest in the main EO, protein abundance of Scn4b was the highest in the SM. Taken together, it is unlikely that Scna could function independently to generate EODs in the EOs as previously suggested. It is probable that different combinations of Scn4aa/Scn4ab and various Scnb isoforms in the three EOs account for the differences in EODs produced in E. electricus. In general, the transcript levels of various scn isoforms in the EOs and the SM were much higher in adult than in juvenile, and the three EOs of the juvenile fish could be functionally indistinct. PMID:27907137

Solution properties of the archaeal CRISPR DNA repeat-binding homeodomain protein Cbp2

PubMed Central

Kenchappa, Chandra S.; Heidarsson, Pétur O.; Kragelund, Birthe B.; Garrett, Roger A.; Poulsen, Flemming M.

2013-01-01

Clustered regularly interspaced short palindromic repeats (CRISPR) form the basis of diverse adaptive immune systems directed primarily against invading genetic elements of archaea and bacteria. Cbp1 of the crenarchaeal thermoacidophilic order Sulfolobales, carrying three imperfect repeats, binds specifically to CRISPR DNA repeats and has been implicated in facilitating production of long transcripts from CRISPR loci. Here, a second related class of CRISPR DNA repeat-binding protein, denoted Cbp2, is characterized that contains two imperfect repeats and is found amongst members of the crenarchaeal thermoneutrophilic order Desulfurococcales. DNA repeat-binding properties of the Hyperthermus butylicus protein Cbp2Hb were characterized and its three-dimensional structure was determined by NMR spectroscopy. The two repeats generate helix-turn-helix structures separated by a basic linker that is implicated in facilitating high affinity DNA binding of Cbp2 by tethering the two domains. Structural studies on mutant proteins provide support for Cys7 and Cys28 enhancing high thermal stability of Cbp2Hb through disulphide bridge formation. Consistent with their proposed CRISPR transcriptional regulatory role, Cbp2Hb and, by inference, other Cbp1 and Cbp2 proteins are closely related in structure to homeodomain proteins with linked helix-turn-helix (HTH) domains, in particular the paired domain Pax and Myb family proteins that are involved in eukaryal transcriptional regulation. PMID:23325851

PARP-2 regulates cell cycle-related genes through histone deacetylation and methylation independently of poly(ADP-ribosyl)ation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, Ya-Chen; Hsu, Chiao-Yu; Yao, Ya-Li

2013-02-01

Highlights: ► PARP-2 acts as a transcription co-repressor independently of PARylation activity. ► PARP-2 recruits HDAC5, 7, and G9a and generates repressive chromatin. ► PARP-2 is recruited to the c-MYC promoter by DNA-binding factor YY1. ► PARP-2 represses cell cycle-related genes and alters cell cycle progression. -- Abstract: Poly(ADP-ribose) polymerase-2 (PARP-2) catalyzes poly(ADP-ribosyl)ation (PARylation) and regulates numerous nuclear processes, including transcription. Depletion of PARP-2 alters the activity of transcription factors and global gene expression. However, the molecular action of how PARP-2 controls the transcription of target promoters remains unclear. Here we report that PARP-2 possesses transcriptional repression activity independently ofmore » its enzymatic activity. PARP-2 interacts and recruits histone deacetylases HDAC5 and HDAC7, and histone methyltransferase G9a to the promoters of cell cycle-related genes, generating repressive chromatin signatures. Our findings propose a novel mechanism of PARP-2 in transcriptional regulation involving specific protein–protein interactions and highlight the importance of PARP-2 in the regulation of cell cycle progression.« less

What's that gene (or protein)? Online resources for exploring functions of genes, transcripts, and proteins

PubMed Central

Hutchins, James R. A.

2014-01-01

The genomic era has enabled research projects that use approaches including genome-scale screens, microarray analysis, next-generation sequencing, and mass spectrometry–based proteomics to discover genes and proteins involved in biological processes. Such methods generate data sets of gene, transcript, or protein hits that researchers wish to explore to understand their properties and functions and thus their possible roles in biological systems of interest. Recent years have seen a profusion of Internet-based resources to aid this process. This review takes the viewpoint of the curious biologist wishing to explore the properties of protein-coding genes and their products, identified using genome-based technologies. Ten key questions are asked about each hit, addressing functions, phenotypes, expression, evolutionary conservation, disease association, protein structure, interactors, posttranslational modifications, and inhibitors. Answers are provided by presenting the latest publicly available resources, together with methods for hit-specific and data set–wide information retrieval, suited to any genome-based analytical technique and experimental species. The utility of these resources is demonstrated for 20 factors regulating cell proliferation. Results obtained using some of these are discussed in more depth using the p53 tumor suppressor as an example. This flexible and universally applicable approach for characterizing experimental hits helps researchers to maximize the potential of their projects for biological discovery. PMID:24723265

Genome-wide regulation of light-controlled seedling morphogenesis by three families of transcription factors.

PubMed

Shi, Hui; Lyu, Mohan; Luo, Yiwen; Liu, Shoucheng; Li, Yue; He, Hang; Wei, Ning; Deng, Xing Wang; Zhong, Shangwei

2018-06-19

Three families of transcription factors have been reported to play key roles in light control of Arabidopsis seedling morphogenesis. Among them, bHLH protein PIFs and plant-specific protein EIN3/EIN3-LIKE 1 (EIN3/EIL1) accumulate in the dark to maintain skotomorphogenesis. On the other hand, HY5 and HY5 HOMOLOG (HYH), two related bZIP proteins, are stabilized in light and promote photomorphogenic development. To systemically investigate the transcriptional regulation of light-controlled seedling morphogenesis, we generated HY5 ox/ pifQein3eil1 , which contained mutations of EIN3/EIL1 and four PIF genes ( pifQein3eil1 ) and overexpression of HY5 Our results show that dark-grown HY5 ox/ pifQein3eil1 seedlings display a photomorphogenesis highly similar to that of wild-type seedlings grown in continuous light, with remarkably enhanced photomorphogenic phenotypes compared with the pifQ mutants. Consistent with the genetic evidence, transcriptome analysis indicated that PIFs, EIN3/EIL1, and HY5 are dominant transcription factors in collectively mediating a wide range of light-caused genome-wide transcriptional changes. Moreover, PIFs and EIN3/EIL1 independently control the expression of light-regulated genes such as HLS1 to cooperatively regulate apical hook formation, hypocotyl elongation, and cotyledon opening and expansion. This study illustrates a comprehensive regulatory network of transcription activities that correspond to specific morphological aspects in seedling skotomorphogenesis and photomorphogenesis.

An Endogenous Accelerator for Viral Gene Expression Confers a Fitness Advantage

PubMed Central

Teng, Melissa W.; Bolovan-Fritts, Cynthia; Dar, Roy D.; Womack, Andrew; Simpson, Michael L.; Shenk, Thomas; Weinberger, Leor S.

2012-01-01

Many signaling circuits face a fundamental tradeoff between accelerating their response speed while maintaining final levels below a cytotoxic threshold. Here, we describe a transcriptional circuitry that dynamically converts signaling inputs into faster rates without amplifying final equilibrium levels. Using time-lapse microscopy, we find that transcriptional activators accelerate human cytomegalovirus (CMV) gene expression in single cells without amplifying steady-state expression levels, and this acceleration generates a significant replication advantage. We map the accelerator to a highly self-cooperative transcriptional negative-feedback loop (Hill coefficient ~ 7) generated by homo-multimerization of the virus’s essential transactivator protein IE2 at nuclear PML bodies. Eliminating the IE2-accelerator circuit reduces transcriptional strength through mislocalization of incoming viral genomes away from PML bodies and carries a heavy fitness cost. In general, accelerators may provide a mechanism for signal-transduction circuits to respond quickly to external signals without increasing steady-state levels of potentially cytotoxic molecules. PMID:23260143

High-throughput annotation of full-length long noncoding RNAs with capture long-read sequencing.

PubMed

Lagarde, Julien; Uszczynska-Ratajczak, Barbara; Carbonell, Silvia; Pérez-Lluch, Sílvia; Abad, Amaya; Davis, Carrie; Gingeras, Thomas R; Frankish, Adam; Harrow, Jennifer; Guigo, Roderic; Johnson, Rory

2017-12-01

Accurate annotation of genes and their transcripts is a foundation of genomics, but currently no annotation technique combines throughput and accuracy. As a result, reference gene collections remain incomplete-many gene models are fragmentary, and thousands more remain uncataloged, particularly for long noncoding RNAs (lncRNAs). To accelerate lncRNA annotation, the GENCODE consortium has developed RNA Capture Long Seq (CLS), which combines targeted RNA capture with third-generation long-read sequencing. Here we present an experimental reannotation of the GENCODE intergenic lncRNA populations in matched human and mouse tissues that resulted in novel transcript models for 3,574 and 561 gene loci, respectively. CLS approximately doubled the annotated complexity of targeted loci, outperforming existing short-read techniques. Full-length transcript models produced by CLS enabled us to definitively characterize the genomic features of lncRNAs, including promoter and gene structure, and protein-coding potential. Thus, CLS removes a long-standing bottleneck in transcriptome annotation and generates manual-quality full-length transcript models at high-throughput scales.

Intron retention and transcript chimerism conserved across mammals: Ly6g5b and Csnk2b-Ly6g5b as examples

PubMed Central

2013-01-01

Background Alternative splicing (AS) is a major mechanism for modulating gene expression of an organism, allowing the synthesis of several structurally and functionally distinct mRNAs and protein isoforms from a unique gene. Related to AS is the Transcription Induced Chimerism (TIC) or Tandem Chimerism, by which chimeric RNAs between adjacent genes can be found, increasing combinatorial complexity of the proteome. The Ly6g5b gene presents particular behaviours in its expression, involving an intron retention event and being capable to form RNA chimera transcripts with the upstream gene Csnk2b. We wanted to characterise these events more deeply in four tissues in six different mammals and analyse their protein products. Results While canonical Csnk2b isoform was widely expressed, Ly6g5b canonical isoform was less ubiquitous, although the Ly6g5b first intron retained transcript was present in all the tissues and species analysed. Csnk2b-Ly6g5b chimeras were present in all the samples analysed, but with restricted expression patterns. Some of these chimeric transcripts maintained correct structural domains from Csnk2b and Ly6g5b. Moreover, we found Csnk2b, Ly6g5b, and Csnk2b-Ly6g5b transcripts that present exon skipping, alternative 5' and 3' splice site and intron retention events. These would generate truncated or aberrant proteins whose role remains unknown. Some chimeric transcripts would encode CSNK2B proteins with an altered C-terminus, which could affect its biological function broadening its substrate specificity. Over-expression of human CSNK2B, LY6G5B, and CSNK2B-LY6G5B proteins, show different patterns of post-translational modifications and cell distribution. Conclusions Ly6g5b intron retention and Csnk2b-Ly6g5b transcript chimerism are broadly distributed in tissues of different mammals. PMID:23521802

Mitochondrial transcription: Lessons from mouse models

PubMed Central

Peralta, Susana; Wang, Xiao; Moraes, Carlos T.

2012-01-01

Mammalian mitochondrial DNA (mtDNA) is a circular double-stranded DNA genome of ∼ 16.5 kilobase pairs (kb) that encodes 13 catalytic proteins of the ATP-producing oxidative phosphorylation system (OXPHOS), and the rRNAs and tRNAs required for the translation of the mtDNA transcripts. All the components needed for transcription and replication of the mtDNA are, therefore, encoded in the nuclear genome, as are the remaining components of the OXPHOS system and the mitochondrial translation machinery. Regulation of mtDNA gene expression is very important for modulating the OXPHOS capacity in response to metabolic requirements and in pathological processes. The combination of in vitro and in vivo studies has allowed the identification of the core machinery required for basal mtDNA transcription in mammals and a few proteins that regulate mtDNA transcription. Specifically, the generation of knockout mouse strains in the last several years, has been key to understanding the basis of mtDNA transcription in vivo. However, it is well accepted that many components of the transcription machinery are still unknown and little is known about mtDNA gene expression regulation under different metabolic requirements or disease processes. In this review we will focus on how the creation of knockout mouse models and the study of their phenotypes have contributed to the understanding of mitochondrial transcription in mammals. PMID:22120174

De Novo Assembly of the Whole Transcriptome of the Wild Embryo, Preleptocephalus, Leptocephalus, and Glass Eel of Anguilla japonica and Deciphering the Digestive and Absorptive Capacities during Early Development.

PubMed

Hsu, Hsiang-Yi; Chen, Shu-Hwa; Cha, Yuh-Ru; Tsukamoto, Katsumi; Lin, Chung-Yen; Han, Yu-San

2015-01-01

Natural stocks of Japanese eel (Anguilla japonica) have decreased drastically because of overfishing, habitat destruction, and changes in the ocean environment over the past few decades. However, to date, artificial mass production of glass eels is far from reality because of the lack of appropriate feed for the eel larvae. In this study, wild glass eel, leptocephali, preleptocephali, and embryos were collected to conduct RNA-seq. Approximately 279 million reads were generated and assembled into 224,043 transcripts. The transcript levels of genes coding for digestive enzymes and nutrient transporters were investigated to estimate the capacities for nutrient digestion and absorption during early development. The results showed that the transcript levels of protein digestion enzymes were higher than those of carbohydrate and lipid digestion enzymes in the preleptocephali and leptocephali, and the transcript levels of amino acid transporters were also higher than those of glucose and fructose transporters and the cholesterol transporter. In addition, the transcript levels of glucose and fructose transporters were significantly raising in the leptocephali. Moreover, the transcript levels of protein, carbohydrate, and lipid digestion enzymes were balanced in the glass eel, but the transcript levels of amino acid transporters were higher than those of glucose and cholesterol transporters. These findings implied that preleptocephali and leptocephali prefer high-protein food, and the nutritional requirements of monosaccharides and lipids for the eel larvae vary with growth. An online database (http://molas.iis.sinica.edu.tw/jpeel/) that will provide the sequences and the annotated results of assembled transcripts was established for the eel research community.

De Novo Assembly of the Whole Transcriptome of the Wild Embryo, Preleptocephalus, Leptocephalus, and Glass Eel of Anguilla japonica and Deciphering the Digestive and Absorptive Capacities during Early Development

PubMed Central

Cha, Yuh-Ru; Tsukamoto, Katsumi; Lin, Chung-Yen; Han, Yu-San

2015-01-01

Natural stocks of Japanese eel (Anguilla japonica) have decreased drastically because of overfishing, habitat destruction, and changes in the ocean environment over the past few decades. However, to date, artificial mass production of glass eels is far from reality because of the lack of appropriate feed for the eel larvae. In this study, wild glass eel, leptocephali, preleptocephali, and embryos were collected to conduct RNA-seq. Approximately 279 million reads were generated and assembled into 224,043 transcripts. The transcript levels of genes coding for digestive enzymes and nutrient transporters were investigated to estimate the capacities for nutrient digestion and absorption during early development. The results showed that the transcript levels of protein digestion enzymes were higher than those of carbohydrate and lipid digestion enzymes in the preleptocephali and leptocephali, and the transcript levels of amino acid transporters were also higher than those of glucose and fructose transporters and the cholesterol transporter. In addition, the transcript levels of glucose and fructose transporters were significantly raising in the leptocephali. Moreover, the transcript levels of protein, carbohydrate, and lipid digestion enzymes were balanced in the glass eel, but the transcript levels of amino acid transporters were higher than those of glucose and cholesterol transporters. These findings implied that preleptocephali and leptocephali prefer high-protein food, and the nutritional requirements of monosaccharides and lipids for the eel larvae vary with growth. An online database (http://molas.iis.sinica.edu.tw/jpeel/) that will provide the sequences and the annotated results of assembled transcripts was established for the eel research community. PMID:26406914

O-GlcNAc transferase regulates transcriptional activity of human Oct4.

PubMed

Constable, Sandii; Lim, Jae-Min; Vaidyanathan, Krithika; Wells, Lance

2017-10-01

O-linked β-N-acetylglucosamine (O-GlcNAc) is a single sugar modification found on many different classes of nuclear and cytoplasmic proteins. Addition of this modification, by the enzyme O-linked N-acetylglucosamine transferase (OGT), is dynamic and inducible. One major class of proteins modified by O-GlcNAc is transcription factors. O-GlcNAc regulates transcription factor properties through a variety of different mechanisms including localization, stability and transcriptional activation. Maintenance of embryonic stem (ES) cell pluripotency requires tight regulation of several key transcription factors, many of which are modified by O-GlcNAc. Octamer-binding protein 4 (Oct4) is one of the key transcription factors required for pluripotency of ES cells and more recently, the generation of induced pluripotent stem (iPS) cells. The action of Oct4 is modulated by the addition of several post-translational modifications, including O-GlcNAc. Previous studies in mice found a single site of O-GlcNAc addition responsible for transcriptional regulation. This study was designed to determine if this mechanism is conserved in humans. We mapped 10 novel sites of O-GlcNAc attachment on human Oct4, and confirmed a role for OGT in transcriptional activation of Oct4 at a site distinct from that found in mouse that allows distinction between different Oct4 target promoters. Additionally, we uncovered a potential new role for OGT that does not include its catalytic function. These results confirm that human Oct4 activity is being regulated by OGT by a mechanism that is distinct from mouse Oct4. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Molecular characterization of the rhesus rhadinovirus (RRV) ORF4 gene and the RRV complement control protein it encodes.

PubMed

Mark, Linda; Spiller, O Brad; Okroj, Marcin; Chanas, Simon; Aitken, Jim A; Wong, Scott W; Damania, Blossom; Blom, Anna M; Blackbourn, David J

2007-04-01

The diversity of viral strategies to modulate complement activation indicates that this component of the immune system has significant antiviral potential. One example is the Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP), which inhibits progression of the complement cascade. Rhesus rhadinovirus (RRV), like KSHV, is a member of the subfamily Gammaherpesvirinae and currently provides the only in vivo model of KSHV pathobiology in primates. In the present study, we characterized the KCP homologue encoded by RRV, RRV complement control protein (RCP). Two strains of RRV have been sequenced to date (H26-95 and 17577), and the RCPs they encode differ substantially in structure: RCP from strain H26-95 has four complement control protein (CCP) domains, whereas RCP from strain 17577 has eight CCP domains. Transcriptional analyses of the RCP gene (ORF4, referred to herein as RCP) in infected rhesus macaque fibroblasts mapped the ends of the transcripts of both strains. They revealed that H26-95 encodes a full-length, unspliced RCP transcript, while 17577 RCP generates a full-length unspliced mRNA and two alternatively spliced transcripts. Western blotting confirmed that infected cells express RCP, and immune electron microscopy disclosed this protein on the surface of RRV virions. Functional studies of RCP encoded by both RRV strains revealed their ability to suppress complement activation by the classical (antibody-mediated) pathway. These data provide the foundation for studies into the biological significance of gammaherpesvirus complement regulatory proteins in a tractable, non-human primate model.

Molecular Characterization of the Rhesus Rhadinovirus (RRV) ORF4 Gene and the RRV Complement Control Protein It Encodes▿

PubMed Central

Mark, Linda; Spiller, O. Brad; Okroj, Marcin; Chanas, Simon; Aitken, Jim A.; Wong, Scott W.; Damania, Blossom; Blom, Anna M.; Blackbourn, David J.

2007-01-01

The diversity of viral strategies to modulate complement activation indicates that this component of the immune system has significant antiviral potential. One example is the Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP), which inhibits progression of the complement cascade. Rhesus rhadinovirus (RRV), like KSHV, is a member of the subfamily Gammaherpesvirinae and currently provides the only in vivo model of KSHV pathobiology in primates. In the present study, we characterized the KCP homologue encoded by RRV, RRV complement control protein (RCP). Two strains of RRV have been sequenced to date (H26-95 and 17577), and the RCPs they encode differ substantially in structure: RCP from strain H26-95 has four complement control protein (CCP) domains, whereas RCP from strain 17577 has eight CCP domains. Transcriptional analyses of the RCP gene (ORF4, referred to herein as RCP) in infected rhesus macaque fibroblasts mapped the ends of the transcripts of both strains. They revealed that H26-95 encodes a full-length, unspliced RCP transcript, while 17577 RCP generates a full-length unspliced mRNA and two alternatively spliced transcripts. Western blotting confirmed that infected cells express RCP, and immune electron microscopy disclosed this protein on the surface of RRV virions. Functional studies of RCP encoded by both RRV strains revealed their ability to suppress complement activation by the classical (antibody-mediated) pathway. These data provide the foundation for studies into the biological significance of gammaherpesvirus complement regulatory proteins in a tractable, non-human primate model. PMID:17287274

Epigenetic and Posttranslational Modifications in Light Signal Transduction and the Circadian Clock in Neurospora crassa

PubMed Central

Proietto, Marco; Bianchi, Michele Maria; Ballario, Paola; Brenna, Andrea

2015-01-01

Blue light, a key abiotic signal, regulates a wide variety of physiological processes in many organisms. One of these phenomena is the circadian rhythm presents in organisms sensitive to the phase-setting effects of blue light and under control of the daily alternation of light and dark. Circadian clocks consist of autoregulatory alternating negative and positive feedback loops intimately connected with the cellular metabolism and biochemical processes. Neurospora crassa provides an excellent model for studying the molecular mechanisms involved in these phenomena. The White Collar Complex (WCC), a blue-light receptor and transcription factor of the circadian oscillator, and Frequency (FRQ), the circadian clock pacemaker, are at the core of the Neurospora circadian system. The eukaryotic circadian clock relies on transcriptional/translational feedback loops: some proteins rhythmically repress their own synthesis by inhibiting the activity of their transcriptional factors, generating self-sustained oscillations over a period of about 24 h. One of the basic mechanisms that perpetuate self-sustained oscillations is post translation modification (PTM). The acronym PTM generically indicates the addition of acetyl, methyl, sumoyl, or phosphoric groups to various types of proteins. The protein can be regulatory or enzymatic or a component of the chromatin. PTMs influence protein stability, interaction, localization, activity, and chromatin packaging. Chromatin modification and PTMs have been implicated in regulating circadian clock function in Neurospora. Research into the epigenetic control of transcription factors such as WCC has yielded new insights into the temporal modulation of light-dependent gene transcription. Here we report on epigenetic and protein PTMs in the regulation of the Neurospora crassa circadian clock. We also present a model that illustrates the molecular mechanisms at the basis of the blue light control of the circadian clock. PMID:26198228

A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription.

PubMed

Völkel, Pamela; Le Faou, Perrine; Vandamme, Julien; Pira, Dorcas; Angrand, Pierre-Olivier

2012-05-01

Polycomb repression controls the expression of hundreds of genes involved in development and is mediated by essentially two classes of chromatin-associated protein complexes. The Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27, an epigenetic mark that serves as a docking site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits: Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph) and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates, thus generating an enormous scope for potential combinatorial diversity. In particular, mammalian genomes encode five Pc family members: CBX2, CBX4, CBX6, CBX7 and CBX8. To complicate matters further, distinct isoforms might arise from single genes. Here, we address the functional role of the two human CBX2 isoforms. Owing to different polyadenylation sites and alternative splicing events, the human CBX2 locus produces two transcripts: a 5-exon transcript that encodes the 532-amino acid CBX2-1 isoform that contains the conserved chromodomain and Pc box and a 4-exon transcript encoding a shorter isoform, CBX2-2, lacking the Pc box but still possessing a chromodomain. Using biochemical approaches and a novel in vivo imaging assay, we show that the short CBX2-2 isoform lacking the Pc box, does not participate in PRC1 protein complexes, but self-associates in vivo and forms complexes of high molecular weight. Furthermore, the CBX2 short isoform is still able to repress transcription, suggesting that Polycomb repression might occur in the absence of PRC1 formation.

Electrostatic study of Alanine mutational effects on transcription: application to GATA-3:DNA interaction complex.

PubMed

El-Assaad, Atlal; Dawy, Zaher; Nemer, Georges

2015-01-01

Protein-DNA interaction is of fundamental importance in molecular biology, playing roles in functions as diverse as DNA transcription, DNA structure formation, and DNA repair. Protein-DNA association is also important in medicine; understanding Protein-DNA binding kinetics can assist in identifying disease root causes which can contribute to drug development. In this perspective, this work focuses on the transcription process by the GATA Transcription Factor (TF). GATA TF binds to DNA promoter region represented by `G,A,T,A' nucleotides sequence, and initiates transcription of target genes. When proper regulation fails due to some mutations on the GATA TF protein sequence or on the DNA promoter sequence (weak promoter), deregulation of the target genes might lead to various disorders. In this study, we aim to understand the electrostatic mechanism behind GATA TF and DNA promoter interactions, in order to predict Protein-DNA binding in the presence of mutations, while elaborating on non-covalent binding kinetics. To generate a family of mutants for the GATA:DNA complex, we replaced every charged amino acid, one at a time, with a neutral amino acid like Alanine (Ala). We then applied Poisson-Boltzmann electrostatic calculations feeding into free energy calculations, for each mutation. These calculations delineate the contribution to binding from each Ala-replaced amino acid in the GATA:DNA interaction. After analyzing the obtained data in view of a two-step model, we are able to identify potential key amino acids in binding. Finally, we applied the model to GATA-3:DNA (crystal structure with PDB-ID: 3DFV) binding complex and validated it against experimental results from the literature.

SASD: the Synthetic Alternative Splicing Database for identifying novel isoform from proteomics

PubMed Central

2013-01-01

Background Alternative splicing is an important and widespread mechanism for generating protein diversity and regulating protein expression. High-throughput identification and analysis of alternative splicing in the protein level has more advantages than in the mRNA level. The combination of alternative splicing database and tandem mass spectrometry provides a powerful technique for identification, analysis and characterization of potential novel alternative splicing protein isoforms from proteomics. Therefore, based on the peptidomic database of human protein isoforms for proteomics experiments, our objective is to design a new alternative splicing database to 1) provide more coverage of genes, transcripts and alternative splicing, 2) exclusively focus on the alternative splicing, and 3) perform context-specific alternative splicing analysis. Results We used a three-step pipeline to create a synthetic alternative splicing database (SASD) to identify novel alternative splicing isoforms and interpret them at the context of pathway, disease, drug and organ specificity or custom gene set with maximum coverage and exclusive focus on alternative splicing. First, we extracted information on gene structures of all genes in the Ensembl Genes 71 database and incorporated the Integrated Pathway Analysis Database. Then, we compiled artificial splicing transcripts. Lastly, we translated the artificial transcripts into alternative splicing peptides. The SASD is a comprehensive database containing 56,630 genes (Ensembl gene IDs), 95,260 transcripts (Ensembl transcript IDs), and 11,919,779 Alternative Splicing peptides, and also covering about 1,956 pathways, 6,704 diseases, 5,615 drugs, and 52 organs. The database has a web-based user interface that allows users to search, display and download a single gene/transcript/protein, custom gene set, pathway, disease, drug, organ related alternative splicing. Moreover, the quality of the database was validated with comparison to other known databases and two case studies: 1) in liver cancer and 2) in breast cancer. Conclusions The SASD provides the scientific community with an efficient means to identify, analyze, and characterize novel Exon Skipping and Intron Retention protein isoforms from mass spectrometry and interpret them at the context of pathway, disease, drug and organ specificity or custom gene set with maximum coverage and exclusive focus on alternative splicing. PMID:24267658

Direct TFIIA-TFIID Protein Contacts Drive Budding Yeast Ribosomal Protein Gene Transcription*

PubMed Central

Layer, Justin H.; Weil, P. Anthony

2013-01-01

We have previously shown that yeast TFIID provides coactivator function on the promoters of ribosomal protein-encoding genes (RPGs) by making direct contact with the transactivator repressor activator protein 1 (Rap1). Further, our structural studies of assemblies generated with purified Rap1, TFIID, and TFIIA on RPG enhancer-promoter DNA indicate that Rap1-TFIID interaction induces dramatic conformational rearrangements of enhancer-promoter DNA and TFIID-bound TFIIA. These data indicate a previously unknown yet critical role for yeast TFIIA in the integration of activator-TFIID contacts with promoter conformation and downstream preinitiation complex formation and/or function. Here we describe the use of systematic mutagenesis to define how specific TFIIA contacts contribute to these processes. We have verified that TFIIA is required for RPG transcription in vivo and in vitro, consistent with the existence of a critical Rap1-TFIIA-TFIID interaction network. We also identified essential points of contact for TFIIA and Rap1 within the Rap1 binding domain of the Taf4 subunit of TFIID. These data suggest a mechanism for how interactions between TFIID, TFIIA, and Rap1 contribute to the high rate of transcription initiation seen on RPGs in vivo. PMID:23814059

Lack of A-factor production induces the expression of nutrient scavenging and stress-related proteins in Streptomyces griseus.

PubMed

Birkó, Zsuzsanna; Swiatek, Magdalena; Szájli, Emília; Medzihradszky, Katalin F; Vijgenboom, Erik; Penyige, András; Keseru, Judit; van Wezel, Gilles P; Biró, Sándor

2009-10-01

The small gamma-butyrolactone A-factor is an important autoregulatory signaling molecule for the soil-inhabiting streptomycetes. Starvation is a major trigger for development, and nutrients are provided by degradation of the vegetative mycelium via a process of programmed cell death, reusing proteins, nucleic acids, and cell wall material. The A-factor regulon includes many extracellular hydrolases. Here we show via proteomics analysis that many nutrient-scavenging and stress-related proteins were overexpressed in an A-factor non-producing mutant of Streptomyces griseus B-2682. Transcript analysis showed that this is primarily due to differential transcription of the target genes during early development. The targets include proteins relating to nutrient stress and environmental stress and an orthologue of the Bacillus sporulation control protein Spo0M. The enhanced expression of these proteins underlines the stress that is generated by the absence of A-factor. Wild-type developmental gene expression was restored to the A-factor non-producing mutant by the signaling protein Factor C in line with our earlier observation that Factor C triggers A-factor production.

Gcn4-Mediator Specificity Is Mediated by a Large and Dynamic Fuzzy Protein-Protein Complex.

PubMed

Tuttle, Lisa M; Pacheco, Derek; Warfield, Linda; Luo, Jie; Ranish, Jeff; Hahn, Steven; Klevit, Rachel E

2018-03-20

Transcription activation domains (ADs) are inherently disordered proteins that often target multiple coactivator complexes, but the specificity of these interactions is not understood. Efficient transcription activation by yeast Gcn4 requires its tandem ADs and four activator-binding domains (ABDs) on its target, the Mediator subunit Med15. Multiple ABDs are a common feature of coactivator complexes. We find that the large Gcn4-Med15 complex is heterogeneous and contains nearly all possible AD-ABD interactions. Gcn4-Med15 forms via a dynamic fuzzy protein-protein interface, where ADs bind the ABDs in multiple orientations via hydrophobic regions that gain helicity. This combinatorial mechanism allows individual low-affinity and specificity interactions to generate a biologically functional, specific, and higher affinity complex despite lacking a defined protein-protein interface. This binding strategy is likely representative of many activators that target multiple coactivators, as it allows great flexibility in combinations of activators that can cooperate to regulate genes with variable coactivator requirements. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Functional dissection of the Hox protein Abdominal-B in Drosophila cell culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhai, Zongzhao; CellNetworks - Cluster of Excellence, Centre for Organismal Studies; Graduate School of Chinese Academy of Sciences, Beijing 100039

2011-11-04

Highlights: Black-Right-Pointing-Pointer ct340 CRM was identified to be the posterior spiracle enhancer of gene cut. Black-Right-Pointing-Pointer ct340 is under the direct transcriptional control of Hox protein Abd-B. Black-Right-Pointing-Pointer An efficient cloning system was developed to assay protein-DNA interaction. Black-Right-Pointing-Pointer New features of Abd-B dependent target gene regulation were detected. -- Abstract: Hox transcription factors regulate the morphogenesis along the anterior-posterior (A/P) body axis through the interaction with small cis-regulatory modules (CRMs) of their target gene, however so far very few Hox CRMs are known and have been analyzed in detail. In this study we have identified a new Hox CRM,more » ct340, which guides the expression of the cell type specification gene cut (ct) in the posterior spiracle under the direct control of the Hox protein Abdominal-B (Abd-B). Using the ct340 enhancer activity as readout, an efficient cloning system to generate VP16 activation domain fusion protein was developed to unambiguously test protein-DNA interaction in Drosophila cell culture. By functionally dissecting the Abd-B protein, new features of Abd-B dependent target gene regulation were detected. Due to its easy adaptability, this system can be generally used to map functional domains within sequence-specific transcriptional factors in Drosophila cell culture, and thus provide preliminary knowledge of the protein functional domain structure for further in vivo analysis.« less

A novel muscle LIM-only protein is generated from the paxillin gene locus in Drosophila.

PubMed

Yagi, R; Ishimaru, S; Yano, H; Gaul, U; Hanafusa, H; Sabe, H

2001-09-01

Paxillin is a protein containing four LIM domains, and functions in integrin signaling. We report here that two transcripts are generated from the paxillin gene locus in Drosophila; one encodes a protein homolog of the vertebrate Paxillin (DPxn37), and the other a protein with only three LIM domains, partly encoded by its own specific exon (PDLP). At the myotendinous junctions of Drosophila embryos where integrins play important roles, both DPxn37 and PDLP are highly expressed with different patterns; DPxn37 is predominantly concentrated at the center of the junctions, whereas PDLP is highly enriched at neighboring sides of the junction centers, primarily expressed in the mesodermal myotubes. Northern blot analysis revealed that DPxn37 is ubiquitously expressed throughout the life cycle, whereas PDLP expression exhibits a biphasic pattern during development, largely concomitant with muscle generation and remodeling. Our results collectively reveal that a unique system exists in Drosophila for the generation of a novel type of LIM-only protein, highly expressed in the embryonic musculature, largely utilizing the Paxillin LIM domains.

Generation and analysis of expression sequence tags from haustoria of the wheat stripe rust fungus Puccinia striiformis f. sp. Tritici

PubMed Central

2009-01-01

Background Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat (Triticum aestivum L.) worldwide. In spite of its agricultural importance, the genomics and genetics of the pathogen are poorly characterized. Pst transcripts from urediniospores and germinated urediniospores have been examined previously, but little is known about genes expressed during host infection. Some genes involved in virulence in other rust fungi have been found to be specifically expressed in haustoria. Therefore, the objective of this study was to generate a cDNA library to characterize genes expressed in haustoria of Pst. Results A total of 5,126 EST sequences of high quality were generated from haustoria of Pst, from which 287 contigs and 847 singletons were derived. Approximately 10% and 26% of the 1,134 unique sequences were homologous to proteins with known functions and hypothetical proteins, respectively. The remaining 64% of the unique sequences had no significant similarities in GenBank. Fifteen genes were predicted to be proteins secreted from Pst haustoria. Analysis of ten genes, including six secreted protein genes, using quantitative RT-PCR revealed changes in transcript levels in different developmental and infection stages of the pathogen. Conclusions The haustorial cDNA library was useful in identifying genes of the stripe rust fungus expressed during the infection process. From the library, we identified 15 genes encoding putative secreted proteins and six genes induced during the infection process. These genes are candidates for further studies to determine their functions in wheat-Pst interactions. PMID:20028560

Transcriptome sequencing and whole genome expression profiling of chrysanthemum under dehydration stress

PubMed Central

2013-01-01

Background Chrysanthemum is one of the most important ornamental crops in the world and drought stress seriously limits its production and distribution. In order to generate a functional genomics resource and obtain a deeper understanding of the molecular mechanisms regarding chrysanthemum responses to dehydration stress, we performed large-scale transcriptome sequencing of chrysanthemum plants under dehydration stress using the Illumina sequencing technology. Results Two cDNA libraries constructed from mRNAs of control and dehydration-treated seedlings were sequenced by Illumina technology. A total of more than 100 million reads were generated and de novo assembled into 98,180 unique transcripts which were further extensively annotated by comparing their sequencing to different protein databases. Biochemical pathways were predicted from these transcript sequences. Furthermore, we performed gene expression profiling analysis upon dehydration treatment in chrysanthemum and identified 8,558 dehydration-responsive unique transcripts, including 307 transcription factors and 229 protein kinases and many well-known stress responsive genes. Gene ontology (GO) term enrichment and biochemical pathway analyses showed that dehydration stress caused changes in hormone response, secondary and amino acid metabolism, and light and photoperiod response. These findings suggest that drought tolerance of chrysanthemum plants may be related to the regulation of hormone biosynthesis and signaling, reduction of oxidative damage, stabilization of cell proteins and structures, and maintenance of energy and carbon supply. Conclusions Our transcriptome sequences can provide a valuable resource for chrysanthemum breeding and research and novel insights into chrysanthemum responses to dehydration stress and offer candidate genes or markers that can be used to guide future studies attempting to breed drought tolerant chrysanthemum cultivars. PMID:24074255

The circadian coordination of cell biology.

PubMed

Chaix, Amandine; Zarrinpar, Amir; Panda, Satchidananda

2016-10-10

Circadian clocks are cell-autonomous timing mechanisms that organize cell functions in a 24-h periodicity. In mammals, the main circadian oscillator consists of transcription-translation feedback loops composed of transcriptional regulators, enzymes, and scaffolds that generate and sustain daily oscillations of their own transcript and protein levels. The clock components and their targets impart rhythmic functions to many gene products through transcriptional, posttranscriptional, translational, and posttranslational mechanisms. This, in turn, temporally coordinates many signaling pathways, metabolic activity, organelles' structure and functions, as well as the cell cycle and the tissue-specific functions of differentiated cells. When the functions of these circadian oscillators are disrupted by age, environment, or genetic mutation, the temporal coordination of cellular functions is lost, reducing organismal health and fitness. © 2016 Chaix et al.

Inventory of high-abundance mRNAs in skeletal muscle of normal men.

PubMed

Welle, S; Bhatt, K; Thornton, C A

1999-05-01

G42875rial analysis of gene expression (SAGE) method was used to generate a catalog of 53,875 short (14 base) expressed sequence tags from polyadenylated RNA obtained from vastus lateralis muscle of healthy young men. Over 12,000 unique tags were detected. The frequency of occurrence of each tag reflects the relative abundance of the corresponding mRNA. The mRNA species that were detected 10 or more times, each comprising >/=0.02% of the mRNA population, accounted for 64% of the mRNA mass but <10% of the total number of mRNA species detected. Almost all of the abundant tags matched mRNA or EST sequences cataloged in GenBank. Mitochondrial transcripts accounted for approximately 20% of the polyadenylated RNA. Transcripts encoding proteins of the myofibrils were the most abundant nuclear-encoded mRNAs. Transcripts encoding ribosomal proteins, and those encoding proteins involved in energy metabolism, also were very abundant. The database can be used as a reference for investigations of alterations in gene expression associated with conditions that influence muscle function, such as muscular dystrophies, aging, and exercise.

Drosophila CLOCK target gene characterization: implications for circadian tissue-specific gene expression

PubMed Central

Abruzzi, Katharine Compton; Rodriguez, Joseph; Menet, Jerome S.; Desrochers, Jennifer; Zadina, Abigail; Luo, Weifei; Tkachev, Sasha; Rosbash, Michael

2011-01-01

CLOCK (CLK) is a master transcriptional regulator of the circadian clock in Drosophila. To identify CLK direct target genes and address circadian transcriptional regulation in Drosophila, we performed chromatin immunoprecipitation (ChIP) tiling array assays (ChIP–chip) with a number of circadian proteins. CLK binding cycles on at least 800 sites with maximal binding in the early night. The CLK partner protein CYCLE (CYC) is on most of these sites. The CLK/CYC heterodimer is joined 4–6 h later by the transcriptional repressor PERIOD (PER), indicating that the majority of CLK targets are regulated similarly to core circadian genes. About 30% of target genes also show cycling RNA polymerase II (Pol II) binding. Many of these generate cycling RNAs despite not being documented in prior RNA cycling studies. This is due in part to different RNA isoforms and to fly head tissue heterogeneity. CLK has specific targets in different tissues, implying that important CLK partner proteins and/or mechanisms contribute to gene-specific and tissue-specific regulation. PMID:22085964

[Analysis of nuclear localization and signal function of MITF protein predisposing to Warrdenburg syndrome].

PubMed

Zhang, Hua; Feng, Juan; Chen, Hongsheng; Li, Jiada; Luo, Hunjin; Feng, Yong

2015-12-01

To study the role of dysfunction of nuclear localization signals (NLS) of MITF protein in the pathogenesis of Waardenburg syndrome. Eukaryotic expression plasmid pCMV-MITF-Flag was used as a template to generate mutant plasmid pCMV-MITF△NLS-Flag by molecular cloning technique in order to design the mutagenic primers. The UACC903 cells were transfected transiently with MITF and MITF△NLS plasmids, and the luciferase activity assays were performed to determine their impact on the transcriptional activities of target gene tyrosinase (TYR). The oligonucleotide 5'-GAACGAAGAAGAAGATTT-3' was subcloned into pEGFP-N1 to generate recombinant eukaryotic expression plasmid pEGFP-N1-MITF-NLS. The NIH3T3 cells were transfected separately with MITF, MITF△NLS, pEGFP-N1 and pEGFP-N1-NLS plasmids, and their subcellular distribution was observed by immunoflorescence assays. Expression plasmids for the mutant MITF△NLS with loss of core NLS sequence and pEGFP-N1-NLS coupled with MITF△NLS were successfully generated. Compared with the wild-type MITF, MITF△NLS was not able to transactivate the transcriptional activities of promoter TYR and did not affect the normal function of MITF. MITF△NLS was only localized in the cytoplasm and pEGFP-N1 was found in both the cytoplasm and nucleus, whereas pEGFP-N1-NLS was mainly located in the nucleus. This study has confirmed the localization function of NLS sequence 213ERRRRF218 within the MITF protein. Mutant MITF with loss of NLS has failed to transactivate the transcriptional activities of target gene TYR, which can result in melanocyte defects and cause WS.

The Chloroplast atpA Gene Cluster in Chlamydomonas reinhardtii1

PubMed Central

Drapier, Dominique; Suzuki, Hideki; Levy, Haim; Rimbault, Blandine; Kindle, Karen L.; Stern, David B.; Wollman, Francis-André

1998-01-01

Most chloroplast genes in vascular plants are organized into polycistronic transcription units, which generate a complex pattern of mono-, di-, and polycistronic transcripts. In contrast, most Chlamydomonas reinhardtii chloroplast transcripts characterized to date have been monocistronic. This paper describes the atpA gene cluster in the C. reinhardtii chloroplast genome, which includes the atpA, psbI, cemA, and atpH genes, encoding the α-subunit of the coupling-factor-1 (CF1) ATP synthase, a small photosystem II polypeptide, a chloroplast envelope membrane protein, and subunit III of the CF0 ATP synthase, respectively. We show that promoters precede the atpA, psbI, and atpH genes, but not the cemA gene, and that cemA mRNA is present only as part of di-, tri-, or tetracistronic transcripts. Deletions introduced into the gene cluster reveal, first, that CF1-α can be translated from di- or polycistronic transcripts, and, second, that substantial reductions in mRNA quantity have minimal effects on protein synthesis rates. We suggest that posttranscriptional mRNA processing is common in C. reinhardtii chloroplasts, permitting the expression of multiple genes from a single promoter. PMID:9625716

Functional analysis of alternative transcripts of the soybean Rj2 gene that restricts nodulation with specific rhizobial strains.

PubMed

Tang, F; Yang, S; Zhu, H

2016-05-01

The Rj2 gene is a TIR-NBS-LRR-type resistance gene in soybean (Glycine max) that restricts root nodule symbiosis with a group of Bradyrhizobium japonicum strains including USDA122. Rj2 generates two distinct transcript variants in its expression profile through alternative splicing. Alternative splicing of Rj2 is caused by the retention of the 86-bp intron 4. Inclusion of intron 4 in mature mRNA introduces an in-frame stop codon; as such, the alternative transcript is predicted to encode a truncated protein consisting of the entire portion of the TIR, NBS and LRR domains but missing the C-terminal domain of the full-length Rj2 protein encoded by the regular transcript. Since alternative splicing has been shown to be essential for full activity of several plant R genes, we attempted to test whether the alternative splicing is required for Rj2-mediated nodulation restriction. Here we demonstrated that the Rj2-mediated nodulation restriction does not require the combined presence of the regular and alternative transcripts, and the expression of the regular transcript alone is sufficient to confer nodulation restriction. © 2016 German Botanical Society and The Royal Botanical Society of the Netherlands.

Redox signaling in pathophysiology of hypertension.

PubMed

Majzunova, Miroslava; Dovinova, Ima; Barancik, Miroslav; Chan, Julie Y H

2013-09-18

Reactive oxygen species (ROS) are products of normal cellular metabolism and derive from various sources in different cellular compartments. Oxidative stress resultant from imbalance between ROS generation and antioxidant defense mechanisms is important in pathogenesis of cardiovascular diseases, such as hypertension, heart failure, atherosclerosis, diabetes, and cardiac hypertrophy. In this review we focus on hypertension and address sources of cellular ROS generation, mechanisms involved in regulation of radical homeostasis, superoxide dismutase isoforms in pathophysiology of hypertension; as well as radical intracellular signaling and phosphorylation processes in proteins of the affected cardiovascular tissues. Finally, we discuss the transcriptional factors involved in redox-sensitive gene transcription and antioxidant response, as well as their roles in hypertension.

Redox signaling in pathophysiology of hypertension

PubMed Central

2013-01-01

Reactive oxygen species (ROS) are products of normal cellular metabolism and derive from various sources in different cellular compartments. Oxidative stress resultant from imbalance between ROS generation and antioxidant defense mechanisms is important in pathogenesis of cardiovascular diseases, such as hypertension, heart failure, atherosclerosis, diabetes, and cardiac hypertrophy. In this review we focus on hypertension and address sources of cellular ROS generation, mechanisms involved in regulation of radical homeostasis, superoxide dismutase isoforms in pathophysiology of hypertension; as well as radical intracellular signaling and phosphorylation processes in proteins of the affected cardiovascular tissues. Finally, we discuss the transcriptional factors involved in redox-sensitive gene transcription and antioxidant response, as well as their roles in hypertension. PMID:24047403

A deep transcriptomic resource for the copepod crustacean Labidocera madurae: A potential indicator species for assessing near shore ecosystem health

PubMed Central

Christie, Andrew E.; Sommer, Stephanie A.; Cieslak, Matthew C.; Hartline, Daniel K.; Lenz, Petra H.

2017-01-01

Coral reef ecosystems of many sub-tropical and tropical marine coastal environments have suffered significant degradation from anthropogenic sources. Research to inform management strategies that mitigate stressors and promote a healthy ecosystem has focused on the ecology and physiology of coral reefs and associated organisms. Few studies focus on the surrounding pelagic communities, which are equally important to ecosystem function. Zooplankton, often dominated by small crustaceans such as copepods, is an important food source for invertebrates and fishes, especially larval fishes. The reef-associated zooplankton includes a sub-neustonic copepod family that could serve as an indicator species for the community. Here, we describe the generation of a de novo transcriptome for one such copepod, Labidocera madurae, a pontellid from an intensively-studied coral reef ecosystem, Kāne‘ohe Bay, Oahu, Hawai‘i. The transcriptome was assembled using high-throughput sequence data obtained from whole organisms. It comprised 211,002 unique transcripts, including 72,391 with coding regions. It was assessed for quality and completeness using multiple workflows. Bench-marking-universal-single-copy-orthologs (BUSCO) analysis identified transcripts for 88% of expected eukaryotic core proteins. Targeted gene-discovery analyses included searches for transcripts coding full-length “giant” proteins (>4,000 amino acids), proteins and splice variants of voltage-gated sodium channels, and proteins involved in the circadian signaling pathway. Four different reference transcriptomes were generated and compared for the detection of differential gene expression between copepodites and adult females; 6,229 genes were consistently identified as differentially expressed between the two regardless of reference. Automated bioinformatics analyses and targeted manual gene curation suggest that the de novo assembled L. madurae transcriptome is of high quality and completeness. This transcriptome provides a new resource for assessing the global physiological status of a planktonic species inhabiting a coral reef ecosystem that is subjected to multiple anthropogenic stressors. The workflows provide a template for generating and assessing transcriptomes in other non-model species. PMID:29065152

A deep transcriptomic resource for the copepod crustacean Labidocera madurae: A potential indicator species for assessing near shore ecosystem health.

PubMed

Roncalli, Vittoria; Christie, Andrew E; Sommer, Stephanie A; Cieslak, Matthew C; Hartline, Daniel K; Lenz, Petra H

2017-01-01

Coral reef ecosystems of many sub-tropical and tropical marine coastal environments have suffered significant degradation from anthropogenic sources. Research to inform management strategies that mitigate stressors and promote a healthy ecosystem has focused on the ecology and physiology of coral reefs and associated organisms. Few studies focus on the surrounding pelagic communities, which are equally important to ecosystem function. Zooplankton, often dominated by small crustaceans such as copepods, is an important food source for invertebrates and fishes, especially larval fishes. The reef-associated zooplankton includes a sub-neustonic copepod family that could serve as an indicator species for the community. Here, we describe the generation of a de novo transcriptome for one such copepod, Labidocera madurae, a pontellid from an intensively-studied coral reef ecosystem, Kāne'ohe Bay, Oahu, Hawai'i. The transcriptome was assembled using high-throughput sequence data obtained from whole organisms. It comprised 211,002 unique transcripts, including 72,391 with coding regions. It was assessed for quality and completeness using multiple workflows. Bench-marking-universal-single-copy-orthologs (BUSCO) analysis identified transcripts for 88% of expected eukaryotic core proteins. Targeted gene-discovery analyses included searches for transcripts coding full-length "giant" proteins (>4,000 amino acids), proteins and splice variants of voltage-gated sodium channels, and proteins involved in the circadian signaling pathway. Four different reference transcriptomes were generated and compared for the detection of differential gene expression between copepodites and adult females; 6,229 genes were consistently identified as differentially expressed between the two regardless of reference. Automated bioinformatics analyses and targeted manual gene curation suggest that the de novo assembled L. madurae transcriptome is of high quality and completeness. This transcriptome provides a new resource for assessing the global physiological status of a planktonic species inhabiting a coral reef ecosystem that is subjected to multiple anthropogenic stressors. The workflows provide a template for generating and assessing transcriptomes in other non-model species.

A transgenic model of transactivation by the Tax protein of HTLV-I.

PubMed

Bieberich, C J; King, C M; Tinkle, B T; Jay, G

1993-09-01

The human T-lymphotropic virus type I (HTLV-I) Tax protein is a transcriptional regulatory protein that has been suggested to play a causal role in the development of several HTLV-I-associated diseases. Tax regulates expression of its own LTR and of certain cellular promoters perhaps by usurping the function of the host transcriptional machinery. We have established a transgenic mouse model system to define the spectrum of tissues in vivo that are capable of supporting Tax-mediated transcriptional transactivation. Transgenic mice carrying the HTLV-I LTR driving expression of the Escherichia coli beta-galactosidase (beta gal) gene were generated, and this LTR-beta gal gene was transcriptionally inactive in all tissues. When LTR-beta gal mice were mated to transgenic mice carrying the same LTR driving expression of the HTLV-I tax gene, mice that carried both transgenes showed restricted expression of the beta gal reporter gene in several tissues including muscle, bone, salivary glands, skin, and nerve. In addition, a dramatic increase in the number of beta gal-expressing cells was seen in response to wounding. These observations provide direct evidence for viral transactivation in vivo, delimit the tissues capable of supporting that transactivation, and provide a model system to study the mechanism of gene regulation by Tax.

Multi-omics approach identifies molecular mechanisms of plant-fungus mycorrhizal interaction

DOE PAGES

Larsen, Peter E.; Sreedasyam, Avinash; Trivedi, Geetika; ...

2016-01-19

In mycorrhizal symbiosis, plant roots form close, mutually beneficial interactions with soil fungi. Before this mycorrhizal interaction can be established however, plant roots must be capable of detecting potential beneficial fungal partners and initiating the gene expression patterns necessary to begin symbiosis. To predict a plant root – mycorrhizal fungi sensor systems, we analyzed in vitro experiments of Populus tremuloides (aspen tree) and Laccaria bicolor (mycorrhizal fungi) interaction and leveraged over 200 previously published transcriptomic experimental data sets, 159 experimentally validated plant transcription factor binding motifs, and more than 120-thousand experimentally validated protein-protein interactions to generate models of pre-mycorrhizal sensormore » systems in aspen root. These sensor mechanisms link extracellular signaling molecules with gene regulation through a network comprised of membrane receptors, signal cascade proteins, transcription factors, and transcription factor biding DNA motifs. Modeling predicted four pre-mycorrhizal sensor complexes in aspen that interact with fifteen transcription factors to regulate the expression of 1184 genes in response to extracellular signals synthesized by Laccaria. Predicted extracellular signaling molecules include common signaling molecules such as phenylpropanoids, salicylate, and, jasmonic acid. Lastly, this multi-omic computational modeling approach for predicting the complex sensory networks yielded specific, testable biological hypotheses for mycorrhizal interaction signaling compounds, sensor complexes, and mechanisms of gene regulation.« less

Multi-omics approach identifies molecular mechanisms of plant-fungus mycorrhizal interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Larsen, Peter E.; Sreedasyam, Avinash; Trivedi, Geetika

In mycorrhizal symbiosis, plant roots form close, mutually beneficial interactions with soil fungi. Before this mycorrhizal interaction can be established however, plant roots must be capable of detecting potential beneficial fungal partners and initiating the gene expression patterns necessary to begin symbiosis. To predict a plant root – mycorrhizal fungi sensor systems, we analyzed in vitro experiments of Populus tremuloides (aspen tree) and Laccaria bicolor (mycorrhizal fungi) interaction and leveraged over 200 previously published transcriptomic experimental data sets, 159 experimentally validated plant transcription factor binding motifs, and more than 120-thousand experimentally validated protein-protein interactions to generate models of pre-mycorrhizal sensormore » systems in aspen root. These sensor mechanisms link extracellular signaling molecules with gene regulation through a network comprised of membrane receptors, signal cascade proteins, transcription factors, and transcription factor biding DNA motifs. Modeling predicted four pre-mycorrhizal sensor complexes in aspen that interact with fifteen transcription factors to regulate the expression of 1184 genes in response to extracellular signals synthesized by Laccaria. Predicted extracellular signaling molecules include common signaling molecules such as phenylpropanoids, salicylate, and, jasmonic acid. Lastly, this multi-omic computational modeling approach for predicting the complex sensory networks yielded specific, testable biological hypotheses for mycorrhizal interaction signaling compounds, sensor complexes, and mechanisms of gene regulation.« less

Neurotrophin Therapy of Neurodegenerative Disorders with Mitochondrial Dysfunction

DTIC Science & Technology

2007-09-01

of the antioxidant transcription factor Nrf2 mediates cytoprotective gene expression in ischemia - reperfusion injury . FASEB J 20: E2166-76. Martin E...anti-apoptotic proteins bax and bcl - 2 . Functionally the Ts16 mitochondria had no change in either calcium uptake capacity or superoxide generation...apoptotic proteins bax and bcl - 2 and of cytochrome c were measured by western blot. Bax is known to redistribute from the cytoplasm to the

DOE Office of Scientific and Technical Information (OSTI.GOV)

Syuhada, O. Nurfarahana; Kalaivani, N.

Sheath blight disease, caused by Rhizoctonia solani 1802/KB was screened on two rice varieties, Oryza sativaindica cultivar MR219 and Oryza sativa indica cultivar UKMRC9. The disease symptom was severe in MR219 compared to UKMRC9. Total RNA from R. solani 1802/KB, infected rice leaves of MR219 and infected rice leaves of UKMRC9 were extracted using TRIzol reagent, purified and sent for small RNA sequencing. Three miRNA libraries were generated and analyzed. The libraries generated 65 805, 78 512 and 81 325 known miRNAs respectively. The structure of miRNA of these samples was predicted. The up-regulated and down-regulated of miRNAs target genemore » prediction and its target functions were discovered and were mainly related to the growth and development of metabolism, protein transport, transcriptional regulation, stress response, and hormone signaling and electron transfer. Sheath blight-induced differential expression of known miRNAs tends to targetMYB transcription factor, F-box proteins, NBS-LRR, leucine-rich repeat receptor protein kinases and zinc finger proteins. Detecting new miRNAs and measuring the expression profiles of known miRNAs is an important tasks required for a better understanding of various biological conditions. Therefore, further analysis using Gene Ontology Slim will be conducted to deduce some biological information from the datasets obtained.« less

Next-generation sequencing-based transcriptomic and proteomic analysis of the common reed, Phragmites australis (Poaceae), reveals genes involved in invasiveness and rhizome specificity.

PubMed

He, Ruifeng; Kim, Min-Jeong; Nelson, William; Balbuena, Tiago S; Kim, Ryan; Kramer, Robin; Crow, John A; May, Greg D; Thelen, Jay J; Soderlund, Carol A; Gang, David R

2012-02-01

The common reed (Phragmites australis), one of the most widely distributed of all angiosperms, uses its rhizomes (underground stems) to invade new territory, making it one of the most successful weedy species worldwide. Characterization of the rhizome transcriptome and proteome is needed to identify candidate genes and proteins involved in rhizome growth, development, metabolism, and invasiveness. We employed next-generation sequencing technologies including 454 and Illumina platforms to characterize the reed rhizome transcriptome and used quantitative proteomics techniques to identify the rhizome proteome. Combining 336514 Roche 454 Titanium reads and 103350802 Illumina paired-end reads in a de novo hybrid assembly yielded 124450 unique transcripts with an average length of 549 bp, of which 54317 were annotated. Rhizome-specific and differentially expressed transcripts were identified between rhizome apical tips (apical meristematic region) and rhizome elongation zones. A total of 1280 nonredundant proteins were identified and quantified using GeLC-MS/MS based label-free proteomics, where 174 and 77 proteins were preferentially expressed in the rhizome elongation zone and apical tip tissues, respectively. Genes involved in allelopathy and in controlling development and potentially invasiveness were identified. In addition to being a valuable sequence and protein data resource for studying plant rhizome species, our results provide useful insights into identifying specific genes and proteins with potential roles in rhizome differentiation, development, and function.

Systemic acquired resistance: turning local infection into global defense.

PubMed

Fu, Zheng Qing; Dong, Xinnian

2013-01-01

Systemic acquired resistance (SAR) is an induced immune mechanism in plants. Unlike vertebrate adaptive immunity, SAR is broad spectrum, with no specificity to the initial infection. An avirulent pathogen causing local programmed cell death can induce SAR through generation of mobile signals, accumulation of the defense hormone salicylic acid, and secretion of the antimicrobial PR (pathogenesis-related) proteins. Consequently, the rest of the plant is protected from secondary infection for a period of weeks to months. SAR can even be passed on to progeny through epigenetic regulation. The Arabidopsis NPR1 (nonexpresser of PR genes 1) protein is a master regulator of SAR. Recent study has shown that salicylic acid directly binds to the NPR1 adaptor proteins NPR3 and NPR4, regulates their interactions with NPR1, and controls NPR1 protein stability. However, how NPR1 interacts with TGA transcription factors to activate defense gene expression is still not well understood. In addition, redox regulators, the mediator complex, WRKY transcription factors, endoplasmic reticulum-resident proteins, and DNA repair proteins play critical roles in SAR.

Deep Sequencing Reveals Uncharted Isoform Heterogeneity of the Protein-Coding Transcriptome in Cerebral Ischemia.

PubMed

Bhattarai, Sunil; Aly, Ahmed; Garcia, Kristy; Ruiz, Diandra; Pontarelli, Fabrizio; Dharap, Ashutosh

2018-06-03

Gene expression in cerebral ischemia has been a subject of intense investigations for several years. Studies utilizing probe-based high-throughput methodologies such as microarrays have contributed significantly to our existing knowledge but lacked the capacity to dissect the transcriptome in detail. Genome-wide RNA-sequencing (RNA-seq) enables comprehensive examinations of transcriptomes for attributes such as strandedness, alternative splicing, alternative transcription start/stop sites, and sequence composition, thus providing a very detailed account of gene expression. Leveraging this capability, we conducted an in-depth, genome-wide evaluation of the protein-coding transcriptome of the adult mouse cortex after transient focal ischemia at 6, 12, or 24 h of reperfusion using RNA-seq. We identified a total of 1007 transcripts at 6 h, 1878 transcripts at 12 h, and 1618 transcripts at 24 h of reperfusion that were significantly altered as compared to sham controls. With isoform-level resolution, we identified 23 splice variants arising from 23 genes that were novel mRNA isoforms. For a subset of genes, we detected reperfusion time-point-dependent splice isoform switching, indicating an expression and/or functional switch for these genes. Finally, for 286 genes across all three reperfusion time-points, we discovered multiple, distinct, simultaneously expressed and differentially altered isoforms per gene that were generated via alternative transcription start/stop sites. Of these, 165 isoforms derived from 109 genes were novel mRNAs. Together, our data unravel the protein-coding transcriptome of the cerebral cortex at an unprecedented depth to provide several new insights into the flexibility and complexity of stroke-related gene transcription and transcript organization.

Novel pre-mRNA splicing of intronically integrated HBV generates oncogenic chimera in hepatocellular carcinoma.

PubMed

Chiu, Yung-Tuen; Wong, John K L; Choi, Shing-Wan; Sze, Karen M F; Ho, Daniel W H; Chan, Lo-Kong; Lee, Joyce M F; Man, Kwan; Cherny, Stacey; Yang, Wan-Ling; Wong, Chun-Ming; Sham, Pak-Chung; Ng, Irene O L

2016-06-01

Hepatitis B virus (HBV) integration is common in HBV-associated hepatocellular carcinoma (HCC) and may play an important pathogenic role through the production of chimeric HBV-human transcripts. We aimed to screen the transcriptome for HBV integrations in HCCs. Transcriptome sequencing was performed on paired HBV-associated HCCs and corresponding non-tumorous liver tissues to identify viral-human chimeric sites. Validation was further performed in an expanded cohort of human HCCs. Here we report the discovery of a novel pre-mRNA splicing mechanism in generating HBV-human chimeric protein. This mechanism was exemplified by the formation of a recurrent HBV-cyclin A2 (CCNA2) chimeric transcript (A2S), as detected in 12.5% (6 of 48) of HCC patients, but in none of the 22 non-HCC HBV-associated cirrhotic liver samples examined. Upon the integration of HBV into the intron of the CCNA2 gene, the mammalian splicing machinery utilized the foreign splice sites at 282nt. and 458nt. of the HBV genome to generate a pseudo-exon, forming an in-frame chimeric fusion with CCNA2. The A2S chimeric protein gained a non-degradable property and promoted cell cycle progression, demonstrating its potential oncogenic functions. A pre-mRNA splicing mechanism is involved in the formation of HBV-human chimeric proteins. This represents a novel and possibly common mechanism underlying the formation of HBV-human chimeric transcripts from intronically integrated HBV genome with functional impact. HBV is involved in the mammalian pre-mRNA splicing machinery in the generation of potential tumorigenic HBV-human chimeras. This study also provided insight on the impact of intronic HBV integration with the gain of splice sites in the development of HBV-associated HCC. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Transcriptome Analysis of Taxillusi chinensis (DC.) Danser Seeds in Response to Water Loss

PubMed Central

Wei, Shugen; Ma, Xiaojun; Pan, Limei; Miao, Jianhua; Fu, Jine; Bai, Longhua; Zhang, Zhonglian; Guan, Yanhong; Mo, Changming; Huang, Hao; Chen, Maoshan

2017-01-01

Background Taxillus chinensis (DC.) Danser, the official species of parasitic loranthus that grows by parasitizing other plants, is used in various traditional Chinese medicine prescriptions. ABA-dependent and ABA-independent pathways are two major pathways in response to drought stress for plants and some genes have been reported to play a key role during the dehydration including dehydration-responsive protein RD22, late embryogenesis abundant (LEA) proteins, and various transcription factors (TFs) like MYB and WRKY. However, genes responding to dehydration are still unknown in loranthus. Methods and Results Initially, loranthus seeds were characterized as recalcitrant seeds. Then, biological replicates of fresh loranthus seeds (CK), and seeds after being dehydrated for 16 hours (Tac-16) and 36 hours (Tac-36) were sequenced by RNA-Seq, generating 386,542,846 high quality reads. A total of 164,546 transcripts corresponding to 114,971 genes were assembled by Trinity and annotated by mapping them to NCBI non-redundant (NR), UniProt, GO, KEGG pathway and COG databases. Transcriptome profiling identified 60,695, 56,027 and 66,389 transcripts (>1 FPKM) in CK, Tac-16 and Tac-36, respectively. Compared to CK, we obtained 2,102 up-regulated and 1,344 down-regulated transcripts in Tac-16 and 1,649 up-regulated and 2,135 down-regulated transcripts in Tac-36 by using edgeR. Among them some have been reported to function in dehydration process, such as RD22, heat shock proteins (HSP) and various TFs (MYB, WRKY and ethylene-responsive transcription factors). Interestingly, transcripts encoding ribosomal proteins peaked in Tac-16. It is indicated that HSPs and ribosomal proteins may function in early response to drought stress. Raw sequencing data can be accessed in NCBI SRA platform under the accession number SRA309567. Conclusions This is the first time to profile transcriptome globally in loranthus seeds. Our findings provide insights into the gene regulations of loranthus seeds in response to water loss and expand our current understanding of drought tolerance and germination of seeds. PMID:28046012

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome

PubMed Central

Camargo, Anamaria A.; Samaia, Helena P. B.; Dias-Neto, Emmanuel; Simão, Daniel F.; Migotto, Italo A.; Briones, Marcelo R. S.; Costa, Fernando F.; Aparecida Nagai, Maria; Verjovski-Almeida, Sergio; Zago, Marco A.; Andrade, Luis Eduardo C.; Carrer, Helaine; El-Dorry, Hamza F. A.; Espreafico, Enilza M.; Habr-Gama, Angelita; Giannella-Neto, Daniel; Goldman, Gustavo H.; Gruber, Arthur; Hackel, Christine; Kimura, Edna T.; Maciel, Rui M. B.; Marie, Suely K. N.; Martins, Elizabeth A. L.; Nóbrega, Marina P.; Paçó-Larson, Maria Luisa; Pardini, Maria Inês M. C.; Pereira, Gonçalo G.; Pesquero, João Bosco; Rodrigues, Vanderlei; Rogatto, Silvia R.; da Silva, Ismael D. C. G.; Sogayar, Mari C.; Sonati, Maria de Fátima; Tajara, Eloiza H.; Valentini, Sandro R.; Alberto, Fernando L.; Amaral, Maria Elisabete J.; Aneas, Ivy; Arnaldi, Liliane A. T.; de Assis, Angela M.; Bengtson, Mário Henrique; Bergamo, Nadia Aparecida; Bombonato, Vanessa; de Camargo, Maria E. R.; Canevari, Renata A.; Carraro, Dirce M.; Cerutti, Janete M.; Corrêa, Maria Lucia C.; Corrêa, Rosana F. R.; Costa, Maria Cristina R.; Curcio, Cyntia; Hokama, Paula O. M.; Ferreira, Ari J. S.; Furuzawa, Gilberto K.; Gushiken, Tsieko; Ho, Paulo L.; Kimura, Elza; Krieger, José E.; Leite, Luciana C. C.; Majumder, Paromita; Marins, Mozart; Marques, Everaldo R.; Melo, Analy S. A.; Melo, Monica; Mestriner, Carlos Alberto; Miracca, Elisabete C.; Miranda, Daniela C.; Nascimento, Ana Lucia T. O.; Nóbrega, Francisco G.; Ojopi, Élida P. B.; Pandolfi, José Rodrigo C.; Pessoa, Luciana G.; Prevedel, Aline C.; Rahal, Paula; Rainho, Claudia A.; Reis, Eduardo M. R.; Ribeiro, Marcelo L.; da Rós, Nancy; de Sá, Renata G.; Sales, Magaly M.; Sant'anna, Simone Cristina; dos Santos, Mariana L.; da Silva, Aline M.; da Silva, Neusa P.; Silva, Wilson A.; da Silveira, Rosana A.; Sousa, Josane F.; Stecconi, Daniella; Tsukumo, Fernando; Valente, Valéria; Soares, Fernando; Moreira, Eloisa S.; Nunes, Diana N.; Correa, Ricardo G.; Zalcberg, Heloisa; Carvalho, Alex F.; Reis, Luis F. L.; Brentani, Ricardo R.; Simpson, Andrew J. G.; de Souza, Sandro J.

2001-01-01

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription–PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning. PMID:11593022

On-Chip Synthesis of Protein Microarrays from DNA Microarrays Via Coupled In Vitro Transcription and Translation for Surface Plasmon Resonance Imaging Biosensor Applications

PubMed Central

Seefeld, Ting H.; Halpern, Aaron R.; Corn, Robert M.

2012-01-01

Protein microarrays are fabricated from double-stranded DNA (dsDNA) microarrays by a one-step, multiplexed enzymatic synthesis in an on-chip microfluidic format and then employed for antibody biosensing measurements with surface plasmon resonance imaging (SPRI). A microarray of dsDNA elements (denoted as generator elements) that encode either a His-tagged green fluorescent protein (GFP) or a His-tagged luciferase protein is utilized to create multiple copies of messenger RNA (mRNA) in a surface RNA polymerase reaction; the mRNA transcripts are then translated into proteins by cell-free protein synthesis in a microfluidic format. The His-tagged proteins diffuse to adjacent Cu(II)-NTA microarray elements (denoted as detector elements) and are specifically adsorbed. The net result is the on-chip, cell-free synthesis of a protein microarray that can be used immediately for SPRI protein biosensing. The dual element format greatly reduces any interference from the nonspecific adsorption of enzyme or proteins. SPRI measurements for the detection of the antibodies anti-GFP and anti-luciferase were used to verify the formation of the protein microarray. This convenient on-chip protein microarray fabrication method can be implemented for multiplexed SPRI biosensing measurements in both clinical and research applications. PMID:22793370

Complementary Proteome and Transcriptome Profiling in Developing Grains of a Notched-Belly Rice Mutant Reveals Key Pathways Involved in Chalkiness Formation

PubMed Central

Lin, Zhaomiao; Wang, Zunxin; Zhang, Xincheng; Li, Ganghua; Wang, Shaohua; Ding, Yanfeng

2017-01-01

Rice grain chalkiness is a highly complex trait involved in multiple metabolic pathways and controlled by polygenes and growth conditions. To uncover novel aspects of chalkiness formation, we performed an integrated profiling of gene activity in the developing grains of a notched-belly rice mutant. Using exhaustive tandem mass spectrometry-based shotgun proteomics and whole-genome RNA sequencing to generate a nearly complete catalog of expressed mRNAs and proteins, we reliably identified 38,476 transcripts and 3,840 proteins. Comparison between the translucent part and chalky part of the notched-belly grains resulted in only a few differently express genes (240) and differently express proteins (363), thus making it possible to focus on ‘core’ genes or common pathways. Several novel key pathways were identified as of relevance to chalkiness formation, in particular the shift of C and N metabolism, the down-regulation of ribosomal proteins and the resulting low abundance of storage proteins especially the 13 kDa prolamin subunit, and the suppressed photosynthetic capacity in the pericarp of the chalky part. Further, genes and proteins as transporters for carbohydrates, amino acid/peptides, proteins, lipids and inorganic ions showed an increasing expression pattern in the chalky part of the notched-belly grains. Similarly, transcripts and proteins of receptors for auxin, ABA, ethylene and brassinosteroid were also up-regulated. In summary, this joint analysis of transcript and protein profiles provides a comprehensive reference map of gene activity regarding the physiological state in the chalky endosperm. PMID:28158863

Transcriptional analysis of the Arabidopsis ovule by massively parallel signature sequencing

PubMed Central

Sánchez-León, Nidia; Arteaga-Vázquez, Mario; Alvarez-Mejía, César; Mendiola-Soto, Javier; Durán-Figueroa, Noé; Rodríguez-Leal, Daniel; Rodríguez-Arévalo, Isaac; García-Campayo, Vicenta; García-Aguilar, Marcelina; Olmedo-Monfil, Vianey; Arteaga-Sánchez, Mario; Martínez de la Vega, Octavio; Nobuta, Kan; Vemaraju, Kalyan; Meyers, Blake C.; Vielle-Calzada, Jean-Philippe

2012-01-01

The life cycle of flowering plants alternates between a predominant sporophytic (diploid) and an ephemeral gametophytic (haploid) generation that only occurs in reproductive organs. In Arabidopsis thaliana, the female gametophyte is deeply embedded within the ovule, complicating the study of the genetic and molecular interactions involved in the sporophytic to gametophytic transition. Massively parallel signature sequencing (MPSS) was used to conduct a quantitative large-scale transcriptional analysis of the fully differentiated Arabidopsis ovule prior to fertilization. The expression of 9775 genes was quantified in wild-type ovules, additionally detecting >2200 new transcripts mapping to antisense or intergenic regions. A quantitative comparison of global expression in wild-type and sporocyteless (spl) individuals resulted in 1301 genes showing 25-fold reduced or null activity in ovules lacking a female gametophyte, including those encoding 92 signalling proteins, 75 transcription factors, and 72 RNA-binding proteins not reported in previous studies based on microarray profiling. A combination of independent genetic and molecular strategies confirmed the differential expression of 28 of them, showing that they are either preferentially active in the female gametophyte, or dependent on the presence of a female gametophyte to be expressed in sporophytic cells of the ovule. Among 18 genes encoding pentatricopeptide-repeat proteins (PPRs) that show transcriptional activity in wild-type but not spl ovules, CIHUATEOTL (At4g38150) is specifically expressed in the female gametophyte and necessary for female gametogenesis. These results expand the nature of the transcriptional universe present in the ovule of Arabidopsis, and offer a large-scale quantitative reference of global expression for future genomic and developmental studies. PMID:22442422

Transcriptional analysis of the Arabidopsis ovule by massively parallel signature sequencing.

PubMed

Sánchez-León, Nidia; Arteaga-Vázquez, Mario; Alvarez-Mejía, César; Mendiola-Soto, Javier; Durán-Figueroa, Noé; Rodríguez-Leal, Daniel; Rodríguez-Arévalo, Isaac; García-Campayo, Vicenta; García-Aguilar, Marcelina; Olmedo-Monfil, Vianey; Arteaga-Sánchez, Mario; de la Vega, Octavio Martínez; Nobuta, Kan; Vemaraju, Kalyan; Meyers, Blake C; Vielle-Calzada, Jean-Philippe

2012-06-01

The life cycle of flowering plants alternates between a predominant sporophytic (diploid) and an ephemeral gametophytic (haploid) generation that only occurs in reproductive organs. In Arabidopsis thaliana, the female gametophyte is deeply embedded within the ovule, complicating the study of the genetic and molecular interactions involved in the sporophytic to gametophytic transition. Massively parallel signature sequencing (MPSS) was used to conduct a quantitative large-scale transcriptional analysis of the fully differentiated Arabidopsis ovule prior to fertilization. The expression of 9775 genes was quantified in wild-type ovules, additionally detecting >2200 new transcripts mapping to antisense or intergenic regions. A quantitative comparison of global expression in wild-type and sporocyteless (spl) individuals resulted in 1301 genes showing 25-fold reduced or null activity in ovules lacking a female gametophyte, including those encoding 92 signalling proteins, 75 transcription factors, and 72 RNA-binding proteins not reported in previous studies based on microarray profiling. A combination of independent genetic and molecular strategies confirmed the differential expression of 28 of them, showing that they are either preferentially active in the female gametophyte, or dependent on the presence of a female gametophyte to be expressed in sporophytic cells of the ovule. Among 18 genes encoding pentatricopeptide-repeat proteins (PPRs) that show transcriptional activity in wild-type but not spl ovules, CIHUATEOTL (At4g38150) is specifically expressed in the female gametophyte and necessary for female gametogenesis. These results expand the nature of the transcriptional universe present in the ovule of Arabidopsis, and offer a large-scale quantitative reference of global expression for future genomic and developmental studies.

IgE recognition of chimeric isoforms of the honeybee (Apis mellifera) venom allergen Api m 10 evaluated by protein array technology.

PubMed

Van Vaerenbergh, Matthias; De Smet, Lina; Rafei-Shamsabadi, David; Blank, Simon; Spillner, Edzard; Ebo, Didier G; Devreese, Bart; Jakob, Thilo; de Graaf, Dirk C

2015-02-01

Api m 10 has recently been established as novel major allergen that is recognized by more than 60% of honeybee venom (HBV) allergic patients. Previous studies suggest Api m 10 protein heterogeneity which may have implications for diagnosis and immunotherapy of HBV allergy. In the present study, RT-PCR revealed the expression of at least nine additional Api m 10 transcript isoforms by the venom glands. Two distinct mechanisms are responsible for the generation of these isoforms: while the previously known variant 2 is produced by an alternative splicing event, novel identified isoforms are intragenic chimeric transcripts. To the best of our knowledge, this is the first report of the identification of chimeric transcripts generated by the honeybee. By a retrospective proteomic analysis we found evidence for the presence of several of these isoforms in the venom proteome. Additionally, we analyzed IgE reactivity to different isoforms by protein array technology using sera from HBV allergic patients, which revealed that IgE recognition of Api m 10 is both isoform- and patient-specific. While it was previously demonstrated that the majority of HBV allergic patients display IgE reactivity to variant 2, our study also shows that some patients lacking IgE antibodies for variant 2 display IgE reactivity to two of the novel identified Api m 10 variants, i.e. variants 3 and 4. Copyright © 2014 Elsevier Ltd. All rights reserved.

Ab Initio Structural Modeling of and Experimental Validation for Chlamydia trachomatis Protein CT296 Reveal Structural Similarity to Fe(II) 2-Oxoglutarate-Dependent Enzymes▿

PubMed Central

Kemege, Kyle E.; Hickey, John M.; Lovell, Scott; Battaile, Kevin P.; Zhang, Yang; Hefty, P. Scott

2011-01-01

Chlamydia trachomatis is a medically important pathogen that encodes a relatively high percentage of proteins with unknown function. The three-dimensional structure of a protein can be very informative regarding the protein's functional characteristics; however, determining protein structures experimentally can be very challenging. Computational methods that model protein structures with sufficient accuracy to facilitate functional studies have had notable successes. To evaluate the accuracy and potential impact of computational protein structure modeling of hypothetical proteins encoded by Chlamydia, a successful computational method termed I-TASSER was utilized to model the three-dimensional structure of a hypothetical protein encoded by open reading frame (ORF) CT296. CT296 has been reported to exhibit functional properties of a divalent cation transcription repressor (DcrA), with similarity to the Escherichia coli iron-responsive transcriptional repressor, Fur. Unexpectedly, the I-TASSER model of CT296 exhibited no structural similarity to any DNA-interacting proteins or motifs. To validate the I-TASSER-generated model, the structure of CT296 was solved experimentally using X-ray crystallography. Impressively, the ab initio I-TASSER-generated model closely matched (2.72-Å Cα root mean square deviation [RMSD]) the high-resolution (1.8-Å) crystal structure of CT296. Modeled and experimentally determined structures of CT296 share structural characteristics of non-heme Fe(II) 2-oxoglutarate-dependent enzymes, although key enzymatic residues are not conserved, suggesting a unique biochemical process is likely associated with CT296 function. Additionally, functional analyses did not support prior reports that CT296 has properties shared with divalent cation repressors such as Fur. PMID:21965559

Ab initio structural modeling of and experimental validation for Chlamydia trachomatis protein CT296 reveal structural similarity to Fe(II) 2-oxoglutarate-dependent enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kemege, Kyle E.; Hickey, John M.; Lovell, Scott

2012-02-13

Chlamydia trachomatis is a medically important pathogen that encodes a relatively high percentage of proteins with unknown function. The three-dimensional structure of a protein can be very informative regarding the protein's functional characteristics; however, determining protein structures experimentally can be very challenging. Computational methods that model protein structures with sufficient accuracy to facilitate functional studies have had notable successes. To evaluate the accuracy and potential impact of computational protein structure modeling of hypothetical proteins encoded by Chlamydia, a successful computational method termed I-TASSER was utilized to model the three-dimensional structure of a hypothetical protein encoded by open reading frame (ORF)more » CT296. CT296 has been reported to exhibit functional properties of a divalent cation transcription repressor (DcrA), with similarity to the Escherichia coli iron-responsive transcriptional repressor, Fur. Unexpectedly, the I-TASSER model of CT296 exhibited no structural similarity to any DNA-interacting proteins or motifs. To validate the I-TASSER-generated model, the structure of CT296 was solved experimentally using X-ray crystallography. Impressively, the ab initio I-TASSER-generated model closely matched (2.72-{angstrom} C{alpha} root mean square deviation [RMSD]) the high-resolution (1.8-{angstrom}) crystal structure of CT296. Modeled and experimentally determined structures of CT296 share structural characteristics of non-heme Fe(II) 2-oxoglutarate-dependent enzymes, although key enzymatic residues are not conserved, suggesting a unique biochemical process is likely associated with CT296 function. Additionally, functional analyses did not support prior reports that CT296 has properties shared with divalent cation repressors such as Fur.« less

Cell-free system for synthesizing membrane proteins cell free method for synthesizing membrane proteins

DOEpatents

Laible, Philip D; Hanson, Deborah K

2013-06-04

The invention provides an in vitro method for producing proteins, membrane proteins, membrane-associated proteins, and soluble proteins that interact with membrane-associated proteins for assembly into an oligomeric complex or that require association with a membrane for proper folding. The method comprises, supplying intracytoplasmic membranes from organisms; modifying protein composition of intracytoplasmic membranes from organism by modifying DNA to delete genes encoding functions of the organism not associated with the formation of the intracytoplasmic membranes; generating appropriate DNA or RNA templates that encode the target protein; and mixing the intracytoplasmic membranes with the template and a transcription/translation-competent cellular extract to cause simultaneous production of the membrane proteins and encapsulation of the membrane proteins within the intracytoplasmic membranes.

Mongolian Almond (Prunus mongolica Maxim): The Morpho-Physiological, Biochemical and Transcriptomic Response to Drought Stress

PubMed Central

Bai, Shulan; Gao, Xiaomin; Liu, Min; Yan, Wei

2015-01-01

Prunus mongolica Maxim, which is widely established in the Gobi Desert, shows extreme tolerance to drought. However, there is a lack of available transcriptomic resources for this species related to its response to water deficiency. To investigate the mechanisms that allow P. mongolica to maintain growth in extremely arid environments, the response of P. mongolica seedlings to drought stress was analyzed using morphological, physiological, biochemical and high-throughput sequencing approaches. We generated 28,713,735 and 26,650,133 raw reads from no-stress control and drought-stressed P. mongolica seedlings, respectively. In total, we obtained 67,352 transcripts with an average length of 874.44 bp. Compared with the no-stress control, 3,365 transcripts were differentially expressed in the drought-stressed seedlings, including 55.75% (1,876 transcripts) up-regulated and 44.25% (1,489 transcripts) down-regulated transcripts. The photosynthesis response showed a decreasing tendency under drought stress, but the changes in the levels of hormones (auxins, cytokinins and abscisic acid) resulted in the closing of stomata and decreased cell enlargement and division; these changes were effective for promoting P. mongolica survival in Gobi Desert. Next, we analyzed the aquaporin and superoxide dismutase gene families due to their importance in plant resistance to drought stress. We found that all of the plasma membrane intrinsic protein transcripts were down-regulated in the drought-stressed treatment, whereas drought did not affect the expression of nodulin intrinsic protein or small basic intrinsic protein transcripts in P. mongolica seedlings. In addition, activation of iron superoxide dismutase transcription and enhanced transcription of manganese superoxide dismutase were observed in P. mongolica to promote tolerance of drought stress. This study identified drought response genes in P. mongolica seedlings. Our results provide a significant contribution to the understanding of how P. mongolica responds to drought stress at the transcriptome level, which may help to elucidate molecular mechanisms associated with the drought response of almond plants. PMID:25893685

Global transcriptomic analysis of induced cardiomyocytes predicts novel regulators for direct cardiac reprogramming.

PubMed

Talkhabi, Mahmood; Razavi, Seyed Morteza; Salari, Ali

2017-06-01

Heart diseases are the most significant cause of morbidity and mortality in the world. De novo generated cardiomyocytes (CMs) are a great cellular source for cell-based therapy and other potential applications. Direct cardiac reprogramming is the newest method to produce CMs, known as induced cardiomyocytes (iCMs). During a direct cardiac reprogramming, also known as transdifferentiation, non-cardiac differentiated adult cells are reprogrammed to cardiac identity by forced expression of cardiac-specific transcription factors (TFs) or microRNAs. To this end, many different combinations of TFs (±microRNAs) have been reported for direct reprogramming of mouse or human fibroblasts to iCMs, although their efficiencies remain very low. It seems that the investigated TFs and microRNAs are not sufficient for efficient direct cardiac reprogramming and other cardiac specific factors may be required for increasing iCM production efficiency, as well as the quality of iCMs. Here, we analyzed gene expression data of cardiac fibroblast (CFs), iCMs and adult cardiomyocytes (aCMs). The up-regulated and down-regulated genes in CMs (aCMs and iCMs) were determined as CM and CF specific genes, respectively. Among CM specific genes, we found 153 transcriptional activators including some cardiac and non-cardiac TFs that potentially activate the expression of CM specific genes. We also identified that 85 protein kinases such as protein kinase D1 (PKD1), protein kinase A (PRKA), calcium/calmodulin-dependent protein kinase (CAMK), protein kinase C (PRKC), and insulin like growth factor 1 receptor (IGF1R) that are strongly involved in establishing CM identity. CM gene regulatory network constructed using protein kinases, transcriptional activators and intermediate proteins predicted some new transcriptional activators such as myocyte enhancer factor 2A (MEF2A) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A), which may be required for qualitatively and quantitatively efficient direct cardiac reprogramming. Taken together, this study provides new insights into the complexity of cell fate conversion and better understanding of the roles of transcriptional activators, signaling pathways and protein kinases in increasing the efficiency of direct cardiac reprogramming and maturity of iCMs.

The use of in vitro transcription to probe regulatory functions of viral protein domains.

PubMed

Loewenstein, Paul M; Song, Chao-Zhong; Green, Maurice

2007-01-01

Adenoviruses (Ads), like other DNA tumor viruses, have evolved specific regulatory genes that facilitate virus replication by controlling the transcription of other viral genes as well as that of key cellular genes. In this regard, the E1A transcription unit contains multiple protein domains that can transcriptionally activate or repress cellular genes involved in the regulation of cell proliferation and cell differentiation. Studies using in vitro transcription have provided a basis for a molecular understanding of the interaction of viral regulatory proteins with the transcriptional machinery of the cell and continue to inform our understanding of transcription regulation. This chapter provides examples of the use of in vitro transcription to analyze transcriptional activation and transcriptional repression by purified, recombinant Ad E1A protein domains and single amino acid substitution mutants as well as the use of protein-affinity chromatography to identify host cell transcription factors involved in viral transcriptional regulation. A detailed description is provided of the methodology to prepare nuclear transcription extract, to prepare biologically active protein domains, to prepare affinity depleted transcription extracts, and to analyze transcription by primer extension and by run-off assay using naked DNA templates.

The Regulation of Transcription in Memory Consolidation

PubMed Central

Alberini, Cristina M.; Kandel, Eric R.

2015-01-01

De novo transcription of DNA is a fundamental requirement for the formation of long-term memory. It is required during both consolidation and reconsolidation, the posttraining and postreactivation phases that change the state of the memory from a fragile into a stable and long-lasting form. Transcription generates both mRNAs that are translated into proteins, which are necessary for the growth of new synaptic connections, as well as noncoding RNA transcripts that have regulatory or effector roles in gene expression. The result is a cascade of events that ultimately leads to structural changes in the neurons that mediate long-term memory storage. The de novo transcription, critical for synaptic plasticity and memory formation, is orchestrated by chromatin and epigenetic modifications. The complexity of transcription regulation, its temporal progression, and the effectors produced all contribute to the flexibility and persistence of long-term memory formation. In this article, we provide an overview of the mechanisms contributing to this transcriptional regulation underlying long-term memory formation. PMID:25475090

Translation initiation events on structured eukaryotic mRNAs generate gene expression noise

PubMed Central

Dacheux, Estelle; Malys, Naglis; Meng, Xiang; Ramachandran, Vinoy; Mendes, Pedro

2017-01-01

Abstract Gene expression stochasticity plays a major role in biology, creating non-genetic cellular individuality and influencing multiple processes, including differentiation and stress responses. We have addressed the lack of knowledge about posttranscriptional contributions to noise by determining cell-to-cell variations in the abundance of mRNA and reporter protein in yeast. Two types of structural element, a stem–loop and a poly(G) motif, not only inhibit translation initiation when inserted into an mRNA 5΄ untranslated region, but also generate noise. The noise-enhancing effect of the stem–loop structure also remains operational when combined with an upstream open reading frame. This has broad significance, since these elements are known to modulate the expression of a diversity of eukaryotic genes. Our findings suggest a mechanism for posttranscriptional noise generation that will contribute to understanding of the generally poor correlation between protein-level stochasticity and transcriptional bursting. We propose that posttranscriptional stochasticity can be linked to cycles of folding/unfolding of a stem–loop structure, or to interconversion between higher-order structural conformations of a G-rich motif, and have created a correspondingly configured computational model that generates fits to the experimental data. Stochastic events occurring during the ribosomal scanning process can therefore feature alongside transcriptional bursting as a source of noise. PMID:28521011

Systematic analysis of transcribed loci in ENCODE regions using RACE sequencing reveals extensive transcription in the human genome.

PubMed

Wu, Jia Qian; Du, Jiang; Rozowsky, Joel; Zhang, Zhengdong; Urban, Alexander E; Euskirchen, Ghia; Weissman, Sherman; Gerstein, Mark; Snyder, Michael

2008-01-03

Recent studies of the mammalian transcriptome have revealed a large number of additional transcribed regions and extraordinary complexity in transcript diversity. However, there is still much uncertainty regarding precisely what portion of the genome is transcribed, the exact structures of these novel transcripts, and the levels of the transcripts produced. We have interrogated the transcribed loci in 420 selected ENCyclopedia Of DNA Elements (ENCODE) regions using rapid amplification of cDNA ends (RACE) sequencing. We analyzed annotated known gene regions, but primarily we focused on novel transcriptionally active regions (TARs), which were previously identified by high-density oligonucleotide tiling arrays and on random regions that were not believed to be transcribed. We found RACE sequencing to be very sensitive and were able to detect low levels of transcripts in specific cell types that were not detectable by microarrays. We also observed many instances of sense-antisense transcripts; further analysis suggests that many of the antisense transcripts (but not all) may be artifacts generated from the reverse transcription reaction. Our results show that the majority of the novel TARs analyzed (60%) are connected to other novel TARs or known exons. Of previously unannotated random regions, 17% were shown to produce overlapping transcripts. Furthermore, it is estimated that 9% of the novel transcripts encode proteins. We conclude that RACE sequencing is an efficient, sensitive, and highly accurate method for characterization of the transcriptome of specific cell/tissue types. Using this method, it appears that much of the genome is represented in polyA+ RNA. Moreover, a fraction of the novel RNAs can encode protein and are likely to be functional.

Zic-Proteins Are Repressors of Dopaminergic Forebrain Fate in Mice and C. elegans.

PubMed

Tiveron, Marie-Catherine; Beclin, Christophe; Murgan, Sabrina; Wild, Stefan; Angelova, Alexandra; Marc, Julie; Coré, Nathalie; de Chevigny, Antoine; Herrera, Eloisa; Bosio, Andreas; Bertrand, Vincent; Cremer, Harold

2017-11-01

In the postnatal forebrain regionalized neural stem cells along the ventricular walls produce olfactory bulb (OB) interneurons with varying neurotransmitter phenotypes and positions. To understand the molecular basis of this region-specific variability we analyzed gene expression in the postnatal dorsal and lateral lineages in mice of both sexes from stem cells to neurons. We show that both lineages maintain transcription factor signatures of their embryonic site of origin, the pallium and subpallium. However, additional factors, including Zic1 and Zic2, are postnatally expressed in the dorsal stem cell compartment and maintained in the lineage that generates calretinin-positive GABAergic neurons for the OB. Functionally, we show that Zic1 and Zic2 induce the generation of calretinin-positive neurons while suppressing dopaminergic fate in the postnatal dorsal lineage. We investigated the evolutionary conservation of the dopaminergic repressor function of Zic proteins and show that it is already present in C. elegans SIGNIFICANCE STATEMENT The vertebrate brain generates thousands of different neuron types. In this work we investigate the molecular mechanisms underlying this variability. Using a genomics approach we identify the transcription factor signatures of defined neural stem cells and neuron populations. Based thereon we show that two related transcription factors, Zic1 and Zic2, are essential to control the balance between two defined neuron types in the postnatal brain. We show that this mechanism is conserved in evolutionary very distant species. Copyright © 2017 the authors 0270-6474/17/3710611-13$15.00/0.

Diversity and functional convergence of small noncoding RNAs in male germ cell differentiation and fertilization

PubMed Central

García-López, Jesús; Alonso, Lola; Cárdenas, David B.; Artaza-Alvarez, Haydeé; Hourcade, Juan de Dios; Martínez, Sergio; Brieño-Enríquez, Miguel A.; del Mazo, Jesús

2015-01-01

The small noncoding RNAs (sncRNAs) are considered as post-transcriptional key regulators of male germ cell development. In addition to microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), other sncRNAs generated from small nucleolar RNAs (snoRNAs), tRNAs, or rRNAs processing may also play important regulatory roles in spermatogenesis. By next-generation sequencing (NGS), we characterized the sncRNA populations detected at three milestone stages in male germ differentiation: primordial germ cells (PGCs), pubertal spermatogonia cells, and mature spermatozoa. To assess their potential transmission through the spermatozoa during fertilization, the sncRNAs of mouse oocytes and zygotes were also analyzed. Both, microRNAs and snoRNA-derived small RNAs are abundantly expressed in PGCs but transiently replaced by piRNAs in spermatozoa and endo-siRNAs in oocytes and zygotes. Exhaustive analysis of miRNA sequence variants also shows an increment of noncanonical microRNA forms along male germ cell differentiation. RNAs-derived from tRNAs and rRNAs interacting with PIWI proteins are not generated by the ping-pong pathway and could be a source of primary piRNAs. Moreover, our results strongly suggest that the small RNAs-derived from tRNAs and rRNAs are interacting with PIWI proteins, and specifically with MILI. Finally, computational analysis revealed their potential involvement in post-transcriptional regulation of mRNA transcripts suggesting functional convergence among different small RNA classes in germ cells and zygotes. PMID:25805854

Transcriptome Profiling of Selectively Bred Pacific Oyster Crassostrea gigas Families that Differ in Tolerance of Heat Shock

PubMed Central

Bayne, Christopher J.; Camara, Mark D.; Cunningham, Charles; Jenny, Matthew J.; Langdon, Christopher J.

2010-01-01

Sessile inhabitants of marine intertidal environments commonly face heat stress, an important component of summer mortality syndrome in the Pacific oyster Crassostrea gigas. Marker-aided selection programs would be useful for developing oyster strains that resist summer mortality; however, there is currently a need to identify candidate genes associated with stress tolerance and to develop molecular markers associated with those genes. To identify candidate genes for further study, we used cDNA microarrays to test the hypothesis that oyster families that had high (>64%) or low (<29%) survival of heat shock (43°C, 1 h) differ in their transcriptional responses to stress. Based upon data generated by the microarray and by real-time quantitative PCR, we found that transcription after heat shock increased for genes putatively encoding heat shock proteins and genes for proteins that synthesize lipids, protect against bacterial infection, and regulate spawning, whereas transcription decreased for genes for proteins that mobilize lipids and detoxify reactive oxygen species. RNAs putatively identified as heat shock protein 27, collagen, peroxinectin, S-crystallin, and two genes with no match in Genbank had higher transcript concentrations in low-surviving families than in high-surviving families, whereas concentration of putative cystatin B mRNA was greater in high-surviving families. These ESTs should be studied further for use in marker-aided selection programs. Low survival of heat shock could result from a complex interaction of cell damage, opportunistic infection, and metabolic exhaustion. PMID:19205802

Identification, cloning, and sequencing of a fragment of Amsacta moorei entomopoxvirus DNA containing the spheroidin gene and three vaccinia virus-related open reading frames.

PubMed Central

Hall, R L; Moyer, R W

1991-01-01

Entomopoxvirus virions are frequently contained within crystalline occlusion bodies, which are composed of primarily a single protein, spheroidin, which is analogous to the polyhedrin protein of baculovirus. The spheroidin gene of Amsacta moorei entomopoxvirus was identified following the microsequencing of polypeptides generated from cyanogen bromide treatment of spheroidin and the subsequent synthesis of oligonucleotide hybridization probes. DNA sequencing of a 6.8-kb region of DNA containing the spheroidin gene showed that the spheroidin protein is derived from a 3.0-kb open reading frame potentially encoding a protein of 115 kDa. Three copies of the heptanucleotide, TTTTTNT, a sequence associated with early gene transcription in the vertebrate poxviruses, and four in-frame translational termination signals were found within 60 bp upstream of the putative spheroidin gene promoter (TAAATG). The spheroidin gene promoter region contains the sequence TAAATG, which is found in many late promoters of the vertebrate poxviruses and which serves as the site of transcriptional initiation, as shown by primer extension. Primer extension experiments also showed that spheroidin gene transcripts contain 5' poly(A) sequences typical of vertebrate poxvirus late transcripts. The 92 bases upstream of the initiating TAAATG are unusually A + T rich and contain only 7 G or C residues. An analysis of open reading frames around the spheroidin gene suggests that the colinear core of "essential genes" typical of the vertebrate poxviruses is absent in A. moorei entomopoxvirus. Images PMID:1942245

Integrated Analysis of Transcriptomic and Proteomic Data

PubMed Central

Haider, Saad; Pal, Ranadip

2013-01-01

Until recently, understanding the regulatory behavior of cells has been pursued through independent analysis of the transcriptome or the proteome. Based on the central dogma, it was generally assumed that there exist a direct correspondence between mRNA transcripts and generated protein expressions. However, recent studies have shown that the correlation between mRNA and Protein expressions can be low due to various factors such as different half lives and post transcription machinery. Thus, a joint analysis of the transcriptomic and proteomic data can provide useful insights that may not be deciphered from individual analysis of mRNA or protein expressions. This article reviews the existing major approaches for joint analysis of transcriptomic and proteomic data. We categorize the different approaches into eight main categories based on the initial algorithm and final analysis goal. We further present analogies with other domains and discuss the existing research problems in this area. PMID:24082820

Integration of multi-omics data of a genome-reduced bacterium: Prevalence of post-transcriptional regulation and its correlation with protein abundances

PubMed Central

Chen, Wei-Hua; van Noort, Vera; Lluch-Senar, Maria; Hennrich, Marco L.; H. Wodke, Judith A.; Yus, Eva; Alibés, Andreu; Roma, Guglielmo; Mende, Daniel R.; Pesavento, Christina; Typas, Athanasios; Gavin, Anne-Claude; Serrano, Luis; Bork, Peer

2016-01-01

We developed a comprehensive resource for the genome-reduced bacterium Mycoplasma pneumoniae comprising 1748 consistently generated ‘-omics’ data sets, and used it to quantify the power of antisense non-coding RNAs (ncRNAs), lysine acetylation, and protein phosphorylation in predicting protein abundance (11%, 24% and 8%, respectively). These factors taken together are four times more predictive of the proteome abundance than of mRNA abundance. In bacteria, post-translational modifications (PTMs) and ncRNA transcription were both found to increase with decreasing genomic GC-content and genome size. Thus, the evolutionary forces constraining genome size and GC-content modify the relative contributions of the different regulatory layers to proteome homeostasis, and impact more genomic and genetic features than previously appreciated. Indeed, these scaling principles will enable us to develop more informed approaches when engineering minimal synthetic genomes. PMID:26773059

Cloning and characterization of a gene encoding Rac/Rop-like monomeric guanosine 5'-triphosphate-binding protein from Scoparia dulcis.

PubMed

Mitamura, Toshiaki; Shite, Masato; Yamamura, Yoshimi; Kurosaki, Fumiya

2009-06-01

A cDNA clone, designated Sd-racrop (969 bp), was isolated from seedlings of Scoparia dulcis. This gene contains an open reading frame encoding the protein of 197 amino acid residues with high homology to Rac/Rop small guanosine 5'-triphosphate-binding proteins from various plant sources. In Southern hybridization analysis, the restriction digests prepared from genomic DNA of S. dulcis showed a main signal together with a few weakly hybridized bands. The transcriptional level of Sd-racrop showed a transient decrease by exposure of the leaf tissues of S. dulcis to the ethylene-generating reagent 2-chloroethylphosphonic acid. However, an appreciable increase in gene expression was reproducibly observed upon treatment of the plant with methyl jasmonate. These results suggest that the Sd-racrop product plays roles in ethylene- and methyl jasmonate-induced responses of S. dulcis accompanying the change in the transcriptional level, however, the cellular events mediated by this protein toward these external stimuli would be regulated by various mechanisms.

A gene network simulator to assess reverse engineering algorithms.

PubMed

Di Camillo, Barbara; Toffolo, Gianna; Cobelli, Claudio

2009-03-01

In the context of reverse engineering of biological networks, simulators are helpful to test and compare the accuracy of different reverse-engineering approaches in a variety of experimental conditions. A novel gene-network simulator is presented that resembles some of the main features of transcriptional regulatory networks related to topology, interaction among regulators of transcription, and expression dynamics. The simulator generates network topology according to the current knowledge of biological network organization, including scale-free distribution of the connectivity and clustering coefficient independent of the number of nodes in the network. It uses fuzzy logic to represent interactions among the regulators of each gene, integrated with differential equations to generate continuous data, comparable to real data for variety and dynamic complexity. Finally, the simulator accounts for saturation in the response to regulation and transcription activation thresholds and shows robustness to perturbations. It therefore provides a reliable and versatile test bed for reverse engineering algorithms applied to microarray data. Since the simulator describes regulatory interactions and expression dynamics as two distinct, although interconnected aspects of regulation, it can also be used to test reverse engineering approaches that use both microarray and protein-protein interaction data in the process of learning. A first software release is available at http://www.dei.unipd.it/~dicamill/software/netsim as an R programming language package.

The EAL domain protein YciR acts as a trigger enzyme in a c-di-GMP signalling cascade in E. coli biofilm control

PubMed Central

Lindenberg, Sandra; Klauck, Gisela; Pesavento, Christina; Klauck, Eberhard; Hengge, Regine

2013-01-01

C-di-GMP—which is produced by diguanylate cyclases (DGC) and degraded by specific phosphodiesterases (PDEs)—is a ubiquitous second messenger in bacterial biofilm formation. In Escherichia coli, several DGCs (YegE, YdaM) and PDEs (YhjH, YciR) and the MerR-like transcription factor MlrA regulate the transcription of csgD, which encodes a biofilm regulator essential for producing amyloid curli fibres of the biofilm matrix. Here, we demonstrate that this system operates as a signalling cascade, in which c-di-GMP controlled by the DGC/PDE pair YegE/YhjH (module I) regulates the activity of the YdaM/YciR pair (module II). Via multiple direct interactions, the two module II proteins form a signalling complex with MlrA. YciR acts as a connector between modules I and II and functions as a trigger enzyme: its direct inhibition of the DGC YdaM is relieved when it binds and degrades c-di-GMP generated by module I. As a consequence, YdaM then generates c-di-GMP and—by direct and specific interaction—activates MlrA to stimulate csgD transcription. Trigger enzymes may represent a general principle in local c-di-GMP signalling. PMID:23708798

Comparative analysis reveals genomic features of stress-induced transcriptional readthrough

PubMed Central

Vilborg, Anna; Sabath, Niv; Wiesel, Yuval; Nathans, Jenny; Levy-Adam, Flonia; Yario, Therese A.; Steitz, Joan A.; Shalgi, Reut

2017-01-01

Transcription is a highly regulated process, and stress-induced changes in gene transcription have been shown to play a major role in stress responses and adaptation. Genome-wide studies reveal prevalent transcription beyond known protein-coding gene loci, generating a variety of RNA classes, most of unknown function. One such class, termed downstream of gene-containing transcripts (DoGs), was reported to result from transcriptional readthrough upon osmotic stress in human cells. However, how widespread the readthrough phenomenon is, and what its causes and consequences are, remain elusive. Here we present a genome-wide mapping of transcriptional readthrough, using nuclear RNA-Seq, comparing heat shock, osmotic stress, and oxidative stress in NIH 3T3 mouse fibroblast cells. We observe massive induction of transcriptional readthrough, both in levels and length, under all stress conditions, with significant, yet not complete, overlap of readthrough-induced loci between different conditions. Importantly, our analyses suggest that stress-induced transcriptional readthrough is not a random failure process, but is rather differentially induced across different conditions. We explore potential regulators and find a role for HSF1 in the induction of a subset of heat shock-induced readthrough transcripts. Analysis of public datasets detected increases in polymerase II occupancy in DoG regions after heat shock, supporting our findings. Interestingly, DoGs tend to be produced in the vicinity of neighboring genes, leading to a marked increase in their antisense-generating potential. Finally, we examine genomic features of readthrough transcription and observe a unique chromatin signature typical of DoG-producing regions, suggesting that readthrough transcription is associated with the maintenance of an open chromatin state. PMID:28928151

Novel noncoding RNA from human Y distal heterochromatic block (Yq12) generates testis-specific chimeric CDC2L2

PubMed Central

Jehan, Zeenath; Vallinayagam, Sambandam; Tiwari, Shrish; Pradhan, Suman; Singh, Lalji; Suresh, Amritha; Reddy, Hemakumar M.; Ahuja, Y.R.; Jesudasan, Rachel A.

2007-01-01

The human Y chromosome, because it is enriched in repetitive DNA, has been very intractable to genetic and molecular analyses. There is no previous evidence for developmental stage- and testis-specific transcription from the male-specific region of the Y (MSY). Here, we present evidence for the first time for a developmental stage- and testis-specific transcription from MSY distal heterochromatic block. We isolated two novel RNAs, which localize to Yq12 in multiple copies, show testis-specific expression, and lack active X-homologs. Experimental evidence shows that one of the above Yq12 noncoding RNAs (ncRNAs) trans-splices with CDC2L2 mRNA from chromosome 1p36.3 locus to generate a testis-specific chimeric β sv13 isoform. This 67-nt 5′UTR provided by the Yq12 transcript contains within it a Y box protein-binding CCAAT motif, indicating translational regulation of the β sv13 isoform in testis. This is also the first report of trans-splicing between a Y chromosomal and an autosomal transcript. PMID:17095710

Activation of HIV Transcription by the Viral Tat Protein Requires a Demethylation Step Mediated by Lysine-specific Demethylase 1 (LSD1/KDM1)

PubMed Central

Sakane, Naoki; Kwon, Hye-Sook; Pagans, Sara; Kaehlcke, Katrin; Mizusawa, Yasuhiro; Kamada, Masafumi; Lassen, Kara G.; Chan, Jonathan; Greene, Warner C.; Schnoelzer, Martina; Ott, Melanie

2011-01-01

The essential transactivator function of the HIV Tat protein is regulated by multiple posttranslational modifications. Although individual modifications are well characterized, their crosstalk and dynamics of occurrence during the HIV transcription cycle remain unclear. We examine interactions between two critical modifications within the RNA-binding domain of Tat: monomethylation of lysine 51 (K51) mediated by Set7/9/KMT7, an early event in the Tat transactivation cycle that strengthens the interaction of Tat with TAR RNA, and acetylation of lysine 50 (K50) mediated by p300/KAT3B, a later process that dissociates the complex formed by Tat, TAR RNA and the cyclin T1 subunit of the positive transcription elongation factor b (P-TEFb). We find K51 monomethylation inhibited in synthetic Tat peptides carrying an acetyl group at K50 while acetylation can occur in methylated peptides, albeit at a reduced rate. To examine whether Tat is subject to sequential monomethylation and acetylation in cells, we performed mass spectrometry on immunoprecipitated Tat proteins and generated new modification-specific Tat antibodies against monomethylated/acetylated Tat. No bimodified Tat protein was detected in cells pointing to a demethylation step during the Tat transactivation cycle. We identify lysine-specific demethylase 1 (LSD1/KDM1) as a Tat K51-specific demethylase, which is required for the activation of HIV transcription in latently infected T cells. LSD1/KDM1 and its cofactor CoREST associates with the HIV promoter in vivo and activate Tat transcriptional activity in a K51-dependent manner. In addition, small hairpin RNAs directed against LSD1/KDM1 or inhibition of its activity with the monoamine oxidase inhibitor phenelzine suppresses the activation of HIV transcription in latently infected T cells. Our data support the model that a LSD1/KDM1/CoREST complex, normally known as a transcriptional suppressor, acts as a novel activator of HIV transcription through demethylation of K51 in Tat. Small molecule inhibitors of LSD1/KDM1 show therapeutic promise by enforcing HIV latency in infected T cells. PMID:21876670

Dynamics simulations for engineering macromolecular interactions

NASA Astrophysics Data System (ADS)

Robinson-Mosher, Avi; Shinar, Tamar; Silver, Pamela A.; Way, Jeffrey

2013-06-01

The predictable engineering of well-behaved transcriptional circuits is a central goal of synthetic biology. The artificial attachment of promoters to transcription factor genes usually results in noisy or chaotic behaviors, and such systems are unlikely to be useful in practical applications. Natural transcriptional regulation relies extensively on protein-protein interactions to insure tightly controlled behavior, but such tight control has been elusive in engineered systems. To help engineer protein-protein interactions, we have developed a molecular dynamics simulation framework that simplifies features of proteins moving by constrained Brownian motion, with the goal of performing long simulations. The behavior of a simulated protein system is determined by summation of forces that include a Brownian force, a drag force, excluded volume constraints, relative position constraints, and binding constraints that relate to experimentally determined on-rates and off-rates for chosen protein elements in a system. Proteins are abstracted as spheres. Binding surfaces are defined radially within a protein. Peptide linkers are abstracted as small protein-like spheres with rigid connections. To address whether our framework could generate useful predictions, we simulated the behavior of an engineered fusion protein consisting of two 20 000 Da proteins attached by flexible glycine/serine-type linkers. The two protein elements remained closely associated, as if constrained by a random walk in three dimensions of the peptide linker, as opposed to showing a distribution of distances expected if movement were dominated by Brownian motion of the protein domains only. We also simulated the behavior of fluorescent proteins tethered by a linker of varying length, compared the predicted Förster resonance energy transfer with previous experimental observations, and obtained a good correspondence. Finally, we simulated the binding behavior of a fusion of two ligands that could simultaneously bind to distinct cell-surface receptors, and explored the landscape of linker lengths and stiffnesses that could enhance receptor binding of one ligand when the other ligand has already bound to its receptor, thus, addressing potential mechanisms for improving targeted signal transduction proteins. These specific results have implications for the design of targeted fusion proteins and artificial transcription factors involving fusion of natural domains. More broadly, the simulation framework described here could be extended to include more detailed system features such as non-spherical protein shapes and electrostatics, without requiring detailed, computationally expensive specifications. This framework should be useful in predicting behavior of engineered protein systems including binding and dissociation reactions.

Dynamics simulations for engineering macromolecular interactions.

PubMed

Robinson-Mosher, Avi; Shinar, Tamar; Silver, Pamela A; Way, Jeffrey

2013-06-01

The predictable engineering of well-behaved transcriptional circuits is a central goal of synthetic biology. The artificial attachment of promoters to transcription factor genes usually results in noisy or chaotic behaviors, and such systems are unlikely to be useful in practical applications. Natural transcriptional regulation relies extensively on protein-protein interactions to insure tightly controlled behavior, but such tight control has been elusive in engineered systems. To help engineer protein-protein interactions, we have developed a molecular dynamics simulation framework that simplifies features of proteins moving by constrained Brownian motion, with the goal of performing long simulations. The behavior of a simulated protein system is determined by summation of forces that include a Brownian force, a drag force, excluded volume constraints, relative position constraints, and binding constraints that relate to experimentally determined on-rates and off-rates for chosen protein elements in a system. Proteins are abstracted as spheres. Binding surfaces are defined radially within a protein. Peptide linkers are abstracted as small protein-like spheres with rigid connections. To address whether our framework could generate useful predictions, we simulated the behavior of an engineered fusion protein consisting of two 20,000 Da proteins attached by flexible glycine/serine-type linkers. The two protein elements remained closely associated, as if constrained by a random walk in three dimensions of the peptide linker, as opposed to showing a distribution of distances expected if movement were dominated by Brownian motion of the protein domains only. We also simulated the behavior of fluorescent proteins tethered by a linker of varying length, compared the predicted Förster resonance energy transfer with previous experimental observations, and obtained a good correspondence. Finally, we simulated the binding behavior of a fusion of two ligands that could simultaneously bind to distinct cell-surface receptors, and explored the landscape of linker lengths and stiffnesses that could enhance receptor binding of one ligand when the other ligand has already bound to its receptor, thus, addressing potential mechanisms for improving targeted signal transduction proteins. These specific results have implications for the design of targeted fusion proteins and artificial transcription factors involving fusion of natural domains. More broadly, the simulation framework described here could be extended to include more detailed system features such as non-spherical protein shapes and electrostatics, without requiring detailed, computationally expensive specifications. This framework should be useful in predicting behavior of engineered protein systems including binding and dissociation reactions.

Bioinformatics Knowledge Map for Analysis of Beta-Catenin Function in Cancer

PubMed Central

Arighi, Cecilia N.; Wu, Cathy H.

2015-01-01

Given the wealth of bioinformatics resources and the growing complexity of biological information, it is valuable to integrate data from disparate sources to gain insight into the role of genes/proteins in health and disease. We have developed a bioinformatics framework that combines literature mining with information from biomedical ontologies and curated databases to create knowledge “maps” of genes/proteins of interest. We applied this approach to the study of beta-catenin, a cell adhesion molecule and transcriptional regulator implicated in cancer. The knowledge map includes post-translational modifications (PTMs), protein-protein interactions, disease-associated mutations, and transcription factors co-activated by beta-catenin and their targets and captures the major processes in which beta-catenin is known to participate. Using the map, we generated testable hypotheses about beta-catenin biology in normal and cancer cells. By focusing on proteins participating in multiple relation types, we identified proteins that may participate in feedback loops regulating beta-catenin transcriptional activity. By combining multiple network relations with PTM proteoform-specific functional information, we proposed a mechanism to explain the observation that the cyclin dependent kinase CDK5 positively regulates beta-catenin co-activator activity. Finally, by overlaying cancer-associated mutation data with sequence features, we observed mutation patterns in several beta-catenin PTM sites and PTM enzyme binding sites that varied by tissue type, suggesting multiple mechanisms by which beta-catenin mutations can contribute to cancer. The approach described, which captures rich information for molecular species from genes and proteins to PTM proteoforms, is extensible to other proteins and their involvement in disease. PMID:26509276

Role of transcription factor Sp1 and RNA binding protein HuR in the down-regulation of Dr+ Escherichia coli receptor protein Decay Accelerating Factor (DAF or CD55) by Nitric oxide

PubMed Central

Banadakoppa, Manu; Liebenthal, Daniel; Nowak, David E; Urvil, Petri; Yallampalli, Uma; Wilson, Gerald M; Kishor, Aparna; Yallampalli, Chandra

2012-01-01

We previously reported that nitric oxide (NO) reduces the rate of bacteremia and maternal mortality in pregnant rats with uterine infection by Escherichia coli expressing the Dr Fimbria (Dr+). The epithelial invasion of Dr+ E. coli is dependent on the expression level of its cellular receptor decay accelerating factor (DAF). NO reduces the rate of bacteremia by down-regulating the expression of DAF. In this study, we elucidated the role of transcription factor Sp1 and RNA binding protein HuR in the down-regulation of human DAF by NO. We generated a series of deletion mutant constructs of DAF gene 5′-untranslated region and mapped NO-response region upstream to the core promoter region of the DAF gene. One of the several Sp1 binding sites in the DAF 5′-untranslated region was located within the NO-response region. The binding of Sp1 to this site was inhibited by NO. Furthermore, NO also promoted the degradation of DAF mRNA. The 3′-untranslated region of DAF harbors an AU-rich element and this element destabilized the mRNA transcript. The NO promoted the rapid degradation of DAF mRNA by inhibiting the binding of mRNA stabilizing protein HuR to this AU-rich region. The inhibition of binding of HuR to AU-rich region was due to the S-nitrosylation of one or more cysteine residues by NO. Thus, these data reveal the molecular mediators of transcriptional and post-transcriptional regulation of DAF by NO with implications in pathophysiology related to DAF. PMID:23176121

Role of transcription factor Sp1 and RNA binding protein HuR in the downregulation of Dr+ Escherichia coli receptor protein decay accelerating factor (DAF or CD55) by nitric oxide.

PubMed

Banadakoppa, Manu; Liebenthal, Daniel; Nowak, David E; Urvil, Petri; Yallampalli, Uma; Wilson, Gerald M; Kishor, Aparna; Yallampalli, Chandra

2013-02-01

We previously reported that nitric oxide (NO) reduces the rate of bacteremia and maternal mortality in pregnant rats with uterine infection by Escherichia coli expressing the Dr Fimbria (Dr(+) ). The epithelial invasion of Dr(+) E. coli is dependent on the expression level of its cellular receptor decay accelerating factor (DAF). NO reduces the rate of bacteremia by downregulating the expression of DAF. In this study, we elucidated the role of transcription factor Sp1 and RNA binding protein HuR in the downregulation of human DAF by NO. We generated a series of deletion mutant constructs of DAF gene 5'-untranslated region and mapped the NO-response region upstream to the core promoter region of the DAF gene. One of the several Sp1 binding sites in the DAF 5'-untranslated region was located within the NO-response region. The binding of Sp1 to this site was inhibited by NO. Furthermore, NO also promoted the degradation of DAF mRNA. The 3'-untranslated region of DAF harbors an AU-rich element and this element destabilized the mRNA transcript. NO promoted the rapid degradation of DAF mRNA by inhibiting the binding of mRNA stabilizing protein HuR to this AU-rich region. The inhibition of binding of HuR to the AU-rich region was due to the S-nitrosylation of one or more cysteine residues by NO. Thus, these data reveal the molecular mediators of transcriptional and post-transcriptional regulation of DAF by NO with implications in pathophysiology related to DAF. © 2012 The Authors Journal compilation © 2012 FEBS.

Parvoviral Left-End Hairpin Ears Are Essential during Infection for Establishing a Functional Intranuclear Transcription Template and for Efficient Progeny Genome Encapsidation

PubMed Central

Li, Lei; Cotmore, Susan F.

2013-01-01

The 121-nucleotide left-end telomere of Minute Virus of Mice (MVM) can be folded into a Y-shaped hairpin with short axial ears that are highly conserved within genus Parvovirus. To explore their potential role(s) during infection, we constructed infectious plasmid clones that lacked one or other ear. Although these were nonviable when transfected into A9 cells, excision of the viral genome and DNA amplification appeared normal, and viral transcripts and proteins were expressed, but progeny virion production was minimal, supporting the idea of a potential role for the ears in genome packaging. To circumvent the absence of progeny that confounded further analysis of these mutants, plasmids were transfected into 293T cells both with and without an adenovirus helper construct, generating single bursts of progeny. These virions bound to A9 cells and were internalized but failed to initiate viral transcription, protein expression, or DNA replication. No defects in mutant virion stability or function could be detected in vitro. Significantly, mutant capsid gene expression and DNA replication could be rescued by coinfection with wild-type virions carrying a replication-competent, capsid-gene-replacement vector. To pinpoint where such complementation occurred, prior transfection of plasmids expressing only MVM nonstructural proteins was explored. NS1 alone, but not NS2, rescued transcription and protein expression from both P4 and P38 promoters, whereas NS1 molecules deleted for their C-terminal transactivation domain did not. These results suggest that the mutant virions reach the nucleus, uncoat, and are converted to duplex DNA but require an intact left-end hairpin structure to form the initiating transcription complex. PMID:23903839

Activity-Dependent Human Brain Coding/Noncoding Gene Regulatory Networks

PubMed Central

Lipovich, Leonard; Dachet, Fabien; Cai, Juan; Bagla, Shruti; Balan, Karina; Jia, Hui; Loeb, Jeffrey A.

2012-01-01

While most gene transcription yields RNA transcripts that code for proteins, a sizable proportion of the genome generates RNA transcripts that do not code for proteins, but may have important regulatory functions. The brain-derived neurotrophic factor (BDNF) gene, a key regulator of neuronal activity, is overlapped by a primate-specific, antisense long noncoding RNA (lncRNA) called BDNFOS. We demonstrate reciprocal patterns of BDNF and BDNFOS transcription in highly active regions of human neocortex removed as a treatment for intractable seizures. A genome-wide analysis of activity-dependent coding and noncoding human transcription using a custom lncRNA microarray identified 1288 differentially expressed lncRNAs, of which 26 had expression profiles that matched activity-dependent coding genes and an additional 8 were adjacent to or overlapping with differentially expressed protein-coding genes. The functions of most of these protein-coding partner genes, such as ARC, include long-term potentiation, synaptic activity, and memory. The nuclear lncRNAs NEAT1, MALAT1, and RPPH1, composing an RNAse P-dependent lncRNA-maturation pathway, were also upregulated. As a means to replicate human neuronal activity, repeated depolarization of SY5Y cells resulted in sustained CREB activation and produced an inverse pattern of BDNF-BDNFOS co-expression that was not achieved with a single depolarization. RNAi-mediated knockdown of BDNFOS in human SY5Y cells increased BDNF expression, suggesting that BDNFOS directly downregulates BDNF. Temporal expression patterns of other lncRNA-messenger RNA pairs validated the effect of chronic neuronal activity on the transcriptome and implied various lncRNA regulatory mechanisms. lncRNAs, some of which are unique to primates, thus appear to have potentially important regulatory roles in activity-dependent human brain plasticity. PMID:22960213

5′ and 3′ Untranslated Regions Strongly Enhance Performance of Geminiviral Replicons in Nicotiana benthamiana Leaves

PubMed Central

Diamos, Andrew G.; Rosenthal, Sun H.; Mason, Hugh S.

2016-01-01

We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5′ untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5′ UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5′ UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of Agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at >20% total soluble protein, corresponding to 1.8 mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody rituximab at 1 mg/g leaf fresh weight. PMID:26941764

N-3 polyunsaturated fatty acid regulation of hepatic gene transcription

PubMed Central

Jump, Donald B.

2009-01-01

Purpose of review The liver plays a central role in whole body lipid metabolism and adapts rapidly to changes in dietary fat composition. This adaption involves changes in the expression of genes involved in glycolysis, de-novo lipogenesis, fatty acid elongation, desaturation and oxidation. This review brings together metabolic and molecular studies that help explain n-3 (omega-3) polyunsaturated fatty acid regulation of hepatic gene transcription. Recent findings Dietary n-3 polyunsaturated fatty acid regulates hepatic gene expression by targeting three major transcriptional regulatory networks: peroxisome proliferator-activated receptor α, sterol regulatory element binding protein-1 and the carbohydrate regulatory element binding protein/Max-like factor X heterodimer. 22 : 6,n-3, the most prominent n-3 polyunsaturated fatty acid in tissues, is a weak activator of peroxisome proliferator-activated receptor α. Hepatic metabolism of 22 : 6,n-3, however, generates 20 : 5,n-3, a strong peroxisome proliferator-activated receptor α activator. In contrast to peroxisome proliferator-activated receptor α, 22 : 6,n-3 is the most potent fatty acid regulator of hepatic sterol regulatory element binding protein-1. 22 : 6,n-3 suppresses sterol regulatory element binding protein-1 gene expression while enhancing degradation of nuclear sterol regulatory element binding protein-1 through 26S proteasome and Erk1/2-dependent mechanisms. Both n-3 and n-6 polyunsaturated fatty acid suppress carbohydrate regulatory element binding protein and Max-like factor X nuclear abundance and interfere with glucose-regulated hepatic metabolism. Summary These studies have revealed unique mechanisms by which specific polyunsaturated fatty acids control peroxisome proliferator activated receptor α, sterol regulatory element binding protein-1 and carbohydrate regulatory element binding protein/Max-like factor X function. As such, specific metabolic and signal transduction pathways contribute significantly to the fatty acid regulation of these transcription factors and their corresponding regulatory networks. PMID:18460914

The laminA/NF-Y protein complex reveals an unknown transcriptional mechanism on cell proliferation

PubMed Central

Mancone, Carmine; Regazzo, Giulia; Spagnuolo, Manuela; Alonzi, Tonino; Carlomosti, Fabrizio; Lucia, Maria Dell’Anna; Dell, Giulia 'Omo; Picardo, Mauro; Ciana, Paolo; Capogrossi, Maurizio C; Tripodi, Marco; Magenta, Alessandra; Giulia, Maria Rizzo; Gurtner, Aymone; Piaggio, Giulia

2017-01-01

Lamin A is a component of the nuclear matrix that also controls proliferation by largely unknown mechanisms. NF-Y is a ubiquitous protein involved in cell proliferation composed of three subunits (-YA -YB -YC) all required for the DNA binding and transactivation activity. To get clues on new NF-Y partner(s) we performed a mass spectrometry screening of proteins that co-precipitate with the regulatory subunit of the complex, NF-YA. By this screening we identified lamin A as a novel putative NF-Y interactor. Co-immunoprecipitation experiments and confocal analysis confirmed the interaction between the two endogenous proteins. Interestingly, this association occurs on euchromatin regions, too. ChIP experiments demonstrate lamin A enrichment in several promoter regions of cell cycle related genes in a NF-Y dependent manner. Gain and loss of function experiments reveal that lamin A counteracts NF-Y transcriptional activity. Taking advantage of a recently generated transgenic reporter mouse, called MITO-Luc, in which an NF-Y–dependent promoter controls luciferase expression, we demonstrate that lamin A counteracts NF-Y transcriptional activity not only in culture cells but also in living animals. Altogether, our data demonstrate the occurrence of lamin A/NF-Y interaction and suggest a possible role of this protein complex in regulation of NF-Y function in cell proliferation. PMID:27793050

In Vitro Transcripts of Wild-Type and Fluorescent Protein-Tagged Triticum mosaic virus (Family Potyviridae) are Biologically Active in Wheat.

PubMed

Tatineni, Satyanarayana; McMechan, Anthony J; Bartels, Melissa; Hein, Gary L; Graybosch, Robert A

2015-11-01

Triticum mosaic virus (TriMV) (genus Poacevirus, family Potyviridae) is a recently described eriophyid mite-transmitted wheat virus. In vitro RNA transcripts generated from full-length cDNA clones of TriMV proved infectious on wheat. Wheat seedlings inoculated with in vitro transcripts elicited mosaic and mottling symptoms similar to the wild-type virus, and the progeny virus was efficiently transmitted by wheat curl mites, indicating that the cloned virus retained pathogenicity, movement, and wheat curl mite transmission characteristics. A series of TriMV-based expression vectors was constructed by engineering a green fluorescent protein (GFP) or red fluorescent protein (RFP) open reading frame with homologous NIa-Pro cleavage peptides between the P1 and HC-Pro cistrons. We found that GFP-tagged TriMV with seven or nine amino acid cleavage peptides efficiently processed GFP from HC-Pro. TriMV-GFP vectors were stable in wheat for more than 120 days and for six serial passages at 14-day intervals by mechanical inoculation and were transmitted by wheat curl mites similarly to the wild-type virus. Fluorescent protein-tagged TriMV was observed in wheat leaves, stems, and crowns. The availability of fluorescent protein-tagged TriMV will facilitate the examination of virus movement and distribution in cereal hosts and the mechanisms of cross protection and synergistic interactions between TriMV and Wheat streak mosaic virus.

The hedgehog regulated oncogenes Gli1 and Gli2 block myoblast differentiation by inhibiting MyoD-mediated transcriptional activation

PubMed Central

Gerber, AN; Wilson, CW; Li, Y-J; Chuang, P-T

2012-01-01

The mechanism by which activation of the Hedgehog (Hh) pathway modulates differentiation and promotes oncogenesis in specific tissues is poorly understood. We therefore, analysed rhabdomyosarcomas from mice that were haploinsufficient for the Hh-binding protein, Hip1, or for the Hh receptor, Patched 1 (Ptch1). Transfection of the Hh-regulated transcription factor Gli1, which is expressed in a subset of mouse and human rhabdomyosarcomas, suppressed differentiation of myogenic rhabdomyosarcoma lines generated from Hip1+/− and Ptch1+/− mice. The closely related factor, Gli2, had similar effects. Gli1 and Gli2 inhibited myogenesis by repressing the capacity of MyoD to activate transcription. Deletion analysis of Gli1 indicated that multiple domains of Gli1 are required for efficient inhibition of MyoD. Gli1 reduced the ability of MyoD to heterodimerize with E12 and bind DNA, providing one mechanism whereby the Gli proteins modulate the activity of MyoD. This novel activity of Gli proteins provides new insights into how Hh signaling modulates terminal differentiation through inhibition of tissue-specific factors such as MyoD. This mechanism may contribute to the broad role of Hh signaling and the Gli proteins in differentiation decisions and cancer formation. PMID:16964293

A model for genesis of transcription systems.

PubMed

Burton, Zachary F; Opron, Kristopher; Wei, Guowei; Geiger, James H

2016-01-01

Repeating sequences generated from RNA gene fusions/ligations dominate ancient life, indicating central importance of building structural complexity in evolving biological systems. A simple and coherent story of life on earth is told from tracking repeating motifs that generate α/β proteins, 2-double-Ψ-β-barrel (DPBB) type RNA polymerases (RNAPs), general transcription factors (GTFs), and promoters. A general rule that emerges is that biological complexity that arises through generation of repeats is often bounded by solubility and closure (i.e., to form a pseudo-dimer or a barrel). Because the first DNA genomes were replicated by DNA template-dependent RNA synthesis followed by RNA template-dependent DNA synthesis via reverse transcriptase, the first DNA replication origins were initially 2-DPBB type RNAP promoters. A simplifying model for evolution of promoters/replication origins via repetition of core promoter elements is proposed. The model can explain why Pribnow boxes in bacterial transcription (i.e., (-12)TATAATG(-6)) so closely resemble TATA boxes (i.e., (-31)TATAAAAG(-24)) in archaeal/eukaryotic transcription. The evolution of anchor DNA sequences in bacterial (i.e., (-35)TTGACA(-30)) and archaeal (BRE(up); BRE for TFB recognition element) promoters is potentially explained. The evolution of BRE(down) elements of archaeal promoters is potentially explained.

Demonstrating In-Cell Target Engagement Using a Pirin Protein Degradation Probe (CCT367766)

PubMed Central

2017-01-01

Demonstrating intracellular protein target engagement is an essential step in the development and progression of new chemical probes and potential small molecule therapeutics. However, this can be particularly challenging for poorly studied and noncatalytic proteins, as robust proximal biomarkers are rarely known. To confirm that our recently discovered chemical probe 1 (CCT251236) binds the putative transcription factor regulator pirin in living cells, we developed a heterobifunctional protein degradation probe. Focusing on linker design and physicochemical properties, we generated a highly active probe 16 (CCT367766) in only three iterations, validating our efficient strategy for degradation probe design against nonvalidated protein targets. PMID:29240418

Protein-protein interactions in the regulation of WRKY transcription factors.

PubMed

Chi, Yingjun; Yang, Yan; Zhou, Yuan; Zhou, Jie; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

2013-03-01

It has been almost 20 years since the first report of a WRKY transcription factor, SPF1, from sweet potato. Great progress has been made since then in establishing the diverse biological roles of WRKY transcription factors in plant growth, development, and responses to biotic and abiotic stress. Despite the functional diversity, almost all analyzed WRKY proteins recognize the TTGACC/T W-box sequences and, therefore, mechanisms other than mere recognition of the core W-box promoter elements are necessary to achieve the regulatory specificity of WRKY transcription factors. Research over the past several years has revealed that WRKY transcription factors physically interact with a wide range of proteins with roles in signaling, transcription, and chromatin remodeling. Studies of WRKY-interacting proteins have provided important insights into the regulation and mode of action of members of the important family of transcription factors. It has also emerged that the slightly varied WRKY domains and other protein motifs conserved within each of the seven WRKY subfamilies participate in protein-protein interactions and mediate complex functional interactions between WRKY proteins and between WRKY and other regulatory proteins in the modulation of important biological processes. In this review, we summarize studies of protein-protein interactions for WRKY transcription factors and discuss how the interacting partners contribute, at different levels, to the establishment of the complex regulatory and functional network of WRKY transcription factors.

Cryptic chromosome 9q34 deletion generates TAF-Ialpha/CAN and TAF-Ibeta/CAN fusion transcripts in acute myeloid leukemia.

PubMed

Rosati, Roberto; La Starza, Roberta; Barba, Gianluca; Gorello, Paolo; Pierini, Valentina; Matteucci, Caterina; Roti, Giovanni; Crescenzi, Barbara; Aloisi, Teresa; Aversa, Franco; Martelli, Massimo Fabrizio; Mecucci, Cristina

2007-02-01

In hematologic malignancies chromosome aberrations generating fusion genes include cryptic deletions. In a patient with acute myeloid leukemia and normal karyo-type we discovered a new cryptic 9q34 deletion and here report the cytogenetic and molecular findings. The 9q34 deletion extends 2.5 megabases and juxtaposes the 5' TAF-I to the 3' CAN producing a TAF-I/CAN fusion gene. TAF-I/CAN transcribes into two fusion proteins bearing either TAF-Ialpha or TAF-Ibeta moieties. We set up molecular assays to monitor the chimeric TAF-Ialpha/CAN and TAF-Ibeta/CAN transcripts which, after hematopoietic stem cell transplantation from an HLA-identical sibling, were no longer detected.

Tuning of RNA editing by ADAR is required in Drosophila

PubMed Central

Keegan, Liam P; Brindle, James; Gallo, Angela; Leroy, Anne; Reenan, Robert A; O'Connell, Mary A

2005-01-01

RNA editing increases during development in more than 20 transcripts encoding proteins involved in rapid synaptic neurotransmission in Drosophila central nervous system and muscle. Adar (adenosine deaminase acting on RNA) mutant flies expressing only genome-encoded, unedited isoforms of ion-channel subunits are viable but show severe locomotion defects. The Adar transcript itself is edited in adult wild-type flies to generate an isoform with a serine to glycine substitution close to the ADAR active site. We show that editing restricts ADAR function since the edited isoform of ADAR is less active in vitro and in vivo than the genome-encoded, unedited isoform. Ubiquitous expression in embryos and larvae of an Adar transcript that is resistant to editing is lethal. Expression of this transcript in embryonic muscle is also lethal, with above-normal, adult-like levels of editing at sites in a transcript encoding a muscle voltage-gated calcium channel. PMID:15920480

INITIATION AND REGULATION OF PARAMYXOVIRUS TRANSCRIPTION AND REPLICATION

PubMed Central

Noton, Sarah L.; Fearns, Rachel

2015-01-01

The paramyxovirus family has a genome consisting of a single strand of negative sense RNA. This genome acts as a template for two distinct processes: transcription to generate subgenomic, capped and polyadenylated mRNAs, and genome replication. These viruses only encode one polymerase. Thus, an intriguing question is, how does the viral polymerase initiate and become committed to either transcription or replication? By answering this we can begin to understand how these two processes are regulated. In this review article, we present recent findings from studies on the paramyxovirus, respiratory syncytial virus, which show how its polymerase is able to initiate transcription and replication from a single promoter. We discuss how these findings apply to other paramyxoviruses. Then, we examine how trans-acting proteins and promoter secondary structure might serve to regulate transcription and replication during different phases of the paramyxovirus replication cycle. PMID:25683441

Initiation and regulation of paramyxovirus transcription and replication.

PubMed

Noton, Sarah L; Fearns, Rachel

2015-05-01

The paramyxovirus family has a genome consisting of a single strand of negative sense RNA. This genome acts as a template for two distinct processes: transcription to generate subgenomic, capped and polyadenylated mRNAs, and genome replication. These viruses only encode one polymerase. Thus, an intriguing question is, how does the viral polymerase initiate and become committed to either transcription or replication? By answering this we can begin to understand how these two processes are regulated. In this review article, we present recent findings from studies on the paramyxovirus, respiratory syncytial virus, which show how its polymerase is able to initiate transcription and replication from a single promoter. We discuss how these findings apply to other paramyxoviruses. Then, we examine how trans-acting proteins and promoter secondary structure might serve to regulate transcription and replication during different phases of the paramyxovirus replication cycle. Copyright © 2015 Elsevier Inc. All rights reserved.

Transcriptomic characterization of cold acclimation in larval zebrafish

PubMed Central

2013-01-01

Background Temperature is one of key environmental parameters that affect the whole life of fishes and an increasing number of studies have been directed towards understanding the mechanisms of cold acclimation in fish. However, the adaptation of larvae to cold stress and the cold-specific transcriptional alterations in fish larvae remain largely unknown. In this study, we characterized the development of cold-tolerance in zebrafish larvae and investigated the transcriptional profiles under cold stress using RNA-seq. Results Pre-exposure of 96 hpf zebrafish larvae to cold stress (16°C) for 24 h significantly increased their survival rates under severe cold stress (12°C). RNA-seq generated 272 million raw reads from six sequencing libraries and about 92% of the processed reads were mapped to the reference genome of zebrafish. Differential expression analysis identified 1,431 up- and 399 down-regulated genes. Gene ontology enrichment analysis of cold-induced genes revealed that RNA splicing, ribosome biogenesis and protein catabolic process were the most highly overrepresented biological processes. Spliceosome, proteasome, eukaryotic ribosome biogenesis and RNA transport were the most highly enriched pathways for genes up-regulated by cold stress. Moreover, alternative splicing of 197 genes and promoter switching of 64 genes were found to be regulated by cold stress. A shorter isoform of stk16 that lacks 67 amino acids at the N-terminus was specifically generated by skipping the second exon in cold-treated larvae. Alternative promoter usage was detected for per3 gene under cold stress, which leading to a highly up-regulated transcript encoding a truncated protein lacking the C-terminal domains. Conclusions These findings indicate that zebrafish larvae possess the ability to build cold-tolerance under mild low temperature and transcriptional and post-transcriptional regulations are extensively involved in this acclimation process. PMID:24024969

The alpha subunit of Go interacts with promyelocytic leukemia zinc finger protein and modulates its functions.

PubMed

Won, Jung Hee; Park, Jung Sik; Ju, Hyun Hee; Kim, Soyeon; Suh-Kim, Haeyoung; Ghil, Sung Ho

2008-05-01

Heterotrimeric GTP-binding proteins (G proteins) mediate signal transduction generated by neurotransmitters and hormones. Go, a member of the Go/Gi family, is the most abundant heterotrimeric G protein in the brain. Most mechanistic analyses on Go activation demonstrate that its action is mediated by the Gbetagamma dimer; downstream effectors for its alpha subunit (Goalpha) have not been clearly defined. Here, we employ the yeast two-hybrid system to screen for Goalpha-interacting partners in a cDNA library from human fetal brain. The transcription factor promyelocytic leukemia zinc finger protein (PLZF) specifically bound to Goalpha. Interactions between PLZF and Goalpha were confirmed using in vitro and in vivo affinity binding assays. Activated Goalpha interacted directly with PLZF, and enhanced its function as a transcriptional and cell growth suppressor. Notably, PLZF activity was additionally promoted by the Go/ialpha-coupled cannabinoid receptor (CB) in HL60 cells endogenously expressing CB and PLZF. These results collectively suggest that Goalpha modulates the function of PLZF via direct interactions. Our novel findings provide insights into the diverse cellular roles of Goalpha and its coupled receptor.

Spliced X-box Binding Protein 1 Couples the Unfolded Protein Response to Hexosamine Biosynthetic Pathway

PubMed Central

Wang, Zhao V.; Deng, Yingfeng; Gao, Ningguo; Pedrozo, Zully; Li, Dan L.; Morales, Cyndi R.; Criollo, Alfredo; Luo, Xiang; Tan, Wei; Jiang, Nan; Lehrman, Mark A.; Rothermel, Beverly A.; Lee, Ann-Hwee; Lavandero, Sergio; Mammen, Pradeep P.A.; Ferdous, Anwarul; Gillette, Thomas G.; Scherer, Philipp E.; Hill, Joseph A.

2014-01-01

SUMMARY The hexosamine biosynthetic pathway (HBP) generates UDP-GlcNAc (uridine diphosphate N-acetylglucosamine) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis, by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress. PMID:24630721

Spliced X-box binding protein 1 couples the unfolded protein response to hexosamine biosynthetic pathway.

PubMed

Wang, Zhao V; Deng, Yingfeng; Gao, Ningguo; Pedrozo, Zully; Li, Dan L; Morales, Cyndi R; Criollo, Alfredo; Luo, Xiang; Tan, Wei; Jiang, Nan; Lehrman, Mark A; Rothermel, Beverly A; Lee, Ann-Hwee; Lavandero, Sergio; Mammen, Pradeep P A; Ferdous, Anwarul; Gillette, Thomas G; Scherer, Philipp E; Hill, Joseph A

2014-03-13

The hexosamine biosynthetic pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress. Copyright © 2014 Elsevier Inc. All rights reserved.

Transcriptional and computational study of expansins differentially expressed in response to inclination in radiata pine.

PubMed

Mateluna, Patricio; Valenzuela-Riffo, Felipe; Morales-Quintana, Luis; Herrera, Raúl; Ramos, Patricio

2017-06-01

Plants have the ability to reorient their vertical growth when exposed to inclination. This response can be as quick as 2 h in inclined young pine (Pinus radiata D. Don) seedlings, with over accumulation of lignin observed after 9 days s. Several studies have identified expansins involved in cell expansion among other developmental processes in plants. Six putative expansin genes were identified in cDNA libraries isolated from inclined pine stems. A differential transcript abundance was observed by qPCR analysis over a time course of inclination. Five genes changed their transcript accumulation in both stem sides in a spatial and temporal manner compared with non-inclined stem. To compare these expansin genes, and to suggest a possible mechanism of action at molecular level, the structures of the predicted proteins were built by comparative modeling methodology. An open groove on the surface of the proteins composed of conserved zresidues was observed. Using a cellulose polymer as ligand the protein-ligand interaction was evaluated, with the results showing differences in the protein-ligand interaction mode. Differences in the binding energy interaction can be explained by changes in some residues that generate differences in electrostatic surface in the open groove region, supporting the participation of six members of multifamily proteins in this specific process. The data suggests participation of different expansin proteins in the dissembling and remodeling of the complex cell wall matrix during the reorientation response to inclination. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

The Pea light-independent photomorphogenesis1 Mutant Results from Partial Duplication of COP1 Generating an Internal Promoter and Producing Two Distinct Transcripts

PubMed Central

Sullivan, James A.; Gray, John C.

2000-01-01

The pea lip1 (light-independent photomorphogenesis1) mutant shows many of the characteristics of light-grown development when grown in continuous darkness. To investigate the identity of LIP1, cDNAs encoding the pea homolog of COP1, a repressor of photomorphogenesis identified in Arabidopsis, were isolated from wild-type and lip1 pea seedlings. lip1 seedlings contained a wild-type COP1 transcript as well as a larger COP1′ transcript that contained an internal in-frame duplication of 894 bp. The COP1′ transcript segregated with the lip1 phenotype in F2 seedlings and could be translated in vitro to produce a protein of ∼100 kD. The COP1 gene in lip1 peas contained a 7.5-kb duplication, consisting of exons 1 to 7 of the wild-type sequence, located 2.5 kb upstream of a region of genomic DNA identical to the wild-type COP1 DNA sequence. Transcription and splicing of the mutant COP1 gene was predicted to produce the COP1′ transcript, whereas transcription from an internal promoter in the 2.5-kb region of DNA located between the duplicated regions of COP1 would produce the wild-type COP1 transcript. The presence of small quantities of wild-type COP1 transcripts may reduce the severity of the phenotype produced by the mutated COP1′ protein. The genomic DNA sequences of the COP1 gene from wild-type and lip1 peas and the cDNA sequences of COP1 and COP1′ transcripts have been submitted to the EMBL database under the EMBL accession numbers AJ276591, AJ276592, AJ289773, and AJ289774, respectively. PMID:11041887

Gene expression in honey bee (Apis mellifera) larvae exposed to pesticides and Varroa mites (Varroa destructor).

PubMed

Gregorc, Aleš; Evans, Jay D; Scharf, Mike; Ellis, James D

2012-08-01

Honey bee (Apis mellifera) larvae reared in vitro were exposed to one of nine pesticides and/or were challenged with the parasitic mite, Varroa destructor. Total RNA was extracted from individual larvae and first strand cDNAs were generated. Gene-expression changes in larvae were measured using quantitative PCR (qPCR) targeting transcripts for pathogens and genes involved in physiological processes, bee health, immunity, and/or xenobiotic detoxification. Transcript levels for Peptidoglycan Recognition Protein (PGRPSC), a pathogen recognition gene, increased in larvae exposed to Varroa mites (P<0.001) and were not changed in pesticide treated larvae. As expected, Varroa-parasitized brood had higher transcripts of Deformed Wing Virus than did control larvae (P<0.001). Varroa parasitism, arguably coupled with virus infection, resulted in significantly higher transcript abundances for the antimicrobial peptides abaecin, hymenoptaecin, and defensin1. Transcript levels for Prophenoloxidase-activating enzyme (PPOact), an immune end product, were elevated in larvae treated with myclobutanil and chlorothalonil (both are fungicides) (P<0.001). Transcript levels for Hexameric storage protein (Hsp70) were significantly upregulated in imidacloprid, fluvalinate, coumaphos, myclobutanil, and amitraz treated larvae. Definitive impacts of pesticides and Varroa parasitism on honey bee larval gene expression were demonstrated. Interactions between larval treatments and gene expression for the targeted genes are discussed. Copyright © 2012 Elsevier Ltd. All rights reserved.

Activation of the Arabidopsis B class homeotic genes by APETALA1.

PubMed

Ng, M; Yanofsky, M F

2001-04-01

Proper development of petals and stamens in Arabidopsis flowers requires the activities of APETALA3 (AP3) and PISTILLATA (PI), whose transcripts can be detected in the petal and stamen primordia. Localized expression of AP3 and PI requires the activities of at least three genes: APETALA1 (AP1), LEAFY (LFY), and UNUSUAL FLORAL ORGANS (UFO). It has been proposed that UFO provides spatial cues and that LFY specifies competence for AP3 and PI expression in the developing flower. To understand the epistatic relationship among AP1, LFY, and UFO in regulating AP3 and PI expression, we generated two versions of AP1 that have strong transcriptional activation potential. Genetic and molecular analyses of transgenic plants expressing these activated AP1 proteins show that the endogenous AP1 protein acts largely as a transcriptional activator in vivo and that AP1 specifies petals by regulating the spatial domains of AP3 and PI expression through UFO.

Blimp-1 controls plasma cell function through the regulation of immunoglobulin secretion and the unfolded protein response.

PubMed

Tellier, Julie; Shi, Wei; Minnich, Martina; Liao, Yang; Crawford, Simon; Smyth, Gordon K; Kallies, Axel; Busslinger, Meinrad; Nutt, Stephen L

2016-03-01

Plasma cell differentiation requires silencing of B cell transcription, while it establishes antibody-secretory function and long-term survival. The transcription factors Blimp-1 and IRF4 are essential for the generation of plasma cells; however, their function in mature plasma cells has remained elusive. We found that while IRF4 was essential for the survival of plasma cells, Blimp-1 was dispensable for this. Blimp-1-deficient plasma cells retained their transcriptional identity but lost the ability to secrete antibody. Blimp-1 regulated many components of the unfolded protein response (UPR), including XBP-1 and ATF6. The overlap in the functions of Blimp-1 and XBP-1 was restricted to that response, with Blimp-1 uniquely regulating activity of the kinase mTOR and the size of plasma cells. Thus, Blimp-1 was required for the unique physiological ability of plasma cells that enables the secretion of protective antibody.

Dynamic integration of splicing within gene regulatory pathways

PubMed Central

Braunschweig, Ulrich; Gueroussov, Serge; Plocik, Alex; Graveley, Brenton R.; Blencowe, Benjamin J.

2013-01-01

Precursor mRNA splicing is one of the most highly regulated processes in metazoan species. In addition to generating vast repertoires of RNAs and proteins, splicing has a profound impact on other gene regulatory layers, including mRNA transcription, turnover, transport and translation. Conversely, factors regulating chromatin and transcription complexes impact the splicing process. This extensive cross-talk between gene regulatory layers takes advantage of dynamic spatial, physical and temporal organizational properties of the cell nucleus, and further emphasizes the importance of developing a multidimensional understanding of splicing control. PMID:23498935

Transcriptome deep-sequencing and clustering of expressed isoforms from Favia corals

PubMed Central

2013-01-01

Background Genomic and transcriptomic sequence data are essential tools for tackling ecological problems. Using an approach that combines next-generation sequencing, de novo transcriptome assembly, gene annotation and synthetic gene construction, we identify and cluster the protein families from Favia corals from the northern Red Sea. Results We obtained 80 million 75 bp paired-end cDNA reads from two Favia adult samples collected at 65 m (Fav1, Fav2) on the Illumina GA platform, and generated two de novo assemblies using ABySS and CAP3. After removing redundancy and filtering out low quality reads, our transcriptome datasets contained 58,268 (Fav1) and 62,469 (Fav2) contigs longer than 100 bp, with N50 values of 1,665 bp and 1,439 bp, respectively. Using the proteome of the sea anemone Nematostella vectensis as a reference, we were able to annotate almost 20% of each dataset using reciprocal homology searches. Homologous clustering of these annotated transcripts allowed us to divide them into 7,186 (Fav1) and 6,862 (Fav2) homologous transcript clusters (E-value ≤ 2e-30). Functional annotation categories were assigned to homologous clusters using the functional annotation of Nematostella vectensis. General annotation of the assembled transcripts was improved 1-3% using the Acropora digitifera proteome. In addition, we screened these transcript isoform clusters for fluorescent proteins (FPs) homologs and identified seven potential FP homologs in Fav1, and four in Fav2. These transcripts were validated as bona fide FP transcripts via robust fluorescence heterologous expression. Annotation of the assembled contigs revealed that 1.34% and 1.61% (in Fav1 and Fav2, respectively) of the total assembled contigs likely originated from the corals’ algal symbiont, Symbiodinium spp. Conclusions Here we present a study to identify the homologous transcript isoform clusters from the transcriptome of Favia corals using a far-related reference proteome. Furthermore, the symbiont-derived transcripts were isolated from the datasets and their contribution quantified. This is the first annotated transcriptome of the genus Favia, a major increase in genomics resources available in this important family of corals. PMID:23937070

MHC class I-associated peptides derive from selective regions of the human genome.

PubMed

Pearson, Hillary; Daouda, Tariq; Granados, Diana Paola; Durette, Chantal; Bonneil, Eric; Courcelles, Mathieu; Rodenbrock, Anja; Laverdure, Jean-Philippe; Côté, Caroline; Mader, Sylvie; Lemieux, Sébastien; Thibault, Pierre; Perreault, Claude

2016-12-01

MHC class I-associated peptides (MAPs) define the immune self for CD8+ T lymphocytes and are key targets of cancer immunosurveillance. Here, the goals of our work were to determine whether the entire set of protein-coding genes could generate MAPs and whether specific features influence the ability of discrete genes to generate MAPs. Using proteogenomics, we have identified 25,270 MAPs isolated from the B lymphocytes of 18 individuals who collectively expressed 27 high-frequency HLA-A,B allotypes. The entire MAP repertoire presented by these 27 allotypes covered only 10% of the exomic sequences expressed in B lymphocytes. Indeed, 41% of expressed protein-coding genes generated no MAPs, while 59% of genes generated up to 64 MAPs, often derived from adjacent regions and presented by different allotypes. We next identified several features of transcripts and proteins associated with efficient MAP production. From these data, we built a logistic regression model that predicts with good accuracy whether a gene generates MAPs. Our results show preferential selection of MAPs from a limited repertoire of proteins with distinctive features. The notion that the MHC class I immunopeptidome presents only a small fraction of the protein-coding genome for monitoring by the immune system has profound implications in autoimmunity and cancer immunology.

MHC class I–associated peptides derive from selective regions of the human genome

PubMed Central

Pearson, Hillary; Granados, Diana Paola; Durette, Chantal; Bonneil, Eric; Courcelles, Mathieu; Rodenbrock, Anja; Laverdure, Jean-Philippe; Côté, Caroline; Thibault, Pierre

2016-01-01

MHC class I–associated peptides (MAPs) define the immune self for CD8+ T lymphocytes and are key targets of cancer immunosurveillance. Here, the goals of our work were to determine whether the entire set of protein-coding genes could generate MAPs and whether specific features influence the ability of discrete genes to generate MAPs. Using proteogenomics, we have identified 25,270 MAPs isolated from the B lymphocytes of 18 individuals who collectively expressed 27 high-frequency HLA-A,B allotypes. The entire MAP repertoire presented by these 27 allotypes covered only 10% of the exomic sequences expressed in B lymphocytes. Indeed, 41% of expressed protein-coding genes generated no MAPs, while 59% of genes generated up to 64 MAPs, often derived from adjacent regions and presented by different allotypes. We next identified several features of transcripts and proteins associated with efficient MAP production. From these data, we built a logistic regression model that predicts with good accuracy whether a gene generates MAPs. Our results show preferential selection of MAPs from a limited repertoire of proteins with distinctive features. The notion that the MHC class I immunopeptidome presents only a small fraction of the protein-coding genome for monitoring by the immune system has profound implications in autoimmunity and cancer immunology. PMID:27841757

RNA Splicing: Regulation and Dysregulation in the Heart.

PubMed

van den Hoogenhof, Maarten M G; Pinto, Yigal M; Creemers, Esther E

2016-02-05

RNA splicing represents a post-transcriptional mechanism to generate multiple functional RNAs or proteins from a single transcript. The evolution of RNA splicing is a prime example of the Darwinian function follows form concept. A mutation that leads to a new mRNA (form) that encodes for a new functional protein (function) is likely to be retained, and this way, the genome has gradually evolved to encode for genes with multiple isoforms, thereby creating an enormously diverse transcriptome. Advances in technologies to characterize RNA populations have led to a better understanding of RNA processing in health and disease. In the heart, alternative splicing is increasingly being recognized as an important layer of post-transcriptional gene regulation. Moreover, the recent identification of several cardiac splice factors, such as RNA-binding motif protein 20 and SF3B1, not only provided important insight into the mechanisms underlying alternative splicing but also revealed how these splicing factors impact functional properties of the heart. Here, we review our current knowledge of alternative splicing in the heart, with a particular focus on the major and minor spliceosome, the factors controlling RNA splicing, and the role of alternative splicing in cardiac development and disease. © 2016 American Heart Association, Inc.

The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro.

PubMed

Kuciak, Monika; Gabus, Caroline; Ivanyi-Nagy, Roland; Semrad, Katharina; Storchak, Roman; Chaloin, Olivier; Muller, Sylviane; Mély, Yves; Darlix, Jean-Luc

2008-06-01

The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44-61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties.

The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro

PubMed Central

Kuciak, Monika; Gabus, Caroline; Ivanyi-Nagy, Roland; Semrad, Katharina; Storchak, Roman; Chaloin, Olivier; Muller, Sylviane; Mély, Yves; Darlix, Jean-Luc

2008-01-01

The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44–61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties. PMID:18442994

A Knockout Screen of ApiAP2 Genes Reveals Networks of Interacting Transcriptional Regulators Controlling the Plasmodium Life Cycle.

PubMed

Modrzynska, Katarzyna; Pfander, Claudia; Chappell, Lia; Yu, Lu; Suarez, Catherine; Dundas, Kirsten; Gomes, Ana Rita; Goulding, David; Rayner, Julian C; Choudhary, Jyoti; Billker, Oliver

2017-01-11

A family of apicomplexa-specific proteins containing AP2 DNA-binding domains (ApiAP2s) was identified in malaria parasites. This family includes sequence-specific transcription factors that are key regulators of development. However, functions for the majority of ApiAP2 genes remain unknown. Here, a systematic knockout screen in Plasmodium berghei identified ten ApiAP2 genes that were essential for mosquito transmission: four were critical for the formation of infectious ookinetes, and three were required for sporogony. We describe non-essential functions for AP2-O and AP2-SP proteins in blood stages, and identify AP2-G2 as a repressor active in both asexual and sexual stages. Comparative transcriptomics across mutants and developmental stages revealed clusters of co-regulated genes with shared cis promoter elements, whose expression can be controlled positively or negatively by different ApiAP2 factors. We propose that stage-specific interactions between ApiAP2 proteins on partly overlapping sets of target genes generate the complex transcriptional network that controls the Plasmodium life cycle. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Structure–function studies of STAR family Quaking proteins bound to their in vivo RNA target sites

PubMed Central

Teplova, Marianna; Hafner, Markus; Teplov, Dmitri; Essig, Katharina; Tuschl, Thomas; Patel, Dinshaw J.

2013-01-01

Mammalian Quaking (QKI) and its Caenorhabditis elegans homolog, GLD-1 (defective in germ line development), are evolutionarily conserved RNA-binding proteins, which post-transcriptionally regulate target genes essential for developmental processes and myelination. We present X-ray structures of the STAR (signal transduction and activation of RNA) domain, composed of Qua1, K homology (KH), and Qua2 motifs of QKI and GLD-1 bound to high-affinity in vivo RNA targets containing YUAAY RNA recognition elements (RREs). The KH and Qua2 motifs of the STAR domain synergize to specifically interact with bases and sugar-phosphate backbones of the bound RRE. Qua1-mediated homodimerization generates a scaffold that enables concurrent recognition of two RREs, thereby plausibly targeting tandem RREs present in many QKI-targeted transcripts. Structure-guided mutations reduced QKI RNA-binding affinity in vitro and in vivo, and expression of QKI mutants in human embryonic kidney cells (HEK293) significantly decreased the abundance of QKI target mRNAs. Overall, our studies define principles underlying RNA target selection by STAR homodimers and provide insights into the post-transcriptional regulatory function of mammalian QKI proteins. PMID:23630077

Kc167, a widely used Drosophila cell line, contains an active primary piRNA pathway.

PubMed

Vrettos, Nicholas; Maragkakis, Manolis; Alexiou, Panagiotis; Mourelatos, Zissimos

2017-01-01

PIWI family proteins bind to small RNAs known as PIWI-interacting RNAs (piRNAs) and play essential roles in the germline by silencing transposons and by promoting germ cell specification and function. Here we report that the widely used Kc167 cell line, derived from Drosophila melanogaster embryos, expresses piRNAs that are loaded to Aub and Piwi. Kc167 piRNAs are produced by a canonical, primary piRNA biogenesis pathway, from phased processing of precursor transcripts by the Zuc endonuclease, Armi helicase, and dGasz mitochondrial scaffold protein. Kc167 piRNAs derive from cytoplasmic transcripts, notably tRNAs and mRNAs, and their abundance correlates with that of parent transcripts. The expression of Aub is robust in Kc167, that of Piwi is modest, while Ago3 is undetectable, explaining the lack of transposon-related piRNA amplification by the Aub-Ago3, ping-pong mechanism. We propose that the default state of the primary piRNA biogenesis machinery is random transcript sampling to allow generation of piRNAs from any transcript, including newly acquired retrotransposons. This state is unmasked in Kc167, likely because they do not express piRNA cluster transcripts in sufficient amounts and do not amplify transposon piRNAs. We use Kc167 to characterize an inactive isoform of Aub protein. Since most Kc167 piRNAs are genic, they can be mapped uniquely to the genome, facilitating computational analyses. Furthermore, because Kc167 is a widely used and well-characterized cell line that is easily amenable to experimental manipulations, we expect that it will serve as an excellent system to study piRNA biogenesis and piRNA-related factors. © 2016 Vrettos et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

A stable transcription factor complex nucleated by oligomeric AML1–ETO controls leukaemogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Xiao-Jian; Wang, Zhanxin; Wang, Lan

2013-06-30

Transcription factors are frequently altered in leukaemia through chromosomal translocation, mutation or aberrant expression. AML1–ETO, a fusion protein generated by the t(8;21) translocation in acute myeloid leukaemia, is a transcription factor implicated in both gene repression and activation. AML1–ETO oligomerization, mediated by the NHR2 domain, is critical for leukaemogenesis, making it important to identify co-regulatory factors that ‘read’ the NHR2 oligomerization and contribute to leukaemogenesis. Here we show that, in human leukaemic cells, AML1–ETO resides in and functions through a stable AML1–ETO-containing transcription factor complex (AETFC) that contains several haematopoietic transcription (co)factors. These AETFC components stabilize the complex through multivalentmore » interactions, provide multiple DNA-binding domains for diverse target genes, co-localize genome wide, cooperatively regulate gene expression, and contribute to leukaemogenesis. Within the AETFC complex, AML1–ETO oligomerization is required for a specific interaction between the oligomerized NHR2 domain and a novel NHR2-binding (N2B) motif in E proteins. Crystallographic analysis of the NHR2–N2B complex reveals a unique interaction pattern in which an N2B peptide makes direct contact with side chains of two NHR2 domains as a dimer, providing a novel model of how dimeric/oligomeric transcription factors create a new protein-binding interface through dimerization/oligomerization. Intriguingly, disruption of this interaction by point mutations abrogates AML1–ETO-induced haematopoietic stem/progenitor cell self-renewal and leukaemogenesis. These results reveal new mechanisms of action of AML1–ETO, and provide a potential therapeutic target in t(8;21)-positive acute myeloid leukaemia.« less

Identification of functional elements and regulatory circuits by Drosophila modENCODE

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roy, Sushmita; Ernst, Jason; Kharchenko, Peter V.

2010-12-22

To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- andmore » tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation. Several years after the complete genetic sequencing of many species, it is still unclear how to translate genomic information into a functional map of cellular and developmental programs. The Encyclopedia of DNA Elements (ENCODE) (1) and model organism ENCODE (modENCODE) (2) projects use diverse genomic assays to comprehensively annotate the Homo sapiens (human), Drosophila melanogaster (fruit fly), and Caenorhabditis elegans (worm) genomes, through systematic generation and computational integration of functional genomic data sets. Previous genomic studies in flies have made seminal contributions to our understanding of basic biological mechanisms and genome functions, facilitated by genetic, experimental, computational, and manual annotation of the euchromatic and heterochromatic genome (3), small genome size, short life cycle, and a deep knowledge of development, gene function, and chromosome biology. The functions of {approx}40% of the protein and nonprotein-coding genes [FlyBase 5.12 (4)] have been determined from cDNA collections (5, 6), manual curation of gene models (7), gene mutations and comprehensive genome-wide RNA interference screens (8-10), and comparative genomic analyses (11, 12). The Drosophila modENCODE project has generated more than 700 data sets that profile transcripts, histone modifications and physical nucleosome properties, general and specific transcription factors (TFs), and replication programs in cell lines, isolated tissues, and whole organisms across several developmental stages (Fig. 1). Here, we computationally integrate these data sets and report (i) improved and additional genome annotations, including full-length proteincoding genes and peptides as short as 21 amino acids; (ii) noncoding transcripts, including 132 candidate structural RNAs and 1608 nonstructural transcripts; (iii) additional Argonaute (Ago)-associated small RNA genes and pathways, including new microRNAs (miRNAs) encoded within protein-coding exons and endogenous small interfering RNAs (siRNAs) from 3-inch untranslated regions; (iv) chromatin 'states' defined by combinatorial patterns of 18 chromatin marks that are associated with distinct functions and properties; (v) regions of high TF occupancy and replication activity with likely epigenetic regulation; (vi)mixed TF and miRNA regulatory networks with hierarchical structure and enriched feed-forward loops; (vii) coexpression- and co-regulation-based functional annotations for nearly 3000 genes; (viii) stage- and tissue-specific regulators; and (ix) predictive models of gene expression levels and regulator function.« less

Novel features of a PIWI-like protein homolog in the parasitic protozoan Leishmania.

PubMed

Padmanabhan, Prasad K; Dumas, Carole; Samant, Mukesh; Rochette, Annie; Simard, Martin J; Papadopoulou, Barbara

2012-01-01

In contrast to nearly all eukaryotes, the Old World Leishmania species L. infantum and L. major lack the bona fide RNAi machinery genes. Interestingly, both Leishmania genomes code for an atypical Argonaute-like protein that possesses a PIWI domain but lacks the PAZ domain found in Argonautes from RNAi proficient organisms. Using sub-cellular fractionation and confocal fluorescence microscopy, we show that unlike other eukaryotes, the PIWI-like protein is mainly localized in the single mitochondrion in Leishmania. To predict PIWI function, we generated a knockout mutant for the PIWI gene in both L. infantum (Lin) and L. major species by double-targeted gene replacement. Depletion of PIWI has no effect on the viability of insect promastigote forms but leads to an important growth defect of the mammalian amastigote lifestage in vitro and significantly delays disease pathology in mice, consistent with a higher expression of the PIWI transcript in amastigotes. Moreover, amastigotes lacking PIWI display a higher sensitivity to apoptosis inducing agents than wild type parasites, suggesting that PIWI may be a sensor for apoptotic stimuli. Furthermore, a whole-genome DNA microarray analysis revealed that loss of LinPIWI in Leishmania amastigotes affects mostly the expression of specific subsets of developmentally regulated genes. Several transcripts encoding surface and membrane-bound proteins were found downregulated in the LinPIWI((-/-)) mutant whereas all histone transcripts were upregulated in the null mutant, supporting the possibility that PIWI plays a direct or indirect role in the stability of these transcripts. Although our data suggest that PIWI is not involved in the biogenesis or the stability of small noncoding RNAs, additional studies are required to gain further insights into the role of this protein on RNA regulation and amastigote development in Leishmania.

Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors

PubMed Central

Hu, Jiabiao; Lei, Yong; Wong, Wing-Ki; Liu, Senquan; Lee, Kai-Chuen; He, Xiangjun; You, Wenxing; Zhou, Rui; Guo, Jun-Tao; Chen, Xiongfong; Peng, Xianlu; Sun, Hao; Huang, He; Zhao, Hui; Feng, Bo

2014-01-01

The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around −120 to −80 bp, while highly effective sgRNAs targeted from −147 to −89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells. PMID:24500196

Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors.

PubMed

Hu, Jiabiao; Lei, Yong; Wong, Wing-Ki; Liu, Senquan; Lee, Kai-Chuen; He, Xiangjun; You, Wenxing; Zhou, Rui; Guo, Jun-Tao; Chen, Xiongfong; Peng, Xianlu; Sun, Hao; Huang, He; Zhao, Hui; Feng, Bo

2014-04-01

The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around -120 to -80 bp, while highly effective sgRNAs targeted from -147 to -89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells.

Introgression of the SbASR-1 Gene Cloned from a Halophyte Salicornia brachiata Enhances Salinity and Drought Endurance in Transgenic Groundnut (Arachis hypogaea) and Acts as a Transcription Factor

PubMed Central

Tiwari, Vivekanand; Chaturvedi, Amit Kumar; Mishra, Avinash; Jha, Bhavanath

2015-01-01

The SbASR-1 gene, cloned from a halophyte Salicornia brachiata, encodes a plant-specific hydrophilic and stress responsive protein. The genome of S. brachiata has two paralogs of the SbASR-1 gene (2549 bp), which is comprised of a single intron of 1611 bp, the largest intron of the  abscisic acid stress ripening [ASR] gene family yet reported. In silico analysis of the 843-bp putative promoter revealed the presence of ABA, biotic stress, dehydration, phytohormone, salinity, and sugar responsive cis-regulatory motifs. The SbASR-1 protein belongs to Group 7 LEA protein family with different amino acid composition compared to their glycophytic homologs. Bipartite Nuclear Localization Signal (NLS) was found on the C-terminal end of protein and localization study confirmed that SbASR-1 is a nuclear protein. Furthermore, transgenic groundnut (Arachis hypogaea) plants over-expressing the SbASR-1 gene constitutively showed enhanced salinity and drought stress tolerance in the T1 generation. Leaves of transgenic lines exhibited higher chlorophyll and relative water contents and lower electrolyte leakage, malondialdehyde content, proline, sugars, and starch accumulation under stress treatments than wild-type (Wt) plants. Also, lower accumulation of H2O2 and O2 .- radicals was detected in transgenic lines compared to Wt plants under stress conditions. Transcript expression of APX (ascorbate peroxidase) and CAT (catalase) genes were higher in Wt plants, whereas the SOD (superoxide dismutase) transcripts were higher in transgenic lines under stress. Electrophoretic mobility shift assay (EMSA) confirmed that the SbASR-1 protein binds at the consensus sequence (C/G/A)(G/T)CC(C/G)(C/G/A)(A/T). Based on results of the present study, it may be concluded that SbASR-1 enhances the salinity and drought stress tolerance in transgenic groundnut by functioning as a LEA (late embryogenesis abundant) protein and a transcription factor. PMID:26158616

A Circadian Clock-Regulated Toggle Switch Explains AtGRP7 and AtGRP8 Oscillations in Arabidopsis thaliana

PubMed Central

Schmal, Christoph; Reimann, Peter; Staiger, Dorothee

2013-01-01

The circadian clock controls many physiological processes in higher plants and causes a large fraction of the genome to be expressed with a 24h rhythm. The transcripts encoding the RNA-binding proteins AtGRP7 (Arabidopsis thaliana Glycine Rich Protein 7) and AtGRP8 oscillate with evening peaks. The circadian clock components CCA1 and LHY negatively affect AtGRP7 expression at the level of transcription. AtGRP7 and AtGRP8, in turn, negatively auto-regulate and reciprocally cross-regulate post-transcriptionally: high protein levels promote the generation of an alternative splice form that is rapidly degraded. This clock-regulated feedback loop has been proposed to act as a molecular slave oscillator in clock output. While mathematical models describing the circadian core oscillator in Arabidopsis thaliana were introduced recently, we propose here the first model of a circadian slave oscillator. We define the slave oscillator in terms of ordinary differential equations and identify the model's parameters by an optimization procedure based on experimental results. The model successfully reproduces the pertinent experimental findings such as waveforms, phases, and half-lives of the time-dependent concentrations. Furthermore, we obtain insights into possible mechanisms underlying the observed experimental dynamics: the negative auto-regulation and reciprocal cross-regulation via alternative splicing could be responsible for the sharply peaking waveforms of the AtGRP7 and AtGRP8 mRNA. Moreover, our results suggest that the AtGRP8 transcript oscillations are subordinated to those of AtGRP7 due to a higher impact of AtGRP7 protein on alternative splicing of its own and of the AtGRP8 pre-mRNA compared to the impact of AtGRP8 protein. Importantly, a bifurcation analysis provides theoretical evidence that the slave oscillator could be a toggle switch, arising from the reciprocal cross-regulation at the post-transcriptional level. In view of this, transcriptional repression of AtGRP7 and AtGRP8 by LHY and CCA1 induces oscillations of the toggle switch, leading to the observed high-amplitude oscillations of AtGRP7 mRNA. PMID:23555221

Establishment of stable cell line for inducing KAP1 protein expression.

PubMed

Liu, Xiaoyan; Khan, Md Asaduzzaman; Cheng, Jingliang; Wei, Chunli; Zhang, Lianmei; Fu, Junjiang

2015-06-01

Generation of the stable cell lines is a highly efficient tool in functional studies of certain genes or proteins, where the particular genes or proteins are inducibly expressed. The KRAB-associated protein-1 (KAP1) is an important transcription regulatory protein, which is investigated in several molecular biological studies. In this study, we have aimed to generate a stable cell line for inducing KAP1 expression. The recombinant plasmid pcDNA5/FRT/TO-KAP1 was constructed at first, which was then transfected into Flp-In™T-REx™-HEK293 cells to establish an inducible pcDNA5/FRT/TO-KAP1-HEK293 cell line. The Western blot analysis showed that the protein level of KAP1 is over-expressed in the established stable cell line by doxycycline induction, both dose and time dependently. Thus we have successfully established stable pcDNA5/FRT/TO-KAP1-HEK293 cell line, which can express KAP1 inducibly. This inducible cell line might be very useful for KAP1 functional studies.

Ubiquitination-Related MdBT Scaffold Proteins Target a bHLH Transcription Factor for Iron Homeostasis1[OPEN

PubMed Central

Zhao, Qiang; Wang, Qing-Jie; Wang, Xiao-Fei; You, Chun-Xiang

2016-01-01

Iron (Fe) homeostasis is crucial for plant growth and development. A network of basic helix-loop-helix (bHLH) transcription factors positively regulates Fe uptake during iron deficiency. However, their up-regulation or overexpression leads to Fe overload and reactive oxygen species generation, thereby damaging the plants. Here, we found that two BTB/TAZ proteins, MdBT1 and MdBT2, interact with the MbHLH104 protein in apple. In addition, the function of MdBT2 was characterized as a regulator of MdbHLH104 degradation via ubiquitination and the 26S proteasome pathway, thereby controlling the activity of plasma membrane H+-ATPases and the acquisition of iron. Furthermore, MdBT2 interacted with MdCUL3 proteins, which were required for the MdBT2-mediated ubiquitination modification of MdbHLH104 and its degradation. In sum, our findings demonstrate that MdBT proteins interact with MdCUL3 to bridge the formation of the MdBTsMdCUL3 complex, which negatively modulates the degradation of the MdbHLH104 protein in response to changes in Fe status to maintain iron homeostasis in plants. PMID:27660166

Spermiogenesis and exchange of basic nuclear proteins are impaired in male germ cells lacking Camk4.

PubMed

Wu, J Y; Ribar, T J; Cummings, D E; Burton, K A; McKnight, G S; Means, A R

2000-08-01

Ca2+/calmodulin-dependent protein kinase IV (Camk4; also known as CaMKIV), a multifunctional serine/threonine protein kinase with limited tissue distribution, has been implicated in transcriptional regulation in lymphocytes, neurons and male germ cells. In the mouse testis, however, Camk4 is expressed in spermatids and associated with chromatin and nuclear matrix. Elongating spermatids are not transcriptionally active, raising the possibility that Camk4 has a novel function in male germ cells. To investigate the role of Camk4 in spermatogenesis, we have generated mice with a targeted deletion of the gene Camk4. Male Camk4-/- mice are infertile with impairment of spermiogenesis in late elongating spermatids. The sequential deposition of sperm basic nuclear proteins on chromatin is disrupted, with a specific loss of protamine-2 and prolonged retention of transition protein-2 (Tnp2) in step-15 spermatids. Protamine-2 is phosphorylated by Camk4 in vitro, implicating a connection between Camk4 signalling and the exchange of basic nuclear proteins in mammalian male germ cells. Defects in protamine-2 have been identified in sperm of infertile men, suggesting that our results may have clinical implications for the understanding of human male infertility.

SclR, a basic helix-loop-helix transcription factor, regulates hyphal morphology and promotes sclerotial formation in Aspergillus oryzae.

PubMed

Jin, Feng Jie; Takahashi, Tadashi; Matsushima, Ken-ichiro; Hara, Seiichi; Shinohara, Yasutomo; Maruyama, Jun-ichi; Kitamoto, Katsuhiko; Koyama, Yasuji

2011-07-01

Most known basic-region helix-loop-helix (bHLH) proteins belong to a superfamily of transcription factors often involved in the control of growth and differentiation. Therefore, inappropriate expression of genes encoding bHLH proteins is frequently associated with developmental dysfunction. In our previously reported study, a novel bHLH protein-encoding gene (AO090011000215) of Aspergillus oryzae was identified. The gene-disrupted strain was found to produce dense conidia, but sparse sclerotia, relative to the parent strain. Here, to further analyze its function, we generated an overexpressing strain using the A. oryzae amyB gene promoter. Genetic overexpression led to a large number of initial hyphal aggregations and then the formation of mature sclerotia; it was therefore designated sclR (sclerotium regulator). At the same time, the sclR-overexpressing strain also displayed both delayed and decreased conidiation. Scanning electron microscopy indicated that the aerial hyphae of the sclR-overexpressing strain were extremely branched and intertwined with each other. In the generation of the SclR-enhanced green fluorescent protein (EGFP) expression strain, the SclR-EGFP protein fusion was conditionally detected in the nuclei. In addition, the loss of sclR function led to rapid protein degradation and cell lysis in dextrin-polypeptone-yeast extract liquid medium. Taken together, these observations indicate that SclR plays an important role in hyphal morphology, asexual conidiospore formation, and the promotion of sclerotial production, even retaining normal cell function, at least in submerged liquid culture.

Synthetic transcripts of double-stranded Birnavirus genome are infectious.

PubMed Central

Mundt, E; Vakharia, V N

1996-01-01

We have developed a system for generation of infectious bursal disease virus (IBDV), a segmented double-stranded RNA virus of the Birnaviridae family, with the use of synthetic transcripts derived from cloned cDNA. Independent full-length cDNA clones were constructed that contained the entire coding and noncoding regions of RNA segments A and B of two distinguishable IBDV strains of serotype I. Segment A encodes all of the structural (VP2, VP4, and VP3) and nonstructural (VP5) proteins, whereas segment B encodes the RNA-dependent RNA polymerase (VP1). Synthetic RNAs of both segments were produced by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of Vero cells with combined plus-sense transcripts of both segments generated infectious virus as early as 36 hr after transfection. The infectivity and specificity of the recovered chimeric virus was ascertained by the appearance of cytopathic effect in chicken embryo cells, by immunofluorescence staining of infected Vero cells with rabbit anti-IBDV serum, and by nucleotide sequence analysis of the recovered virus, respectively. In addition, transfectant viruses containing genetically tagged sequences in either segment A or segment B of IBDV were generated to confirm the feasibility of this system. The development of a reverse genetics system for double-stranded RNA viruses will greatly facilitate studies of the regulation of viral gene expression, pathogenesis, and design of a new generation of live vaccines. Images Fig. 2 Fig. 3 Fig. 4 PMID:8855321

The DVR-1 (Vg1) transcript of zebrafish is maternally supplied and distributed throughout the embryo.

PubMed

Helde, K A; Grunwald, D J

1993-10-01

It is not known how region- or tissue-specific differences are generated in the zebrafish embryo. To look at the potential role of maternal transcripts in generating cell diversity, we have isolated and characterized the zebrafish homologue of Xenopus DVR-1 (Vg1), a maternally supplied RNA that encodes a member of the transforming growth factor-beta superfamily. The zebrafish DVR-1 RNA is maternally supplied and its protein product shares a high degree of sequence identity with Xenopus DVR-1. These conserved features indicate that DVR-1 is likely to have an essential function in early embryogenesis. However, unlike the frog transcript, which is restricted to vegetal cells, DVR-1 RNA is distributed equally among all zebrafish blastomeres. We suggest that the ubiquitous distribution of DVR-1 RNA reflects a significant aspect of the developmental strategy of the zebrafish in which each blastomere retains an equivalent developmental potential throughout the cleavage period.

Generation of Marker-free Transgenic Plants Concurrently Resistant to a DNA Geminivirus and a RNA Tospovirus

PubMed Central

Yang, Ching-Fu; Chen, Kuan-Chun; Cheng, Ying-Hui; Raja, Joseph A. J.; Huang, Ya-Ling; Chien, Wan-Chu; Yeh, Shyi-Dong

2014-01-01

Global threats of ssDNA geminivirus and ss(-)RNA tospovirus on crops necessitate the development of transgenic resistance. Here, we constructed a two-T DNA vector carrying a hairpin of the intergenic region (IGR) of Ageratum yellow vein virus (AYVV), residing in an intron inserted in an untranslatable nucleocapsid protein (NP) fragment of Melon yellow spot virus (MYSV). Transgenic tobacco lines highly resistant to AYVV and MYSV were generated. Accumulation of 24-nt siRNA, higher methylation levels on the IGR promoters of the transgene, and suppression of IGR promoter activity of invading AYVV indicate that AYVV resistance is mediated by transcriptional gene silencing. Lack of NP transcript and accumulation of corresponding siRNAs indicate that MYSV resistance is mediated through post-transcriptional gene silencing. Marker-free progenies with concurrent resistance to both AYVV and MYSV, stably inherited as dominant nuclear traits, were obtained. Hence, we provide a novel way for concurrent control of noxious DNA and RNA viruses with less biosafety concerns. PMID:25030413

A Small RNA-Catalytic Argonaute Pathway Tunes Germline Transcript Levels to Ensure Embryonic Divisions

PubMed Central

Gerson-Gurwitz, Adina; Wang, Shaohe; Sathe, Shashank; Green, Rebecca; Yeo, Gene W.; Oegema, Karen; Desai, Arshad

2016-01-01

SUMMARY Multiple division cycles without growth are a characteristic feature of early embryogenesis. The female germline loads proteins and RNAs into oocytes to support these divisions, which lack many quality control mechanisms operating in somatic cells undergoing growth. Here we describe a small RNA-Argonaute pathway that ensures early embryonic divisions in C. elegans by employing catalytic slicing activity to broadly tune, instead of silence, germline gene expression. Misregulation of one target, a kinesin-13 microtubule depolymerase, underlies a major phenotype associated with pathway loss. Tuning of target transcript levels is guided by density of homologous small RNAs, whose generation must ultimately be related to target sequence. Thus, the tuning action of a small RNA-catalytic Argonaute pathway generates oocytes capable of supporting embryogenesis. We speculate that the specialized nature of germline chromatin led to emergence of small RNA-catalytic Argonaute pathways in the female germline as a post-transcriptional control layer to optimize oocyte composition. PMID:27020753

Defining pancreatic endocrine precursors and their descendants.

PubMed

White, Peter; May, Catherine Lee; Lamounier, Rodrigo N; Brestelli, John E; Kaestner, Klaus H

2008-03-01

The global incidence of diabetes continues to increase. Cell replacement therapy and islet transplantation offer hope, especially for severely affected patients. Efforts to differentiate insulin-producing beta-cells from progenitor or stem cells require knowledge of the transcriptional programs that regulate the development of the endocrine pancreas. Differentiation toward the endocrine lineage is dependent on the transcription factor Neurogenin 3 (Neurog3, Ngn3). We utilize a Neurog3-enhanced green fluorescent protein knock-in mouse model to isolate endocrine progenitor cells from embryonic pancreata (embryonic day [E]13.5 through E17.5). Using advanced genomic approaches, we generate a comprehensive gene expression profile of these progenitors and their immediate descendants. A total of 1,029 genes were identified as being temporally regulated in the endocrine lineage during fetal development, 237 of which are transcriptional regulators. Through pathway analysis, we have modeled regulatory networks involving these proteins that highlight the complex transcriptional hierarchy governing endocrine differentiation. We have been able to accurately capture the gene expression profile of the pancreatic endocrine progenitors and their descendants. The list of temporally regulated genes identified in fetal endocrine precursors and their immediate descendants provides a novel and important resource for developmental biologists and diabetes researchers alike.

Dramatic expansion of the black widow toxin arsenal uncovered by multi-tissue transcriptomics and venom proteomics.

PubMed

Haney, Robert A; Ayoub, Nadia A; Clarke, Thomas H; Hayashi, Cheryl Y; Garb, Jessica E

2014-06-11

Animal venoms attract enormous interest given their potential for pharmacological discovery and understanding the evolution of natural chemistries. Next-generation transcriptomics and proteomics provide unparalleled, but underexploited, capabilities for venom characterization. We combined multi-tissue RNA-Seq with mass spectrometry and bioinformatic analyses to determine venom gland specific transcripts and venom proteins from the Western black widow spider (Latrodectus hesperus) and investigated their evolution. We estimated expression of 97,217 L. hesperus transcripts in venom glands relative to silk and cephalothorax tissues. We identified 695 venom gland specific transcripts (VSTs), many of which BLAST and GO term analyses indicate may function as toxins or their delivery agents. ~38% of VSTs had BLAST hits, including latrotoxins, inhibitor cystine knot toxins, CRISPs, hyaluronidases, chitinase, and proteases, and 59% of VSTs had predicted protein domains. Latrotoxins are venom toxins that cause massive neurotransmitter release from vertebrate or invertebrate neurons. We discovered ≥ 20 divergent latrotoxin paralogs expressed in L. hesperus venom glands, significantly increasing this biomedically important family. Mass spectrometry of L. hesperus venom identified 49 proteins from VSTs, 24 of which BLAST to toxins. Phylogenetic analyses showed venom gland specific gene family expansions and shifts in tissue expression. Quantitative expression analyses comparing multiple tissues are necessary to identify venom gland specific transcripts. We present a black widow venom specific exome that uncovers a trove of diverse toxins and associated proteins, suggesting a dynamic evolutionary history. This justifies a reevaluation of the functional activities of black widow venom in light of its emerging complexity.

Regulation of nitrate assimilation in cyanobacteria.

PubMed

Ohashi, Yoshitake; Shi, Wei; Takatani, Nobuyuki; Aichi, Makiko; Maeda, Shin-ichi; Watanabe, Satoru; Yoshikawa, Hirofumi; Omata, Tatsuo

2011-02-01

Nitrate assimilation by cyanobacteria is inhibited by the presence of ammonium in the growth medium. Both nitrate uptake and transcription of the nitrate assimilatory genes are regulated. The major intracellular signal for the regulation is, however, not ammonium or glutamine, but 2-oxoglutarate (2-OG), whose concentration changes according to the change in cellular C/N balance. When nitrogen is limiting growth, accumulation of 2-OG activates the transcription factor NtcA to induce transcription of the nitrate assimilation genes. Ammonium inhibits transcription by quickly depleting the 2-OG pool through its metabolism via the glutamine synthetase/glutamate synthase cycle. The P(II) protein inhibits the ABC-type nitrate transporter, and also nitrate reductase in some strains, by an unknown mechanism(s) when the cellular 2-OG level is low. Upon nitrogen limitation, 2-OG binds to P(II) to prevent the protein from inhibiting nitrate assimilation. A pathway-specific transcriptional regulator NtcB activates the nitrate assimilation genes in response to nitrite, either added to the medium or generated intracellularly by nitrate reduction. It plays an important role in selective activation of the nitrate assimilation pathway during growth under a limited supply of nitrate. P(II) was recently shown to regulate the activity of NtcA negatively by binding to PipX, a small coactivator protein of NtcA. On the basis of accumulating genome information from a variety of cyanobacteria and the molecular genetic data obtained from the representative strains, common features and group- or species-specific characteristics of the response of cyanobacteria to nitrogen is summarized and discussed in terms of ecophysiological significance.

The LMNA mutation p.Arg321Ter associated with dilated cardiomyopathy leads to reduced expression and a skewed ratio of lamin A and lamin C proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Al-Saaidi, Rasha; Rasmussen, Torsten B.; Palmfeldt, Johan

2013-11-15

Dilated cardiomyopathy (DCM) is a disease of the heart muscle characterized by cardiac chamber enlargement and reduced systolic function of the left ventricle. Mutations in the LMNA gene represent the most frequent known genetic cause of DCM associated with disease of the conduction systems. The LMNA gene generates two major transcripts encoding the nuclear lamina major components lamin A and lamin C by alternative splicing. Both haploinsuffiency and dominant negative effects have been proposed as disease mechanism for premature termination codon (PTC) mutations in LMNA. These mechanisms however are still not clearly established. In this study, we used a representativemore » LMNA nonsense mutation, p.Arg321Ter, to shed light on the molecular disease mechanisms. Cultured fibroblasts from three DCM patients carrying this mutation were analyzed. Quantitative reverse transcriptase PCR and sequencing of these PCR products indicated that transcripts from the mutant allele were degraded by the nonsense-mediated mRNA decay (NMD) mechanism. The fact that no truncated mutant protein was detectable in western blot (WB) analysis strengthens the notion that the mutant transcript is efficiently degraded. Furthermore, WB analysis showed that the expression of lamin C protein was reduced by the expected approximately 50%. Clearly decreased lamin A and lamin C levels were also observed by immunofluorescence microscopy analysis. However, results from both WB and nano-liquid chromatography/mass spectrometry demonstrated that the levels of lamin A protein were more reduced suggesting an effect on expression of lamin A from the wild type allele. PCR analysis of the ratio of lamin A to lamin C transcripts showed unchanged relative amounts of lamin A transcript suggesting that the effect on the wild type allele was operative at the protein level. Immunofluorescence microscopy analysis showed no abnormal nuclear morphology of patient fibroblast cells. Based on these data, we propose that heterozygosity for the nonsense mutation causes NMD degradation of the mutant transcripts blocking expression of the truncated mutant protein and an additional trans effect on lamin A protein levels expressed from the wild type allele. We discuss the possibility that skewing of the lamin A to lamin C ratio may contribute to ensuing processes that destabilize cardiomyocytes and trigger cardiomyopathy - Highlights: • We study disease mechanisms in DCM patients carrying PTC mutations in the LMNA gene. • The mutant transcript is degraded by the nonsense mediated mRNA decay system. • Skewed lamin A to lamin C protein ratio expressed from the wild type allele. • We suggest a combined pathomechanism: haploinsuffiency plus lamin A/C imbalance.« less

Transgenerationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing

PubMed Central

Le Thomas, Adrien; Stuwe, Evelyn; Li, Sisi; Marinov, Georgi; Rozhkov, Nikolay; Chen, Yung-Chia Ariel; Luo, Yicheng; Sachidanandam, Ravi; Toth, Katalin Fejes; Patel, Dinshaw; Aravin, Alexei A.

2014-01-01

Small noncoding RNAs that associate with Piwi proteins, called piRNAs, serve as guides for repression of diverse transposable elements in germ cells of metazoa. In Drosophila, the genomic regions that give rise to piRNAs, the so-called piRNA clusters, are transcribed to generate long precursor molecules that are processed into mature piRNAs. How genomic regions that give rise to piRNA precursor transcripts are differentiated from the rest of the genome and how these transcripts are specifically channeled into the piRNA biogenesis pathway are not known. We found that transgenerationally inherited piRNAs provide the critical trigger for piRNA production from homologous genomic regions in the next generation by two different mechanisms. First, inherited piRNAs enhance processing of homologous transcripts into mature piRNAs by initiating the ping-pong cycle in the cytoplasm. Second, inherited piRNAs induce installment of the histone 3 Lys9 trimethylation (H3K9me3) mark on genomic piRNA cluster sequences. The heterochromatin protein 1 (HP1) homolog Rhino binds to the H3K9me3 mark through its chromodomain and is enriched over piRNA clusters. Rhino recruits the piRNA biogenesis factor Cutoff to piRNA clusters and is required for efficient transcription of piRNA precursors. We propose that transgenerationally inherited piRNAs act as an epigenetic memory for identification of substrates for piRNA biogenesis on two levels: by inducing a permissive chromatin environment for piRNA precursor synthesis and by enhancing processing of these precursors. PMID:25085419

Phenotypes on demand via switchable target protein degradation in multicellular organisms

PubMed Central

Faden, Frederik; Ramezani, Thomas; Mielke, Stefan; Almudi, Isabel; Nairz, Knud; Froehlich, Marceli S.; Höckendorff, Jörg; Brandt, Wolfgang; Hoehenwarter, Wolfgang; Dohmen, R. Jürgen; Schnittger, Arp; Dissmeyer, Nico

2016-01-01

Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest are invaluable tools to analyse and engineer biological processes. While temperature-sensitive alleles are frequently used as conditional mutants in microorganisms, they are usually difficult to identify in multicellular species. Here we present a versatile and transferable, genetically stable system based on a low-temperature-controlled N-terminal degradation signal (lt-degron) that allows reversible and switch-like tuning of protein levels under physiological conditions in vivo. Thereby, developmental effects can be triggered and phenotypes on demand generated. The lt-degron was established to produce conditional and cell-type-specific phenotypes and is generally applicable in a wide range of organisms, from eukaryotic microorganisms to plants and poikilothermic animals. We have successfully applied this system to control the abundance and function of transcription factors and different enzymes by tunable protein accumulation. PMID:27447739

Protein kinase inhibitor SU6668 attenuates positive regulation of Gli proteins in cancer and multipotent progenitor cells.

PubMed

Piirsoo, Alla; Kasak, Lagle; Kauts, Mari-Liis; Loog, Mart; Tints, Kairit; Uusen, Piia; Neuman, Toomas; Piirsoo, Marko

2014-04-01

Observations that Glioma-associated transcription factors Gli1 and Gli2 (Gli1/2), executers of the Sonic Hedgehog (Shh) signaling pathway and targets of the Transforming Growth Factor β (TGF-β) signaling axis, are involved in numerous developmental and pathological processes unveil them as attractive pharmaceutical targets. Unc-51-like serine/threonine kinase Ulk3 has been suggested to play kinase activity dependent and independent roles in the control of Gli proteins in the context of the Shh signaling pathway. This study aimed at investigating whether the mechanism of generation of Gli1/2 transcriptional activators has similarities regardless of the signaling cascade evoking their activation. We also elucidate further the role of Ulk3 kinase in regulation of Gli1/2 proteins and examine SU6668 as an inhibitor of Ulk3 catalytic activity and a compound targeting Gli1/2 proteins in different cell-based experimental models. Here we demonstrate that Ulk3 is required not only for maintenance of basal levels of Gli1/2 proteins but also for TGF-β or Shh dependent activation of endogenous Gli1/2 proteins in human adipose tissue derived multipotent stromal cells (ASCs) and mouse immortalized progenitor cells, respectively. We show that cultured ASCs possess the functional Shh signaling axis and differentiate towards osteoblasts in response to Shh. Also, we demonstrate that similarly to Ulk3 RNAi, SU6668 prevents de novo expression of Gli1/2 proteins and antagonizes the Gli-dependent activation of the gene expression programs induced by either Shh or TGF-β. Our data suggest SU6668 as an efficient inhibitor of Ulk3 kinase allowing manipulation of the Gli-dependent transcriptional outcome. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Benzoate-mediated changes on expression profile of soluble proteins in Serratia sp. DS001.

PubMed

Pandeeti, E V P; Chinnaboina, M R; Siddavattam, D

2009-05-01

To assess differences in protein expression profile associated with shift in carbon source from succinate to benzoate in Serratia sp. DS001 using a proteomics approach. A basic proteome map was generated for the soluble proteins extracted from Serratia sp. DS001 grown in succinate and benzoate. The differently and differentially expressed proteins were identified using ImageMaster 2D Platinum software (GE Healthcare). The identity of the proteins was determined by employing MS or MS/MS. Important enzymes such as Catechol 1,2 dioxygenase and transcriptional regulators that belong to the LysR superfamily were identified. Nearly 70 proteins were found to be differentially expressed when benzoate was used as carbon source. Based on the protein identity and degradation products generated from benzoate it is found that ortho pathway is operational in Serratia sp. DS001. Expression profile of the soluble proteins associated with shift in carbon source was mapped. The study also elucidates degradation pathway of benzoate in Serratia sp. DS001 by correlating the proteomics data with the catabolites of benzoate.

Mechanism of short-term ERK activation by electromagnetic fields at mobile phone frequencies.

PubMed

Friedman, Joseph; Kraus, Sarah; Hauptman, Yirmi; Schiff, Yoni; Seger, Rony

2007-08-01

The exposure to non-thermal microwave electromagnetic fields generated by mobile phones affects the expression of many proteins. This effect on transcription and protein stability can be mediated by the MAPK (mitogen-activated protein kinase) cascades, which serve as central signalling pathways and govern essentially all stimulated cellular processes. Indeed, long-term exposure of cells to mobile phone irradiation results in the activation of p38 as well as the ERK (extracellular-signal-regulated kinase) MAPKs. In the present study, we have studied the immediate effect of irradiation on the MAPK cascades, and found that ERKs, but not stress-related MAPKs, are rapidly activated in response to various frequencies and intensities. Using signalling inhibitors, we delineated the mechanism that is involved in this activation. We found that the first step is mediated in the plasma membrane by NADH oxidase, which rapidly generates ROS (reactive oxygen species). These ROS then directly stimulate MMPs (matrix metalloproteinases) and allow them to cleave and release Hb-EGF [heparin-binding EGF (epidermal growth factor)]. This secreted factor activates the EGF receptor, which in turn further activates the ERK cascade. Thus this study demonstrates for the first time a detailed molecular mechanism by which electromagnetic irradiation from mobile phones induces the activation of the ERK cascade and thereby induces transcription and other cellular processes.

RNA-Seq Analysis Using De Novo Transcriptome Assembly as a Reference for the Salmon Louse Caligus rogercresseyi

PubMed Central

Gallardo-Escárate, Cristian; Valenzuela-Muñoz, Valentina; Nuñez-Acuña, Gustavo

2014-01-01

Despite the economic and environmental impacts that sea lice infestations have on salmon farming worldwide, genomic data generated by high-throughput transcriptome sequencing for different developmental stages, sexes, and strains of sea lice is still limited or unknown. In this study, RNA-seq analysis was performed using de novo transcriptome assembly as a reference for evidenced transcriptional changes from six developmental stages of the salmon louse Caligus rogercresseyi. EST-datasets were generated from the nauplius I, nauplius II, copepodid and chalimus stages and from female and male adults using MiSeq Illumina sequencing. A total of 151,788,682 transcripts were yielded, which were assembled into 83,444 high quality contigs and subsequently annotated into roughly 24,000 genes based on known proteins. To identify differential transcription patterns among salmon louse stages, cluster analyses were performed using normalized gene expression values. Herein, four clusters were differentially expressed between nauplius I–II and copepodid stages (604 transcripts), five clusters between copepodid and chalimus stages (2,426 transcripts), and six clusters between female and male adults (2,478 transcripts). Gene ontology analysis revealed that the nauplius I–II, copepodid and chalimus stages are mainly annotated to aminoacid transfer/repair/breakdown, metabolism, molting cycle, and nervous system development. Additionally, genes showing differential transcription in female and male adults were highly related to cytoskeletal and contractile elements, reproduction, cell development, morphogenesis, and transcription-translation processes. The data presented in this study provides the most comprehensive transcriptome resource available for C. rogercresseyi, which should be used for future genomic studies linked to host-parasite interactions. PMID:24691066

RNA-Seq analysis using de novo transcriptome assembly as a reference for the salmon louse Caligus rogercresseyi.

PubMed

Gallardo-Escárate, Cristian; Valenzuela-Muñoz, Valentina; Nuñez-Acuña, Gustavo

2014-01-01

Despite the economic and environmental impacts that sea lice infestations have on salmon farming worldwide, genomic data generated by high-throughput transcriptome sequencing for different developmental stages, sexes, and strains of sea lice is still limited or unknown. In this study, RNA-seq analysis was performed using de novo transcriptome assembly as a reference for evidenced transcriptional changes from six developmental stages of the salmon louse Caligus rogercresseyi. EST-datasets were generated from the nauplius I, nauplius II, copepodid and chalimus stages and from female and male adults using MiSeq Illumina sequencing. A total of 151,788,682 transcripts were yielded, which were assembled into 83,444 high quality contigs and subsequently annotated into roughly 24,000 genes based on known proteins. To identify differential transcription patterns among salmon louse stages, cluster analyses were performed using normalized gene expression values. Herein, four clusters were differentially expressed between nauplius I-II and copepodid stages (604 transcripts), five clusters between copepodid and chalimus stages (2,426 transcripts), and six clusters between female and male adults (2,478 transcripts). Gene ontology analysis revealed that the nauplius I-II, copepodid and chalimus stages are mainly annotated to aminoacid transfer/repair/breakdown, metabolism, molting cycle, and nervous system development. Additionally, genes showing differential transcription in female and male adults were highly related to cytoskeletal and contractile elements, reproduction, cell development, morphogenesis, and transcription-translation processes. The data presented in this study provides the most comprehensive transcriptome resource available for C. rogercresseyi, which should be used for future genomic studies linked to host-parasite interactions.

Quantitative evaluation of hepatic cytochrome P4501A transcript, protein, and catalytic activity in the striped sea bream (Lithognathus mormyrus).

PubMed

Tom, Moshe; Shmul, Merav; Shefer, Edna; Chen, Nir; Slor, Hanoch; Rinkevich, Baruch; Herut, Barak

2003-09-01

Hepatic cytochrome P4501A (CYP1A) expression was partially characterized in the striped sea bream (Lithognathus mormyrus) from the Mediterranean coast of Israel as part of the process of establishing the CYP1A gene as an environmental biomarker. Reverse transcription-competitive polymerase chain reaction, competitive enzyme-linked immunosorbent assay, and ethoxyresorufin O-deethylase (EROD) assay were used for evaluating transcript, protein, and catalytic activity levels, respectively, in absolute units. Highest elucidated transcript, protein, and catalytic activity levels were 0.264 +/- SD 0.084 fmol/microg total RNA, 0.88 +/- 0.52 pmol/microg total protein, and 1.11 +/- 0.52 pmol resorufin/min/microg total protein, respectively, and the lower levels were 0.009 +/- 0.007 fmol/microg total RNA, 0.17 +/- 0.08 pmol/microg total protein, and 0.11 +/- 0.06 pmol resorufin/min/microg total protein, respectively, demonstrating substantial induction potential. All alternate pairs of seven examined field samples, revealing a transcript-level ratio higher than 1.7, also demonstrated a significant difference between their transcript levels, indicating a potential to detect relatively small biomarker changes (1.7-fold) caused by environmental effects. Simultaneous triple measurements of transcript, protein, and catalytic activity were carried out in individuals from two field samples and during a 318-d decay experiment. Fish from the field samples revealed significant alternate bivariate correlation between transcript, protein, and enzymatic activity. Conflicting results were found when analyzing the decay experiment, in which both protein and catalytic activity levels decreased significantly to basal levels, in contrast to no significant change in transcript levels throughout the experiment. No significant difference was observed between males and females regarding the levels of CYP1A transcript, protein, and EROD.

Transcriptome and proteome data reveal candidate genes for pollinator attraction in sexually deceptive orchids.

PubMed

Sedeek, Khalid E M; Qi, Weihong; Schauer, Monica A; Gupta, Alok K; Poveda, Lucy; Xu, Shuqing; Liu, Zhong-Jian; Grossniklaus, Ueli; Schiestl, Florian P; Schlüter, Philipp M

2013-01-01

Sexually deceptive orchids of the genus Ophrys mimic the mating signals of their pollinator females to attract males as pollinators. This mode of pollination is highly specific and leads to strong reproductive isolation between species. This study aims to identify candidate genes responsible for pollinator attraction and reproductive isolation between three closely related species, O. exaltata, O. sphegodes and O. garganica. Floral traits such as odour, colour and morphology are necessary for successful pollinator attraction. In particular, different odour hydrocarbon profiles have been linked to differences in specific pollinator attraction among these species. Therefore, the identification of genes involved in these traits is important for understanding the molecular basis of pollinator attraction by sexually deceptive orchids. We have created floral reference transcriptomes and proteomes for these three Ophrys species using a combination of next-generation sequencing (454 and Solexa), Sanger sequencing, and shotgun proteomics (tandem mass spectrometry). In total, 121 917 unique transcripts and 3531 proteins were identified. This represents the first orchid proteome and transcriptome from the orchid subfamily Orchidoideae. Proteome data revealed proteins corresponding to 2644 transcripts and 887 proteins not observed in the transcriptome. Candidate genes for hydrocarbon and anthocyanin biosynthesis were represented by 156 and 61 unique transcripts in 20 and 7 genes classes, respectively. Moreover, transcription factors putatively involved in the regulation of flower odour, colour and morphology were annotated, including Myb, MADS and TCP factors. Our comprehensive data set generated by combining transcriptome and proteome technologies allowed identification of candidate genes for pollinator attraction and reproductive isolation among sexually deceptive orchids. This includes genes for hydrocarbon and anthocyanin biosynthesis and regulation, and the development of floral morphology. These data will serve as an invaluable resource for research in orchid floral biology, enabling studies into the molecular mechanisms of pollinator attraction and speciation.

Transcriptome and Proteome Data Reveal Candidate Genes for Pollinator Attraction in Sexually Deceptive Orchids

PubMed Central

Sedeek, Khalid E. M.; Qi, Weihong; Schauer, Monica A.; Gupta, Alok K.; Poveda, Lucy; Xu, Shuqing; Liu, Zhong-Jian; Grossniklaus, Ueli; Schiestl, Florian P.; Schlüter, Philipp M.

2013-01-01

Background Sexually deceptive orchids of the genus Ophrys mimic the mating signals of their pollinator females to attract males as pollinators. This mode of pollination is highly specific and leads to strong reproductive isolation between species. This study aims to identify candidate genes responsible for pollinator attraction and reproductive isolation between three closely related species, O. exaltata, O. sphegodes and O. garganica. Floral traits such as odour, colour and morphology are necessary for successful pollinator attraction. In particular, different odour hydrocarbon profiles have been linked to differences in specific pollinator attraction among these species. Therefore, the identification of genes involved in these traits is important for understanding the molecular basis of pollinator attraction by sexually deceptive orchids. Results We have created floral reference transcriptomes and proteomes for these three Ophrys species using a combination of next-generation sequencing (454 and Solexa), Sanger sequencing, and shotgun proteomics (tandem mass spectrometry). In total, 121 917 unique transcripts and 3531 proteins were identified. This represents the first orchid proteome and transcriptome from the orchid subfamily Orchidoideae. Proteome data revealed proteins corresponding to 2644 transcripts and 887 proteins not observed in the transcriptome. Candidate genes for hydrocarbon and anthocyanin biosynthesis were represented by 156 and 61 unique transcripts in 20 and 7 genes classes, respectively. Moreover, transcription factors putatively involved in the regulation of flower odour, colour and morphology were annotated, including Myb, MADS and TCP factors. Conclusion Our comprehensive data set generated by combining transcriptome and proteome technologies allowed identification of candidate genes for pollinator attraction and reproductive isolation among sexually deceptive orchids. This includes genes for hydrocarbon and anthocyanin biosynthesis and regulation, and the development of floral morphology. These data will serve as an invaluable resource for research in orchid floral biology, enabling studies into the molecular mechanisms of pollinator attraction and speciation. PMID:23734209

The C-module-binding factor supports amplification of TRE5-A retrotransposons in the Dictyostelium discoideum genome.

PubMed

Bilzer, Annika; Dölz, Heike; Reinhardt, Alexander; Schmith, Anika; Siol, Oliver; Winckler, Thomas

2011-01-01

Retrotransposable elements are molecular parasites that have invaded the genomes of virtually all organisms. Although retrotransposons encode essential proteins to mediate their amplification, they also require assistance by host cell-encoded machineries that perform functions such as DNA transcription and repair. The retrotransposon TRE5-A of the social amoeba Dictyostelium discoideum generates a notable amount of both sense and antisense RNAs, which are generated from element-internal promoters, located in the A module and the C module, respectively. We observed that TRE5-A retrotransposons depend on the C-module-binding factor (CbfA) to maintain high steady-state levels of TRE5-A transcripts and that CbfA supports the retrotransposition activity of TRE5-A elements. The carboxy-terminal domain of CbfA was found to be required and sufficient to mediate the accumulation of TRE5-A transcripts, but it did not support productive retrotransposition of TRE5-A. This result suggests different roles for CbfA protein domains in the regulation of TRE5-A retrotransposition frequency in D. discoideum cells. Although CbfA binds to the C module in vitro, the factor regulates neither C-module nor A-module promoter activity in vivo. We speculate that CbfA supports the amplification of TRE5-A retrotransposons by suppressing the expression of an as yet unidentified component of the cellular posttranscriptional gene silencing machinery.

WRKY transcription factors.

PubMed

Rushton, Paul J; Somssich, Imre E; Ringler, Patricia; Shen, Qingxi J

2010-05-01

WRKY transcription factors are one of the largest families of transcriptional regulators in plants and form integral parts of signalling webs that modulate many plant processes. Here, we review recent significant progress in WRKY transcription factor research. New findings illustrate that WRKY proteins often act as repressors as well as activators, and that members of the family play roles in both the repression and de-repression of important plant processes. Furthermore, it is becoming clear that a single WRKY transcription factor might be involved in regulating several seemingly disparate processes. Mechanisms of signalling and transcriptional regulation are being dissected, uncovering WRKY protein functions via interactions with a diverse array of protein partners, including MAP kinases, MAP kinase kinases, 14-3-3 proteins, calmodulin, histone deacetylases, resistance proteins and other WRKY transcription factors. WRKY genes exhibit extensive autoregulation and cross-regulation that facilitates transcriptional reprogramming in a dynamic web with built-in redundancy. 2010 Elsevier Ltd. All rights reserved.

Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome.

PubMed

Szymula, Agnieszka; Palermo, Richard D; Bayoumy, Amr; Groves, Ian J; Ba Abdullah, Mohammed; Holder, Beth; White, Robert E

2018-02-01

The Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is the first viral latency-associated protein produced after EBV infection of resting B cells. Its role in B cell transformation is poorly defined, but it has been reported to enhance gene activation by the EBV protein EBNA2 in vitro. We generated EBNA-LP knockout (LPKO) EBVs containing a STOP codon within each repeat unit of internal repeat 1 (IR1). EBNA-LP-mutant EBVs established lymphoblastoid cell lines (LCLs) from adult B cells at reduced efficiency, but not from umbilical cord B cells, which died approximately two weeks after infection. Adult B cells only established EBNA-LP-null LCLs with a memory (CD27+) phenotype. Quantitative PCR analysis of virus gene expression after infection identified both an altered ratio of the EBNA genes, and a dramatic reduction in transcript levels of both EBNA2-regulated virus genes (LMP1 and LMP2) and the EBNA2-independent EBER genes in the first 2 weeks. By 30 days post infection, LPKO transcription was the same as wild-type EBV. In contrast, EBNA2-regulated cellular genes were induced efficiently by LPKO viruses. Chromatin immunoprecipitation revealed that EBNA2 and the host transcription factors EBF1 and RBPJ were delayed in their recruitment to all viral latency promoters tested, whereas these same factors were recruited efficiently to several host genes, which exhibited increased EBNA2 recruitment. We conclude that EBNA-LP does not simply co-operate with EBNA2 in activating gene transcription, but rather facilitates the recruitment of several transcription factors to the viral genome, to enable transcription of virus latency genes. Additionally, our findings suggest that EBNA-LP is essential for the survival of EBV-infected naïve B cells.

Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome

PubMed Central

Szymula, Agnieszka; Palermo, Richard D.; Bayoumy, Amr; Groves, Ian J.

2018-01-01

The Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is the first viral latency-associated protein produced after EBV infection of resting B cells. Its role in B cell transformation is poorly defined, but it has been reported to enhance gene activation by the EBV protein EBNA2 in vitro. We generated EBNA-LP knockout (LPKO) EBVs containing a STOP codon within each repeat unit of internal repeat 1 (IR1). EBNA-LP-mutant EBVs established lymphoblastoid cell lines (LCLs) from adult B cells at reduced efficiency, but not from umbilical cord B cells, which died approximately two weeks after infection. Adult B cells only established EBNA-LP-null LCLs with a memory (CD27+) phenotype. Quantitative PCR analysis of virus gene expression after infection identified both an altered ratio of the EBNA genes, and a dramatic reduction in transcript levels of both EBNA2-regulated virus genes (LMP1 and LMP2) and the EBNA2-independent EBER genes in the first 2 weeks. By 30 days post infection, LPKO transcription was the same as wild-type EBV. In contrast, EBNA2-regulated cellular genes were induced efficiently by LPKO viruses. Chromatin immunoprecipitation revealed that EBNA2 and the host transcription factors EBF1 and RBPJ were delayed in their recruitment to all viral latency promoters tested, whereas these same factors were recruited efficiently to several host genes, which exhibited increased EBNA2 recruitment. We conclude that EBNA-LP does not simply co-operate with EBNA2 in activating gene transcription, but rather facilitates the recruitment of several transcription factors to the viral genome, to enable transcription of virus latency genes. Additionally, our findings suggest that EBNA-LP is essential for the survival of EBV-infected naïve B cells. PMID:29462212

Analyses of advanced rice anther transcriptomes reveal global tapetum secretory functions and potential proteins for lipid exine formation.

PubMed

Huang, Ming-Der; Wei, Fu-Jin; Wu, Cheng-Cheih; Hsing, Yue-Ie Caroline; Huang, Anthony H C

2009-02-01

The anthers in flowers perform important functions in sexual reproduction. Several recent studies used microarrays to study anther transcriptomes to explore genes controlling anther development. To analyze the secretion and other functions of the tapetum, we produced transcriptomes of anthers of rice (Oryza sativa subsp. japonica) at six progressive developmental stages and pollen with sequencing-by-synthesis technology. The transcriptomes included at least 18,000 unique transcripts, about 25% of which had antisense transcripts. In silico anther-minus-pollen subtraction produced transcripts largely unique to the tapetum; these transcripts include all the reported tapetum-specific transcripts of orthologs in other species. The differential developmental profiles of the transcripts and their antisense transcripts signify extensive regulation of gene expression in the anther, especially the tapetum, during development. The transcriptomes were used to dissect two major cell/biochemical functions of the tapetum. First, we categorized and charted the developmental profiles of all transcripts encoding secretory proteins present in the cellular exterior; these transcripts represent about 12% and 30% of the those transcripts having more than 100 and 1,000 transcripts per million, respectively. Second, we successfully selected from hundreds of transcripts several transcripts encoding potential proteins for lipid exine synthesis during early anther development. These proteins include cytochrome P450, acyltransferases, and lipid transfer proteins in our hypothesized mechanism of exine synthesis in and export from the tapetum. Putative functioning of these proteins in exine formation is consistent with proteins and metabolites detected in the anther locule fluid obtained by micropipetting.

Rye B chromosomes encode a functional Argonaute-like protein with in vitro slicer activities similar to its A chromosome paralog.

PubMed

Ma, Wei; Gabriel, Tobias Sebastian; Martis, Mihaela Maria; Gursinsky, Torsten; Schubert, Veit; Vrána, Jan; Doležel, Jaroslav; Grundlach, Heidrun; Altschmied, Lothar; Scholz, Uwe; Himmelbach, Axel; Behrens, Sven-Erik; Banaei-Moghaddam, Ali Mohammad; Houben, Andreas

2017-01-01

B chromosomes (Bs) are supernumerary, dispensable parts of the nuclear genome, which appear in many different species of eukaryote. So far, Bs have been considered to be genetically inert elements without any functional genes. Our comparative transcriptome analysis and the detection of active RNA polymerase II (RNAPII) in the proximity of B chromatin demonstrate that the Bs of rye (Secale cereale) contribute to the transcriptome. In total, 1954 and 1218 B-derived transcripts with an open reading frame were expressed in generative and vegetative tissues, respectively. In addition to B-derived transposable element transcripts, a high percentage of short transcripts without detectable similarity to known proteins and gene fragments from A chromosomes (As) were found, suggesting an ongoing gene erosion process. In vitro analysis of the A- and B-encoded AGO4B protein variants demonstrated that both possess RNA slicer activity. These data demonstrate unambiguously the presence of a functional AGO4B gene on Bs and that these Bs carry both functional protein coding genes and pseudogene copies. Thus, B-encoded genes may provide an additional level of gene control and complexity in combination with their related A-located genes. Hence, physiological effects, associated with the presence of Bs, may partly be explained by the activity of B-located (pseudo)genes. © 2016 IPK Gatersleben. New Phytologist © 2016 New Phytologist Trust.

Insight into the transcriptome of Arthrobotrys conoides using high throughput sequencing.

PubMed

Ramesh, Pandit; Reena, Patel; Amitbikram, Mohapatra; Chaitanya, Joshi; Anju, Kunjadia

2015-12-01

Arthrobotrys conoides is a nematode-trapping fungus belonging to Orbiliales, Ascomycota group, and traps prey nematodes by means of adhesive network. Fungus has a potential to be used as a biocontrol agent against plant parasitic nematodes. In the present study, we characterized the transcriptome of A. conoides using high-throughput sequencing technology and characterized its virulence unigenes. Total 7,255 cDNA contigs with an average length of 425 bp were generated and 6184 (61.81%) transcripts were functionally annotated and characterized. Majority of unigenes were found analogous to the genes of plant pathogenic fungi. A total of 1749 transcripts were found to be orthologous with eukaryotic proteins of KOG database. Several carbohydrate active enzymes and peptidases were identified. We also analyzed classically and nonclassically secreted proteins and confirmed by BLASTP against fungal secretome database. A total of 916 contigs were analogous to 556 unique proteins of Pathogen Host Interaction (PHI) database. Further, we identified 91 unigenes homologous to the database of fungal virulence factor (DFVF). A total of 104 putative protein kinases coding transcripts were identified by BLASTP against KinBase database, which are major players in signaling pathways. This study provides a comprehensive look at the transcriptome of A. conoides and the identified unigenes might have a role in catching and killing prey nematodes by A. conoides. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dynamics simulations for engineering macromolecular interactions

PubMed Central

Robinson-Mosher, Avi; Shinar, Tamar; Silver, Pamela A.; Way, Jeffrey

2013-01-01

The predictable engineering of well-behaved transcriptional circuits is a central goal of synthetic biology. The artificial attachment of promoters to transcription factor genes usually results in noisy or chaotic behaviors, and such systems are unlikely to be useful in practical applications. Natural transcriptional regulation relies extensively on protein-protein interactions to insure tightly controlled behavior, but such tight control has been elusive in engineered systems. To help engineer protein-protein interactions, we have developed a molecular dynamics simulation framework that simplifies features of proteins moving by constrained Brownian motion, with the goal of performing long simulations. The behavior of a simulated protein system is determined by summation of forces that include a Brownian force, a drag force, excluded volume constraints, relative position constraints, and binding constraints that relate to experimentally determined on-rates and off-rates for chosen protein elements in a system. Proteins are abstracted as spheres. Binding surfaces are defined radially within a protein. Peptide linkers are abstracted as small protein-like spheres with rigid connections. To address whether our framework could generate useful predictions, we simulated the behavior of an engineered fusion protein consisting of two 20 000 Da proteins attached by flexible glycine/serine-type linkers. The two protein elements remained closely associated, as if constrained by a random walk in three dimensions of the peptide linker, as opposed to showing a distribution of distances expected if movement were dominated by Brownian motion of the protein domains only. We also simulated the behavior of fluorescent proteins tethered by a linker of varying length, compared the predicted Förster resonance energy transfer with previous experimental observations, and obtained a good correspondence. Finally, we simulated the binding behavior of a fusion of two ligands that could simultaneously bind to distinct cell-surface receptors, and explored the landscape of linker lengths and stiffnesses that could enhance receptor binding of one ligand when the other ligand has already bound to its receptor, thus, addressing potential mechanisms for improving targeted signal transduction proteins. These specific results have implications for the design of targeted fusion proteins and artificial transcription factors involving fusion of natural domains. More broadly, the simulation framework described here could be extended to include more detailed system features such as non-spherical protein shapes and electrostatics, without requiring detailed, computationally expensive specifications. This framework should be useful in predicting behavior of engineered protein systems including binding and dissociation reactions. PMID:23822508

Defining the consequences of genetic variation on a proteome–wide scale

PubMed Central

Chick, Joel M.; Munger, Steven C.; Simecek, Petr; Huttlin, Edward L.; Choi, Kwangbom; Gatti, Daniel M.; Raghupathy, Narayanan; Svenson, Karen L.; Churchill, Gary A.; Gygi, Steven P.

2016-01-01

Genetic variation modulates protein expression through both transcriptional and post-transcriptional mechanisms. To characterize the consequences of natural genetic diversity on the proteome, here we combine a multiplexed, mass spectrometry-based method for protein quantification with an emerging outbred mouse model containing extensive genetic variation from eight inbred founder strains. By measuring genome-wide transcript and protein expression in livers from 192 Diversity outbred mice, we identify 2,866 protein quantitative trait loci (pQTL) with twice as many local as distant genetic variants. These data support distinct transcriptional and post-transcriptional models underlying the observed pQTL effects. Using a sensitive approach to mediation analysis, we often identified a second protein or transcript as the causal mediator of distant pQTL. Our analysis reveals an extensive network of direct protein–protein interactions. Finally, we show that local genotype can provide accurate predictions of protein abundance in an independent cohort of collaborative cross mice. PMID:27309819

Measles virus minigenomes encoding two autofluorescent proteins reveal cell-to-cell variation in reporter expression dependent on viral sequences between the transcription units.

PubMed

Rennick, Linda J; Duprex, W Paul; Rima, Bert K

2007-10-01

Transcription from morbillivirus genomes commences at a single promoter in the 3' non-coding terminus, with the six genes being transcribed sequentially. The 3' and 5' untranslated regions (UTRs) of the genes (mRNA sense), together with the intergenic trinucleotide spacer, comprise the non-coding sequences (NCS) of the virus and contain the conserved gene end and gene start signals, respectively. Bicistronic minigenomes containing transcription units (TUs) encoding autofluorescent reporter proteins separated by measles virus (MV) NCS were used to give a direct estimation of gene expression in single, living cells by assessing the relative amounts of each fluorescent protein in each cell. Initially, five minigenomes containing each of the MV NCS were generated. Assays were developed to determine the amount of each fluorescent protein in cells at both cell population and single-cell levels. This revealed significant variations in gene expression between cells expressing the same NCS-containing minigenome. The minigenome containing the M/F NCS produced significantly lower amounts of fluorescent protein from the second TU (TU2), compared with the other minigenomes. A minigenome with a truncated F 5' UTR had increased expression from TU2. This UTR is 524 nt longer than the other MV 5' UTRs. Insertions into the 5' UTR of the enhanced green fluorescent protein gene in the minigenome containing the N/P NCS showed that specific sequences, rather than just the additional length of F 5' UTR, govern this decreased expression from TU2.

High-efficiency generation of induced pluripotent mesenchymal stem cells from human dermal fibroblasts using recombinant proteins.

PubMed

Chen, Fanfan; Zhang, Guoqiang; Yu, Ling; Feng, Yanye; Li, Xianghui; Zhang, Zhijun; Wang, Yongting; Sun, Dapeng; Pradhan, Sriharsa

2016-07-30

Induced pluripotent mesenchymal stem cells (iPMSCs) are novel candidates for drug screening, regenerative medicine, and cell therapy. However, introduction of transcription factor encoding genes for induced pluripotent stem cell (iPSC) generation which could be used to generate mesenchymal stem cells is accompanied by the risk of insertional mutations in the target cell genome. We demonstrate a novel method using an inactivated viral particle to package and deliver four purified recombinant Yamanaka transcription factors (Sox2, Oct4, Klf4, and c-Myc) resulting in reprogramming of human primary fibroblasts. Whole genome bisulfite sequencing was used to analyze genome-wide CpG methylation of human iPMSCs. Western blot, quantitative PCR, immunofluorescence, and in-vitro differentiation were used to assess the pluripotency of iPMSCs. The resulting reprogrammed fibroblasts show high-level expression of stem cell markers. The human fibroblast-derived iPMSC genome showed gains in DNA methylation in low to medium methylated regions and concurrent loss of methylation in previously hypermethylated regions. Most of the differentially methylated regions are close to transcription start sites and many of these genes are pluripotent pathway associated. We found that DNA methylation of these genes is regulated by the four iPSC transcription factors, which functions as an epigenetic switch during somatic reprogramming as reported previously. These iPMSCs successfully differentiate into three embryonic germ layer cells, both in vitro and in vivo. Following multipotency induction in our study, the delivered transcription factors were degraded, leading to an improved efficiency of subsequent programmed differentiation. Recombinant transcription factor based reprogramming and derivatization of iPMSC offers a novel high-efficiency approach for regenerative medicine from patient-derived cells.

Late-onset spastic paraplegia: Aberrant SPG11 transcripts generated by a novel splice site donor mutation.

PubMed

Kawarai, Toshitaka; Miyamoto, Ryosuke; Mori, Atsuko; Oki, Ryosuke; Tsukamoto-Miyashiro, Ai; Matsui, Naoko; Miyazaki, Yoshimichi; Orlacchio, Antonio; Izumi, Yuishin; Nishida, Yoshihiko; Kaji, Ryuji

2015-12-15

We identified a novel homozygous mutation in the splice site donor (SSD) of intron 30 (c.5866+1G>A) in consanguineous Japanese SPG11 siblings showing late-onset spastic paraplegia using the whole-exome sequencing. Phenotypic variability was observed, including age-at-onset, dysarthria and pes cavus. Coding DNA sequencing revealed that the mutation affected the recognition of the constitutive SSD of intron 30, splicing upstream onto a nearby cryptic SSD in exon 30. The use of constitutive splice sites of intron 29 was confirmed by sequencing. The mutant transcripts are mostly subject to degradation by the nonsense-mediated mRNA decay system. SPG11 transcripts, escaping from the nonsense-mediated mRNA decay pathway, would generate a truncated protein (p.Tyr1900Phefs5X) containing the first 1899 amino acids and followed by 4 aberrant amino acids. This study showed a successful clinical application of whole-exome sequencing in spastic paraplegia and demonstrated a further evidence of allelic heterogeneity in SPG11. The confirmation of aberrant transcript by splice site mutation is a prerequisite for a more precise molecular diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Efficiency of VIGS and gene expression in a novel bipartite potexvirus vector delivery system as a function of strength of TGB1 silencing suppression.

PubMed

Lim, Hyoun-Sub; Vaira, Anna Maria; Domier, Leslie L; Lee, Sung Chul; Kim, Hong Gi; Hammond, John

2010-06-20

We have developed plant virus-based vectors for virus-induced gene silencing (VIGS) and protein expression, based on Alternanthera mosaic virus (AltMV), for infection of a wide range of host plants including Nicotiana benthamiana and Arabidopsis thaliana by either mechanical inoculation of in vitro transcripts or via agroinfiltration. In vivo transcripts produced by co-agroinfiltration of bacteriophage T7 RNA polymerase resulted in T7-driven AltMV infection from a binary vector in the absence of the Cauliflower mosaic virus 35S promoter. An artificial bipartite viral vector delivery system was created by separating the AltMV RNA-dependent RNA polymerase and Triple Gene Block (TGB)123-Coat protein (CP) coding regions into two constructs each bearing the AltMV 5' and 3' non-coding regions, which recombined in planta to generate a full-length AltMV genome. Substitution of TGB1 L(88)P, and equivalent changes in other potexvirus TGB1 proteins, affected RNA silencing suppression efficacy and suitability of the vectors from protein expression to VIGS. Published by Elsevier Inc.

P-class pentatricopeptide repeat proteins are required for efficient 5′ end formation of plant mitochondrial transcripts

PubMed Central

Binder, Stefan; Stoll, Katrin; Stoll, Birgit

2013-01-01

It is well recognized that flowering plants maintain a particularly broad spectrum of factors to support gene expression in mitochondria. Many of these factors are pentatricopeptide repeat (PPR) proteins that participate in virtually all processes dealing with RNA. One of these processes is the post-transcriptional generation of mature 5′ termini of RNA. Several PPR proteins are required for efficient 5′ maturation of mitochondrial mRNA and rRNA. These so-called RNA PROCESSING FACTORs (RPF) exclusively represent P-class PPR proteins, mainly composed of canonical PPR motifs without any extra domains. Applying the recent PPR-nucleotide recognition code, binding sites of RPF are predicted on the 5′ leader sequences. The sequence-specific interaction of an RPF with one or a few RNA substrates probably directly or indirectly recruits an as-yet-unidentified endonuclease to the processing site(s). The identification and characterization of RPF is a major step toward the understanding of the role of 5′ end maturation in flowering plant mitochondria. PMID:24184847

Islet-to-LMO stoichiometries control the function of transcription complexes that specify motor neuron and V2a interneuron identity

PubMed Central

Song, Mi-Ryoung; Sun, Yunfu; Bryson, Ami; Gill, Gordon N.; Evans, Sylvia M.; Pfaff, Samuel L.

2009-01-01

Summary LIM transcription factors bind to nuclear LIM interactor (Ldb/NLI/Clim) in specific ratios to form higher-order complexes that regulate gene expression. Here we examined how the dosage of LIM homeodomain proteins Isl1 and Isl2 and LIM-only protein Lmo4 influences the assembly and function of complexes involved in the generation of spinal motor neurons (MNs) and V2a interneurons (INs). Reducing the levels of Islet proteins using a graded series of mutations favored V2a IN differentiation at the expense of MN formation. Although LIM-only proteins (LMOs) are predicted to antagonize the function of Islet proteins, we found that the presence or absence of Lmo4 had little influence on MN or V2a IN specification. We did find, however, that the loss of MNs resulting from reduced Islet levels was rescued by eliminating Lmo4, unmasking a functional interaction between these proteins. Our findings demonstrate that MN and V2a IN fates are specified by distinct complexes that are sensitive to the relative stoichiometries of the constituent factors and we present a model to explain how LIM domain proteins modulate these complexes and, thereby, this binary-cell-fate decision. PMID:19666821

Highly efficient intracellular transduction in three-dimensional gradients for programming cell fate.

PubMed

Eltaher, Hoda M; Yang, Jing; Shakesheff, Kevin M; Dixon, James E

2016-09-01

Fundamental behaviour such as cell fate, growth and death are mediated through the control of key genetic transcriptional regulators. These regulators are activated or repressed by the integration of multiple signalling molecules in spatio-temporal gradients. Engineering these gradients is complex but considered key in controlling tissue formation in regenerative medicine approaches. Direct programming of cells using exogenously delivered transcription factors can by-pass growth factor complexity but there is still a requirement to deliver such activity spatio-temporally. We previously developed a technology termed GAG-binding enhanced transduction (GET) to efficiently deliver a variety of cargoes intracellularly using GAG-binding domains to promote cell targeting, and cell penetrating peptides (CPPs) to allow cell entry. Herein we demonstrate that GET can be used in a three dimensional (3D) hydrogel matrix to produce gradients of intracellular transduction of mammalian cells. Using a compartmentalised diffusion model with a source-gel-sink (So-G-Si) assembly, we created gradients of reporter proteins (mRFP1-tagged) and a transcription factor (TF, myogenic master regulator MyoD) and showed that GET can be used to deliver molecules into cells spatio-temporally by monitoring intracellular transduction and gene expression programming as a function of location and time. The ability to spatio-temporally control the intracellular delivery of functional proteins will allow the establishment of gradients of cell programming in hydrogels and approaches to direct cellular behaviour for many regenerative medicine applications. Regenerative medicine aims to reform functional biological tissues by controlling cell behaviour. Growth factors (GFs) are soluble cues presented to cells in spatio-temporal gradients and play important roles programming cell fate and gene expression. The efficient transduction of cells by GET (Glycosaminoglycan-enhanced transducing)-tagged transcription factors (TFs) can be used to by-pass GF-stimulation and directly program cells. For the first time we demonstrate diffusion of GET proteins generate stable protein transduction gradients. We demonstrated the feasibility of creating spatio-temporal gradients of GET-MyoD and show differential programing of myogenic differentiation. We believe that GET could provide a powerful tool to program cell behaviour using gradients of recombinant proteins that allow tissue generation directly by programming gene expression with TFs. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Comparative analysis of response to selection with three insecticides in the dengue mosquito Aedes aegypti using mRNA sequencing

PubMed Central

2014-01-01

Background Mosquito control programmes using chemical insecticides are increasingly threatened by the development of resistance. Such resistance can be the consequence of changes in proteins targeted by insecticides (target site mediated resistance), increased insecticide biodegradation (metabolic resistance), altered transport, sequestration or other mechanisms. As opposed to target site resistance, other mechanisms are far from being fully understood. Indeed, insecticide selection often affects a large number of genes and various biological processes can hypothetically confer resistance. In this context, the aim of the present study was to use RNA sequencing (RNA-seq) for comparing transcription level and polymorphism variations associated with adaptation to chemical insecticides in the mosquito Aedes aegypti. Biological materials consisted of a parental susceptible strain together with three child strains selected across multiple generations with three insecticides from different classes: the pyrethroid permethrin, the neonicotinoid imidacloprid and the carbamate propoxur. Results After ten generations, insecticide-selected strains showed elevated resistance levels to the insecticides used for selection. RNA-seq data allowed detecting over 13,000 transcripts, of which 413 were differentially transcribed in insecticide-selected strains as compared to the susceptible strain. Among them, a significant enrichment of transcripts encoding cuticle proteins, transporters and enzymes was observed. Polymorphism analysis revealed over 2500 SNPs showing > 50% allele frequency variations in insecticide-selected strains as compared to the susceptible strain, affecting over 1000 transcripts. Comparing gene transcription and polymorphism patterns revealed marked differences among strains. While imidacloprid selection was linked to the over transcription of many genes, permethrin selection was rather linked to polymorphism variations. Focusing on detoxification enzymes revealed that permethrin selection strongly affected the polymorphism of several transcripts encoding cytochrome P450 monooxygenases likely involved in insecticide biodegradation. Conclusions The present study confirmed the power of RNA-seq for identifying concomitantly quantitative and qualitative transcriptome changes associated with insecticide resistance in mosquitoes. Our results suggest that transcriptome modifications can be selected rapidly by insecticides and affect multiple biological functions. Previously neglected by molecular screenings, polymorphism variations of detoxification enzymes may play an important role in the adaptive response of mosquitoes to insecticides. PMID:24593293

Comparative analysis of response to selection with three insecticides in the dengue mosquito Aedes aegypti using mRNA sequencing.

PubMed

David, Jean-Philippe; Faucon, Frédéric; Chandor-Proust, Alexia; Poupardin, Rodolphe; Riaz, Muhammad Asam; Bonin, Aurélie; Navratil, Vincent; Reynaud, Stéphane

2014-03-05

Mosquito control programmes using chemical insecticides are increasingly threatened by the development of resistance. Such resistance can be the consequence of changes in proteins targeted by insecticides (target site mediated resistance), increased insecticide biodegradation (metabolic resistance), altered transport, sequestration or other mechanisms. As opposed to target site resistance, other mechanisms are far from being fully understood. Indeed, insecticide selection often affects a large number of genes and various biological processes can hypothetically confer resistance. In this context, the aim of the present study was to use RNA sequencing (RNA-seq) for comparing transcription level and polymorphism variations associated with adaptation to chemical insecticides in the mosquito Aedes aegypti. Biological materials consisted of a parental susceptible strain together with three child strains selected across multiple generations with three insecticides from different classes: the pyrethroid permethrin, the neonicotinoid imidacloprid and the carbamate propoxur. After ten generations, insecticide-selected strains showed elevated resistance levels to the insecticides used for selection. RNA-seq data allowed detecting over 13,000 transcripts, of which 413 were differentially transcribed in insecticide-selected strains as compared to the susceptible strain. Among them, a significant enrichment of transcripts encoding cuticle proteins, transporters and enzymes was observed. Polymorphism analysis revealed over 2500 SNPs showing > 50% allele frequency variations in insecticide-selected strains as compared to the susceptible strain, affecting over 1000 transcripts. Comparing gene transcription and polymorphism patterns revealed marked differences among strains. While imidacloprid selection was linked to the over transcription of many genes, permethrin selection was rather linked to polymorphism variations. Focusing on detoxification enzymes revealed that permethrin selection strongly affected the polymorphism of several transcripts encoding cytochrome P450 monooxygenases likely involved in insecticide biodegradation. The present study confirmed the power of RNA-seq for identifying concomitantly quantitative and qualitative transcriptome changes associated with insecticide resistance in mosquitoes. Our results suggest that transcriptome modifications can be selected rapidly by insecticides and affect multiple biological functions. Previously neglected by molecular screenings, polymorphism variations of detoxification enzymes may play an important role in the adaptive response of mosquitoes to insecticides.

A transgenerational role of the germline nuclear RNAi pathway in repressing heat stress-induced transcriptional activation in C. elegans.

PubMed

Ni, Julie Zhouli; Kalinava, Natallia; Chen, Esteban; Huang, Alex; Trinh, Thi; Gu, Sam Guoping

2016-01-01

Environmental stress-induced transgenerational epigenetic effects have been observed in various model organisms and human. The capacity and mechanism of such phenomena are poorly understood. In C. elegans, siRNA mediates transgenerational gene silencing through the germline nuclear RNAi pathway. This pathway is also required to maintain the germline immortality when C. elegans is under heat stress. However, the underlying molecular mechanism is unknown. In this study, we investigated the impact of heat stress on chromatin, transcription, and siRNAs at the whole-genome level, and whether any of the heat-induced effects is transgenerationally heritable in either the wild-type or the germline nuclear RNAi mutant animals. We performed 12-generation temperature-shift experiments using the wild-type C. elegans and a mutant strain that lacks the germline-specific nuclear Argonaute protein HRDE-1/WAGO-9. By examining the mRNA, small RNA, RNA polymerase II, and H3K9 trimethylation profiles at the whole-genome level, we revealed an epigenetic role of HRDE-1 in repressing heat stress-induced transcriptional activation of over 280 genes. Many of these genes are in or near LTR (long-terminal repeat) retrotransposons. Strikingly, for some of these genes, the heat stress-induced transcriptional activation in the hrde-1 mutant intensifies in the late generations under the heat stress and is heritable for at least two generations after the mutant animals are shifted back to lower temperature. hrde-1 mutation also leads to siRNA expression changes of many genes. This effect on siRNA is dependent on both the temperature and generation. Our study demonstrated that a large number of the endogenous targets of the germline nuclear RNAi pathway in C. elegans are sensitive to heat-induced transcriptional activation. This effect at certain genomic loci including LTR retrotransposons is transgenerational. Germline nuclear RNAi antagonizes this temperature effect at the transcriptional level and therefore may play a key role in heat stress response in C. elegans.

Transcriptome-Wide Identification of Preferentially Expressed Genes in the Hypothalamus and Pituitary Gland

PubMed Central

St-Amand, Jonny; Yoshioka, Mayumi; Tanaka, Keitaro; Nishida, Yuichiro

2012-01-01

To identify preferentially expressed genes in the central endocrine organs of the hypothalamus and pituitary gland, we generated transcriptome-wide mRNA profiles of the hypothalamus, pituitary gland, and parietal cortex in male mice (12–15 weeks old) using serial analysis of gene expression (SAGE). Total counts of SAGE tags for the hypothalamus, pituitary gland, and parietal cortex were 165824, 126688, and 161045 tags, respectively. This represented 59244, 45151, and 55131 distinct tags, respectively. Comparison of these mRNA profiles revealed that 22 mRNA species, including three potential novel transcripts, were preferentially expressed in the hypothalamus. In addition to well-known hypothalamic transcripts, such as hypocretin, several genes involved in hormone function, intracellular transduction, metabolism, protein transport, steroidogenesis, extracellular matrix, and brain disease were identified as preferentially expressed hypothalamic transcripts. In the pituitary gland, 106 mRNA species, including 60 potential novel transcripts, were preferentially expressed. In addition to well-known pituitary genes, such as growth hormone and thyroid stimulating hormone beta, a number of genes classified to function in transport, amino acid metabolism, intracellular transduction, cell adhesion, disulfide bond formation, stress response, transcription, protein synthesis, and turnover, cell differentiation, the cell cycle, and in the cytoskeleton and extracellular matrix were also preferentially expressed. In conclusion, the current study identified not only well-known hypothalamic and pituitary transcripts but also a number of new candidates likely to be involved in endocrine homeostatic systems regulated by the hypothalamus and pituitary gland. PMID:22649398

Transcriptome-wide identification of preferentially expressed genes in the hypothalamus and pituitary gland.

PubMed

St-Amand, Jonny; Yoshioka, Mayumi; Tanaka, Keitaro; Nishida, Yuichiro

2011-01-01

To identify preferentially expressed genes in the central endocrine organs of the hypothalamus and pituitary gland, we generated transcriptome-wide mRNA profiles of the hypothalamus, pituitary gland, and parietal cortex in male mice (12-15 weeks old) using serial analysis of gene expression (SAGE). Total counts of SAGE tags for the hypothalamus, pituitary gland, and parietal cortex were 165824, 126688, and 161045 tags, respectively. This represented 59244, 45151, and 55131 distinct tags, respectively. Comparison of these mRNA profiles revealed that 22 mRNA species, including three potential novel transcripts, were preferentially expressed in the hypothalamus. In addition to well-known hypothalamic transcripts, such as hypocretin, several genes involved in hormone function, intracellular transduction, metabolism, protein transport, steroidogenesis, extracellular matrix, and brain disease were identified as preferentially expressed hypothalamic transcripts. In the pituitary gland, 106 mRNA species, including 60 potential novel transcripts, were preferentially expressed. In addition to well-known pituitary genes, such as growth hormone and thyroid stimulating hormone beta, a number of genes classified to function in transport, amino acid metabolism, intracellular transduction, cell adhesion, disulfide bond formation, stress response, transcription, protein synthesis, and turnover, cell differentiation, the cell cycle, and in the cytoskeleton and extracellular matrix were also preferentially expressed. In conclusion, the current study identified not only well-known hypothalamic and pituitary transcripts but also a number of new candidates likely to be involved in endocrine homeostatic systems regulated by the hypothalamus and pituitary gland.

Generation of the bovine viral diarrhea virus e0 protein in transgenic astragalus and its immunogenicity in sika deer.

PubMed

Gao, Yugang; Zhao, Xueliang; Zang, Pu; Liu, Qun; Wei, Gongqing; Zhang, Lianxue

2014-01-01

The bovine viral diarrhea virus (BVDV), a single-stranded RNA virus, can cause fatal diarrhea syndrome, respiratory problems, and reproductive disorders in herds. Over the past few years, it has become clear that the BVDV infection rates are increasing and it is likely that an effective vaccine for BVDV will be needed. In this study, transgenic Astragalus was used as an alternative productive platform for the expression of glycoprotein E0. The immunogenicity of glycoprotein E0 expressed in transgenic Astragalus was detected in deer. The presence of pBI121-E0 was confirmed by polymerase chain reaction (PCR), transcription was verified by reverse transcription- (RT-) PCR, and recombinant protein expression was confirmed by ELISA and Western blot analyses. Deer that were immunized subcutaneously with the transgenic plant vaccine developed specific humoral and cell-mediated immune responses against BVDV. This study provides a new method for a protein with weak immunogenicity to be used as part of a transgenic plant vaccine.

Generation of the Bovine Viral Diarrhea Virus E0 Protein in Transgenic Astragalus and Its Immunogenicity in Sika Deer

PubMed Central

Gao, Yugang; Zang, Pu; Liu, Qun; Wei, Gongqing

2014-01-01

The bovine viral diarrhea virus (BVDV), a single-stranded RNA virus, can cause fatal diarrhea syndrome, respiratory problems, and reproductive disorders in herds. Over the past few years, it has become clear that the BVDV infection rates are increasing and it is likely that an effective vaccine for BVDV will be needed. In this study, transgenic Astragalus was used as an alternative productive platform for the expression of glycoprotein E0. The immunogenicity of glycoprotein E0 expressed in transgenic Astragalus was detected in deer. The presence of pBI121-E0 was confirmed by polymerase chain reaction (PCR), transcription was verified by reverse transcription- (RT-) PCR, and recombinant protein expression was confirmed by ELISA and Western blot analyses. Deer that were immunized subcutaneously with the transgenic plant vaccine developed specific humoral and cell-mediated immune responses against BVDV. This study provides a new method for a protein with weak immunogenicity to be used as part of a transgenic plant vaccine. PMID:24963321

Annexin A2-S100A10 heterotetramer is upregulated by PML/RARα fusion protein and promotes plasminogen-dependent fibrinolysis and matrix invasion in acute promyelocytic leukemia.

PubMed

Huang, Dan; Yang, Yan; Sun, Jian; Dong, Xiaorong; Wang, Jiao; Liu, Hongchen; Lu, Chengquan; Chen, Xueyu; Shao, Jing; Yan, Jinsong

2017-09-01

Aberrant expression of annexin A2-S100A10 heterotetramer (AIIt) associated with PML/RARα fusion protein causes lethal hyperfibrinolysis in acute promyelocytic leukemia (APL), but the mechanism is unclear. To facilitate the investigation of regulatory association between ANXA2 and promyelocytic leukemia/retinoic acid receptor a (PML/RARα) fusion protein, this work was performed to determine the transcription start site of ANXA2 promoter with rapid amplification of 5'-cDNA ends analysis. Zinc-induced U937/PR9 cells expressed PML/RARα fusion protein, and resultant increases in ANXA2 transcripts and translational expressions of both ANXA2 and S100A10, while S100A10 transcripts remained constitutive. The transactivation of ANXA2 promoter by PML/RARα fusion protein was 3.29 ± 0.13 fold higher than that by control pSG5 vector or wild-type RARα. The overexpression of ANXA2 in U937 transfected with full-length ANXA2 cDNA was associated with increased S100A10 subunit, although S100A10 transcripts remained constitutive. The tPA-dependent initial rate of plasmin generation (IRPG) in zinc-treated U937/PR9 increased by 2.13-fold, and cell invasiveness increased by 27.6%. Antibodies against ANXA2, S100A10, or combination of both all remarkably inhibited the IRPG and invasiveness in U937/PR9 and NB4. Treatment of zinc-induced U937/PR9 or circulating APL blasts with all-trans retinoic acid (ATRA) significantly reduced cell surface ANXA2 and S100A10 and associated reductions in IRPG and invasiveness. Thus, PML/RARα fusion protein transactivated the ANXA2 promoter to upregulate ANXA2 and accumulate S100A10. Increased AIIt promoted IRPG and invasiveness, both of which were partly abolished by antibodies against ANXA2 and S100A10 or by ATRA.

Identification of a New Human Adenovirus Protein Encoded by a Novel Late l-Strand Transcription Unit▿

PubMed Central

Tollefson, Ann E.; Ying, Baoling; Doronin, Konstantin; Sidor, Peter D.; Wold, William S. M.

2007-01-01

A short open reading frame named the “U exon,” located on the adenovirus (Ad) l-strand (for leftward transcription) between the early E3 region and the fiber gene, is conserved in mastadenoviruses. We have observed that Ad5 mutants with large deletions in E3 that infringe on the U exon display a mild growth defect, as well as an aberrant Ad E2 DNA-binding protein (DBP) intranuclear localization pattern and an apparent failure to organize replication centers during late infection. Mutants in which the U exon DNA is reconstructed have a reversed phenotype. Chow et al. (L. T. Chow et al., J. Mol. Biol. 134:265-303, 1979) described mRNAs initiating in the region of the U exon and spliced to downstream sequences in the late DBP mRNA leader and the DBP-coding region. We have cloned this mRNA (as cDNA) from Ad5 late mRNA; the predicted protein is 217 amino acids, initiating in the U exon and continuing in frame in the DBP leader and in the DBP-coding region but in a different reading frame from DBP. Polyclonal and monoclonal antibodies generated against the predicted U exon protein (UXP) showed that UXP is ∼24K in size by immunoblot and is a late protein. At 18 to 24 h postinfection, UXP is strongly associated with nucleoli and is found throughout the nucleus; later, UXP is associated with the periphery of replication centers, suggesting a function relevant to Ad DNA replication or RNA transcription. UXP is expressed by all four species C Ads. When expressed in transient transfections, UXP complements the aberrant DBP localization pattern of UXP-negative Ad5 mutants. Our data indicate that UXP is a previously unrecognized protein derived from a novel late l-strand transcription unit. PMID:17881437

Rsr1 Focuses Cdc42 Activity at Hyphal Tips and Promotes Maintenance of Hyphal Development in Candida albicans

PubMed Central

Pulver, Rebecca; Heisel, Timothy; Gonia, Sara; Robins, Robert; Norton, Jennifer; Haynes, Paula

2013-01-01

The extremely elongated morphology of fungal hyphae is dependent on the cell's ability to assemble and maintain polarized growth machinery over multiple cell cycles. The different morphologies of the fungus Candida albicans make it an excellent model organism in which to study the spatiotemporal requirements for constitutive polarized growth and the generation of different cell shapes. In C. albicans, deletion of the landmark protein Rsr1 causes defects in morphogenesis that are not predicted from study of the orthologous protein in the related yeast Saccharomyces cerevisiae, thus suggesting that Rsr1 has expanded functions during polarized growth in C. albicans. Here, we show that Rsr1 activity localizes to hyphal tips by the differential localization of the Rsr1 GTPase-activating protein (GAP), Bud2, and guanine nucleotide exchange factor (GEF), Bud5. In addition, we find that Rsr1 is needed to maintain the focused localization of hyphal polarity structures and proteins, including Bem1, a marker of the active GTP-bound form of the Rho GTPase, Cdc42. Further, our results indicate that tip-localized Cdc42 clusters are associated with the cell's ability to express a hyphal transcriptional program and that the ability to generate a focused Cdc42 cluster in early hyphae (germ tubes) is needed to maintain hyphal morphogenesis over time. We propose that in C. albicans, Rsr1 “fine-tunes” the distribution of Cdc42 activity and that self-organizing (Rsr1-independent) mechanisms of polarized growth are not sufficient to generate narrow cell shapes or to provide feedback to the transcriptional program during hyphal morphogenesis. PMID:23223038

Alteration of lysine 178 in the hinge region of the Escherichia coli ada protein interferes with activation of ada, but not alkA, transcription.

PubMed

Saget, B M; Shevell, D E; Walker, G C

1995-03-01

The ada gene of Escherichia coli K-12 encodes the 39-kDa Ada protein, which consists of two domains joined by a hinge region that is sensitive to proteolytic cleavage in vitro. The amino-terminal domain has a DNA methyltransferase activity that repairs the S-diastereoisomer of methylphosphotriesters while the carboxyl-terminal domain has a DNA methyltransferase activity that repairs O6-methylguanine and O4-methylthymine lesions. Transfer of a methyl group to Cys-69 by repair of a methylphosphotriester lesion converts Ada into a transcriptional activator of the ada and alkA genes. Activation of ada, but not alkA, requires elements contained within the carboxyl-terminal domain of Ada. In addition, physiologically relevant concentrations of the unmethylated form of Ada specifically inhibit methylated Ada-promoted ada transcription both in vitro and in vivo and it has been suggested that this phenomenon plays a pivotal role in the down-regulation of the adaptive response. A set of site-directed mutations were generated within the hinge region, changing the lysine residue at position 178 to leucine, valine, glycine, tyrosine, arginine, cysteine, proline, and serine. All eight mutant proteins have deficiencies in their ability to activate ada transcription in the presence or absence of a methylating agent but are proficient in alkA activation. AdaK178P (lysine 178 changed to proline) is completely defective for the transcriptional activation function of ada while it is completely proficient for transcriptional activation of alkA. In addition, AdaK178P possesses both classes of DNA repair activities both in vitro and in vivo. Transcriptional activation of ada does not occur if both the amino- and carboxyl-terminal domains are produced separately within the same cell. The mutation at position 178 might interfere with activation of ada transcription by changing a critical contact with RNA polymerase, by causing a conformational change of Ada, or by interfering with the communication of conformational information between the amino- and the carboxyl-terminal domains. These results indicate that the hinge region of Ada is important for ada but not alkA transcription and further support the notion that the mechanism(s) by which Ada activates ada transcription differs from that by which it activates transcription at alkA.

Sumoylation of CCAAT/enhancer-binding protein α is implicated in hematopoietic stem/progenitor cell development through regulating runx1 in zebrafish.

PubMed

Yuan, Hao; Zhang, Tao; Liu, Xiaohui; Deng, Min; Zhang, Wenqing; Wen, Zilong; Chen, Saijuan; Chen, Zhu; de The, Hugues; Zhou, Jun; Zhu, Jun

2015-03-11

The small ubiquitin-related modifier (SUMO) participates in various cellular processes, including maintenance of genome integrity, nuclear transport, transcription and signal transduction. However, the biological function of sumoylation in hematopoiesis has not been fully explored. We show here that definitive hematopoietic stem/progenitor cells (HSPCs) are depleted in SUMO-deficient zebrafish embryos. Impairment of sumoylation attenuates HSPC generation and proliferation. The hyposumoylation triggered HSPC defects are CCAAT/enhancer-binding protein α (C/ebpα) dependent. Critically, a SUMO-C/ebpα fusion rescues the defective hematopoiesis in SUMO-deficient embryos, at least in part through restored runx1 expression. While C/ebpα-dependent transcription is involved in myeloid differentiation, our studies here reveal that C/ebpα sumoylation is essential for HSPC development during definitive hematopoiesis.

Sumoylation of CCAAT/enhancer-binding protein α is implicated in hematopoietic stem/progenitor cell development through regulating runx1 in zebrafish

PubMed Central

Yuan, Hao; Zhang, Tao; Liu, Xiaohui; Deng, Min; Zhang, Wenqing; Wen, Zilong; Chen, Saijuan; Chen, Zhu; de The, Hugues; Zhou, Jun; Zhu, Jun

2015-01-01

The small ubiquitin-related modifier (SUMO) participates in various cellular processes, including maintenance of genome integrity, nuclear transport, transcription and signal transduction. However, the biological function of sumoylation in hematopoiesis has not been fully explored. We show here that definitive hematopoietic stem/progenitor cells (HSPCs) are depleted in SUMO-deficient zebrafish embryos. Impairment of sumoylation attenuates HSPC generation and proliferation. The hyposumoylation triggered HSPC defects are CCAAT/enhancer-binding protein α (C/ebpα) dependent. Critically, a SUMO-C/ebpα fusion rescues the defective hematopoiesis in SUMO-deficient embryos, at least in part through restored runx1 expression. While C/ebpα-dependent transcription is involved in myeloid differentiation, our studies here reveal that C/ebpα sumoylation is essential for HSPC development during definitive hematopoiesis. PMID:25757417

Diversity and specificity: auxin perception and signaling through the TIR1/AFB pathway

DOE PAGES

Wang, Renhou; Estelle, Mark

2014-07-15

Auxin is a versatile plant hormone that plays an essential role in most aspects of plant growth and development. Auxin regulates various growth processes by modulating gene transcription through a SCF TIR1/AFB-Aux/IAA-ARF nuclear signaling module. Recent work has generated clues as to how multiple layers of regulation of the auxin signaling components may result in diverse and specific response outputs. Finally, in particular, interaction and structural studies of key auxin signaling proteins have produced novel insights into the molecular basis of auxin-regulated transcription and may lead to a refined auxin signaling model.

Tapping the RNA world for therapeutics.

PubMed

Lieberman, Judy

2018-05-01

A recent revolution in RNA biology has led to the identification of new RNA classes with unanticipated functions, new types of RNA modifications, an unexpected multiplicity of alternative transcripts and widespread transcription of extragenic regions. This development in basic RNA biology has spawned a corresponding revolution in RNA-based strategies to generate new types of therapeutics. Here, I review RNA-based drug design and discuss barriers to broader applications and possible ways to overcome them. Because they target nucleic acids rather than proteins, RNA-based drugs promise to greatly extend the domain of 'druggable' targets beyond what can be achieved with small molecules and biologics.

Lab-on-a-chip in vitro compartmentalization technologies for protein studies.

PubMed

Zhu, Yonggang; Power, Barbara E

2008-01-01

In vitro compartmentalization (IVC) is a powerful tool for studying protein-protein reactions, due to its high capacity and the versatility of droplet technologies. IVC bridges the gap between chemistry and biology as it enables the incorporation of unnatural amino acids with modifications into biological systems, through protein transcription and translation reactions, in a cell-like microdrop environment. The quest for the ultimate chip for protein studies using IVC is the drive for the development of various microfluidic droplet technologies to enable these unusual biochemical reactions to occur. These techniques have been shown to generate precise microdrops with a controlled size. Various chemical and physical phenomena have been utilized for on-chip manipulation to allow the droplets to be generated, fused, and split. Coupled with detection techniques, droplets can be sorted and selected. These capabilities allow directed protein evolution to be carried out on a microchip. With further technological development of the detection module, factors such as addressable storage, transport and interfacing technologies, could be integrated and thus provide platforms for protein studies with high efficiency and accuracy that conventional laboratories cannot achieve.

Impact of agriculture on the selection of insecticide resistance in the malaria vector Anopheles gambiae: a multigenerational study in controlled conditions.

PubMed

Nkya, Theresia Estomih; Poupardin, Rodolphe; Laporte, Frederic; Akhouayri, Idir; Mosha, Franklin; Magesa, Stephen; Kisinza, William; David, Jean-Philippe

2014-10-16

Resistance of mosquitoes to insecticides is mainly attributed to their adaptation to vector control interventions. Although pesticides used in agriculture have been frequently mentioned as an additional force driving the selection of resistance, only a few studies were dedicated to validate this hypothesis and characterise the underlying mechanisms. While insecticide resistance is rising dramatically in Africa, deciphering how agriculture affects resistance is crucial for improving resistance management strategies. In this context, the multigenerational effect of agricultural pollutants on the selection of insecticide resistance was examined in Anopheles gambiae. An urban Tanzanian An. gambiae population displaying a low resistance level was used as a parental strain for a selection experiment across 20 generations. At each generation larvae were selected with a mixture containing pesticides and herbicides classically used in agriculture in Africa. The resistance levels of adults to deltamethrin, DDT and bendiocarb were compared between the selected and non-selected strains across the selection process together with the frequency of kdr mutations. A microarray approach was used for pinpointing transcription level variations selected by the agricultural pesticide mixture at the adult stage. A gradual increase of adult resistance to all insecticides was observed across the selection process. The frequency of the L1014S kdr mutation rose from 1.6% to 12.5% after 20 generations of selection. Microarray analysis identified 90 transcripts over-transcribed in the selected strain as compared to the parental and the non-selected strains. Genes encoding cuticle proteins, detoxification enzymes, proteins linked to neurotransmitter activity and transcription regulators were mainly affected. RT-qPCR transcription profiling of candidate genes across multiple generations supported their link with insecticide resistance. This study confirms the potency of agriculture in selecting for insecticide resistance in malaria vectors. We demonstrated that the recurrent exposure of larvae to agricultural pollutants can select for resistance mechanisms to vector control insecticides at the adult stage. Our data suggest that in addition to selected target-site resistance mutations, agricultural pollutants may also favor cuticle, metabolic and synaptic transmission-based resistance mechanisms. These results emphasize the need for integrated resistance management strategies taking into account agriculture activities.

Macaca specific exon creation event generates a novel ZKSCAN5 transcript.

PubMed

Kim, Young-Hyun; Choe, Se-Hee; Song, Bong-Seok; Park, Sang-Je; Kim, Myung-Jin; Park, Young-Ho; Yoon, Seung-Bin; Lee, Youngjeon; Jin, Yeung Bae; Sim, Bo-Woong; Kim, Ji-Su; Jeong, Kang-Jin; Kim, Sun-Uk; Lee, Sang-Rae; Park, Young-Il; Huh, Jae-Won; Chang, Kyu-Tae

2016-02-15

ZKSCAN5 (also known as ZFP95) is a zinc-finger protein belonging to the Krűppel family. ZKSCAN5 contains a SCAN box and a KRAB A domain and is proposed to play a distinct role during spermatogenesis. In humans, alternatively spliced ZKSCAN5 transcripts with different 5'-untranslated regions (UTRs) have been identified. However, investigation of our Macaca UniGene Database revealed novel alternative ZKSCAN5 transcripts that arose due to an exon creation event. Therefore, in this study, we identified the full-length sequences of ZKSCAN5 and its alternative transcripts in Macaca spp. Additionally, we investigated different nonhuman primate sequences to elucidate the molecular mechanism underlying the exon creation event. We analyzed the evolutionary features of the ZKSCAN5 transcripts by reverse transcription polymerase chain reaction (RT-PCR) and genomic PCR, and by sequencing various nonhuman primate DNA and RNA samples. The exon-created transcript was only detected in the Macaca lineage (crab-eating monkey and rhesus monkey). Full-length sequence analysis by rapid amplification of cDNA ends (RACE) identified ten full-length transcripts and four functional isoforms of ZKSCAN5. Protein sequence analyses revealed the presence of two groups of isoforms that arose because of differences in start-codon usage. Together, our results demonstrate that there has been specific selection for a discrete set of ZKSCAN5 variants in the Macaca lineage. Furthermore, study of this locus (and perhaps others) in Macaca spp. might facilitate our understanding of the evolutionary pressures that have shaped the mechanism of exon creation in primates. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Staying alive in adversity: transcriptome dynamics in the stress-resistant dauer larva.

PubMed

Holt, Suzan J

2006-10-01

In response to food depletion and overcrowding, the soil nematode Caenorhabditis elegans can arrest development and form an alternate third larval stage called the dauer. Though nonfeeding, the dauer larva is long lived and stress resistant. Metabolic and transcription rates are lowered but the transcriptome of the dauer is complex. In this study, distribution analysis of transcript profiles generated by Serial Analysis of Gene Expression (SAGE) in dauer larvae and in mixed developmental stages is presented. An inverse relationship was observed between frequency and abundance/copy number of SAGE tag types (transcripts) in both profiles. In the dauer profile, a relatively greater proportion of highly abundant transcripts was counterbalanced by a smaller fraction of low to moderately abundant transcripts. Comparisons of abundant tag counts between the two profiles revealed relative enrichment in the dauer profile of transcripts with predicted or known involvement in ribosome biogenesis and protein synthesis, membrane transport, and immune responses. Translation-coupled mRNA decay is proposed as part of an immune-like stress response in the dauer larva. An influence of genomic region on transcript level may reflect the coordination of transcription and mRNA turnover.

Engineering microbial phenotypes through rewiring of genetic networks

PubMed Central

Rodrigues, Rui T.L.; Lee, Sangjin; Haines, Matthew

2017-01-01

Abstract The ability to program cellular behaviour is a major goal of synthetic biology, with applications in health, agriculture and chemicals production. Despite efforts to build ‘orthogonal’ systems, interactions between engineered genetic circuits and the endogenous regulatory network of a host cell can have a significant impact on desired functionality. We have developed a strategy to rewire the endogenous cellular regulatory network of yeast to enhance compatibility with synthetic protein and metabolite production. We found that introducing novel connections in the cellular regulatory network enabled us to increase the production of heterologous proteins and metabolites. This strategy is demonstrated in yeast strains that show significantly enhanced heterologous protein expression and higher titers of terpenoid production. Specifically, we found that the addition of transcriptional regulation between free radical induced signalling and nitrogen regulation provided robust improvement of protein production. Assessment of rewired networks revealed the importance of key topological features such as high betweenness centrality. The generation of rewired transcriptional networks, selection for specific phenotypes, and analysis of resulting library members is a powerful tool for engineering cellular behavior and may enable improved integration of heterologous protein and metabolite pathways. PMID:28369627

Characterization of Bunyamwera virus S RNA that is transcribed and replicated by the L protein expressed from recombinant vaccinia virus.

PubMed

Jin, H; Elliott, R M

1993-03-01

Analysis of the 5' termini of Bunyamwera virus S segment mRNAs by cloning and sequence analysis revealed the presence of nonviral, heterogeneous sequences 12 to 17 bases long. This is similar to reports for other members of the family Bunyaviridae and is taken to indicate that mRNA transcription is primed by a "cap-snatching" mechanism. The 3' end of the Bunyamwera virus S mRNA was mapped, by using an RNase protection assay, to 100 to 110 nucleotides upstream of the 3' end of the template. Previously we reported expression of the Bunyamwera virus L (polymerase) protein by recombinant vaccinia virus and demonstrated that the recombinant L protein was functional in terms of RNA synthesis activity in a nucleocapsid transfection assay (H. Jin and R. M. Elliott, J. Virol. 65: 4182-4189, 1991). In the present study we further analyze the RNAs made by using this system and show that positive-sense RNAs contain 5' nonviral sequences. Hence the initiation of mRNA transcription by the recombinant L protein resembles that seen during authentic bunyavirus infection and suggests that the L protein has the endonuclease activity which generates the primers. Some of these positive-sense transcripts terminated at the mRNA termination site, but the majority read through to the end of the template. No primer sequences were found at the 5' terminal of negative-sense RNAs. The recombinant L protein was able to replicate negative-sense RNA supplied by transfected virion-derived nucleocapsids, and both positive- and negative-sense RNAs were synthesized. These results indicate that the recombinant L protein has both transcriptase and replicase activities.

Characterization of Bunyamwera virus S RNA that is transcribed and replicated by the L protein expressed from recombinant vaccinia virus.

PubMed Central

Jin, H; Elliott, R M

1993-01-01

Analysis of the 5' termini of Bunyamwera virus S segment mRNAs by cloning and sequence analysis revealed the presence of nonviral, heterogeneous sequences 12 to 17 bases long. This is similar to reports for other members of the family Bunyaviridae and is taken to indicate that mRNA transcription is primed by a "cap-snatching" mechanism. The 3' end of the Bunyamwera virus S mRNA was mapped, by using an RNase protection assay, to 100 to 110 nucleotides upstream of the 3' end of the template. Previously we reported expression of the Bunyamwera virus L (polymerase) protein by recombinant vaccinia virus and demonstrated that the recombinant L protein was functional in terms of RNA synthesis activity in a nucleocapsid transfection assay (H. Jin and R. M. Elliott, J. Virol. 65: 4182-4189, 1991). In the present study we further analyze the RNAs made by using this system and show that positive-sense RNAs contain 5' nonviral sequences. Hence the initiation of mRNA transcription by the recombinant L protein resembles that seen during authentic bunyavirus infection and suggests that the L protein has the endonuclease activity which generates the primers. Some of these positive-sense transcripts terminated at the mRNA termination site, but the majority read through to the end of the template. No primer sequences were found at the 5' terminal of negative-sense RNAs. The recombinant L protein was able to replicate negative-sense RNA supplied by transfected virion-derived nucleocapsids, and both positive- and negative-sense RNAs were synthesized. These results indicate that the recombinant L protein has both transcriptase and replicase activities. Images PMID:8437222

Effect of point mutations on Herbaspirillum seropedicae NifA activity.

PubMed

Aquino, B; Stefanello, A A; Oliveira, M A S; Pedrosa, F O; Souza, E M; Monteiro, R A; Chubatsu, L S

2015-08-01

NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

Effect of point mutations on Herbaspirillum seropedicae NifA activity

PubMed Central

Aquino, B.; Stefanello, A.A.; Oliveira, M.A.S.; Pedrosa, F.O.; Souza, E.M.; Monteiro, R.A.; Chubatsu, L.S.

2015-01-01

NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain. PMID:26176311

Opposite GC skews at the 5' and 3' ends of genes in unicellular fungi

PubMed Central

2011-01-01

Background GC-skews have previously been linked to transcription in some eukaryotes. They have been associated with transcription start sites, with the coding strand G-biased in mammals and C-biased in fungi and invertebrates. Results We show a consistent and highly significant pattern of GC-skew within genes of almost all unicellular fungi. The pattern of GC-skew is asymmetrical: the coding strand of genes is typically C-biased at the 5' ends but G-biased at the 3' ends, with intermediate skews at the middle of genes. Thus, the initiation, elongation, and termination phases of transcription are associated with different skews. This pattern influences the encoded proteins by generating differential usage of amino acids at the 5' and 3' ends of genes. These biases also affect fourfold-degenerate positions and extend into promoters and 3' UTRs, indicating that skews cannot be accounted by selection for protein function or translation. Conclusions We propose two explanations, the mutational pressure hypothesis, and the adaptive hypothesis. The mutational pressure hypothesis is that different co-factors bind to RNA pol II at different phases of transcription, producing different mutational regimes. The adaptive hypothesis is that cytidine triphosphate deficiency may lead to C-avoidance at the 3' ends of transcripts to control the flow of RNA pol II molecules and reduce their frequency of collisions. PMID:22208287

A ‘resource allocator’ for transcription based on a highly fragmented T7 RNA polymerase

PubMed Central

Segall-Shapiro, Thomas H; Meyer, Adam J; Ellington, Andrew D; Sontag, Eduardo D; Voigt, Christopher A

2014-01-01

Synthetic genetic systems share resources with the host, including machinery for transcription and translation. Phage RNA polymerases (RNAPs) decouple transcription from the host and generate high expression. However, they can exhibit toxicity and lack accessory proteins (σ factors and activators) that enable switching between different promoters and modulation of activity. Here, we show that T7 RNAP (883 amino acids) can be divided into four fragments that have to be co-expressed to function. The DNA-binding loop is encoded in a C-terminal 285-aa ‘σ fragment’, and fragments with different specificity can direct the remaining 601-aa ‘core fragment’ to different promoters. Using these parts, we have built a resource allocator that sets the core fragment concentration, which is then shared by multiple σ fragments. Adjusting the concentration of the core fragment sets the maximum transcriptional capacity available to a synthetic system. Further, positive and negative regulation is implemented using a 67-aa N-terminal ‘α fragment’ and a null (inactivated) σ fragment, respectively. The α fragment can be fused to recombinant proteins to make promoters responsive to their levels. These parts provide a toolbox to allocate transcriptional resources via different schemes, which we demonstrate by building a system which adjusts promoter activity to compensate for the difference in copy number of two plasmids. PMID:25080493

RHON1 mediates a Rho-like activity for transcription termination in plastids of Arabidopsis thaliana.

PubMed

Chi, Wei; He, Baoye; Manavski, Nikolay; Mao, Juan; Ji, Daili; Lu, Congming; Rochaix, Jean David; Meurer, Jörg; Zhang, Lixin

2014-12-01

Although transcription termination is essential to generate functional RNAs, its underlying molecular mechanisms are still poorly understood in plastids of vascular plants. Here, we show that the RNA binding protein RHON1 participates in transcriptional termination of rbcL (encoding large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase) in Arabidopsis thaliana. Inactivation of RHON1 leads to enhanced rbcL read-through transcription and to aberrant accD (encoding β-subunit of the acetyl-CoA carboxylase) transcriptional initiation, which may result from inefficient transcription termination of rbcL. RHON1 can bind to the mRNA as well as to single-stranded DNA of rbcL, displays an RNA-dependent ATPase activity, and terminates transcription of rbcL in vitro. These results suggest that RHON1 terminates rbcL transcription using an ATP-driven mechanism similar to that of Rho of Escherichia coli. This RHON1-dependent transcription termination occurs in Arabidopsis but not in rice (Oryza sativa) and appears to reflect a fundamental difference between plastomes of dicotyledonous and monocotyledonous plants. Our results point to the importance and significance of plastid transcription termination and provide insights into its machinery in an evolutionary context. © 2014 American Society of Plant Biologists. All rights reserved.

The Ebola Virus Nucleoprotein Recruits the Host PP2A-B56 Phosphatase to Activate Transcriptional Support Activity of VP30.

PubMed

Kruse, Thomas; Biedenkopf, Nadine; Hertz, Emil Peter Thrane; Dietzel, Erik; Stalmann, Gertrud; López-Méndez, Blanca; Davey, Norman E; Nilsson, Jakob; Becker, Stephan

2018-01-04

Transcription of the Ebola virus genome depends on the viral transcription factor VP30 in its unphosphorylated form, but the underlying molecular mechanism of VP30 dephosphorylation is unknown. Here we show that the Ebola virus nucleoprotein (NP) recruits the host PP2A-B56 protein phosphatase through a B56-binding LxxIxE motif and that this motif is essential for VP30 dephosphorylation and viral transcription. The LxxIxE motif and the binding site of VP30 in NP are in close proximity, and both binding sites are required for the dephosphorylation of VP30. We generate a specific inhibitor of PP2A-B56 and show that it suppresses Ebola virus transcription and infection. This work dissects the molecular mechanism of VP30 dephosphorylation by PP2A-B56, and it pinpoints this phosphatase as a potential target for therapeutic intervention. Copyright © 2017 Elsevier Inc. All rights reserved.

Transcriptional regulation of human eosinophil RNases by an evolutionary- conserved sequence motif in primate genome

PubMed Central

Wang, Hsiu-Yu; Chang, Hao-Teng; Pai, Tun-Wen; Wu, Chung-I; Lee, Yuan-Hung; Chang, Yen-Hsin; Tai, Hsiu-Ling; Tang, Chuan-Yi; Chou, Wei-Yao; Chang, Margaret Dah-Tsyr

2007-01-01

Background Human eosinophil-derived neurotoxin (edn) and eosinophil cationic protein (ecp) are members of a subfamily of primate ribonuclease (rnase) genes. Although they are generated by gene duplication event, distinct edn and ecp expression profile in various tissues have been reported. Results In this study, we obtained the upstream promoter sequences of several representative primate eosinophil rnases. Bioinformatic analysis revealed the presence of a shared 34-nucleotide (nt) sequence stretch located at -81 to -48 in all edn promoters and macaque ecp promoter. Such a unique sequence motif constituted a region essential for transactivation of human edn in hepatocellular carcinoma cells. Gel electrophoretic mobility shift assay, transient transfection and scanning mutagenesis experiments allowed us to identify binding sites for two transcription factors, Myc-associated zinc finger protein (MAZ) and SV-40 protein-1 (Sp1), within the 34-nt segment. Subsequent in vitro and in vivo binding assays demonstrated a direct molecular interaction between this 34-nt region and MAZ and Sp1. Interestingly, overexpression of MAZ and Sp1 respectively repressed and enhanced edn promoter activity. The regulatory transactivation motif was mapped to the evolutionarily conserved -74/-65 region of the edn promoter, which was guanidine-rich and critical for recognition by both transcription factors. Conclusion Our results provide the first direct evidence that MAZ and Sp1 play important roles on the transcriptional activation of the human edn promoter through specific binding to a 34-nt segment present in representative primate eosinophil rnase promoters. PMID:17927842

Recombinant modified vaccinia virus Ankara generating excess early double-stranded RNA transiently activates protein kinase R and triggers enhanced innate immune responses.

PubMed

Wolferstätter, Michael; Schweneker, Marc; Späth, Michaela; Lukassen, Susanne; Klingenberg, Marieken; Brinkmann, Kay; Wielert, Ursula; Lauterbach, Henning; Hochrein, Hubertus; Chaplin, Paul; Suter, Mark; Hausmann, Jürgen

2014-12-01

Double-stranded RNA (dsRNA) is an important molecular pattern associated with viral infection and is detected by various extra- and intracellular recognition molecules. Poxviruses have evolved to avoid producing dsRNA early in infection but generate significant amounts of dsRNA late in infection due to convergent transcription of late genes. Protein kinase R (PKR) is activated by dsRNA and triggers major cellular defenses against viral infection, including protein synthesis shutdown, apoptosis, and type I interferon (IFN-I) production. The poxviral E3 protein binds and sequesters viral dsRNA and is a major antagonist of the PKR pathway. We found that the highly replication-restricted modified vaccinia virus Ankara (MVA) engineered to produce excess amounts of dsRNA early in infection showed enhanced induction of IFN-β in murine and human cells in the presence of an intact E3L gene. IFN-β induction required a minimum overlap length of 300 bp between early complementary transcripts and was strongly PKR dependent. Excess early dsRNA produced by MVA activated PKR early but transiently in murine cells and induced enhanced systemic levels of IFN-α, IFN-γ, and other cytokines and chemokines in mice in a largely PKR-dependent manner. Replication-competent chorioallantois vaccinia virus Ankara (CVA) generating excess early dsRNA also enhanced IFN-I production and was apathogenic in mice even at very high doses but showed no in vitro host range defect. Thus, genetically adjuvanting MVA and CVA to generate excess early dsRNA is an effective method to enhance innate immune stimulation by orthopoxvirus vectors and to attenuate replicating vaccinia virus in vivo. Efficient cellular sensing of pathogen-specific components, including double-stranded RNA (dsRNA), is an important prerequisite of an effective antiviral immune response. The prototype poxvirus vaccinia virus (VACV) and its derivative modified vaccinia virus Ankara (MVA) produce dsRNA as a by-product of viral transcription. We found that inhibition of cellular dsRNA recognition established by the virus-encoded proteins E3 and K3 can be overcome by directing viral overexpression of dsRNA early in infection without compromising replication of MVA in permissive cells. Early dsRNA induced transient activation of the cellular dsRNA sensor protein kinase R (PKR), resulting in enhanced production of interferons and cytokines in cells and mice. Enhancing the capacity of MVA to activate the innate immune system is an important approach to further improve the immunogenicity of this promising vaccine vector. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Profilin Is Required for Optimal Actin-Dependent Transcription of Respiratory Syncytial Virus Genome RNA

PubMed Central

Burke, Emily; Mahoney, Nicole M.; Almo, Steven C.; Barik, Sailen

2000-01-01

Transcription of human respiratory syncytial virus (RSV) genome RNA exhibited an obligatory need for the host cytoskeletal protein actin. Optimal transcription, however, required the participation of another cellular protein that was characterized as profilin by a number of criteria. The amino acid sequence of the protein, purified on the basis of its transcription-optimizing activity in vitro, exactly matched that of profilin. RSV transcription was inhibited 60 to 80% by antiprofilin antibody or poly-l-proline, molecules that specifically bind profilin. Native profilin, purified from extracts of lung epithelial cells by affinity binding to a poly-l-proline matrix, stimulated the actin-saturated RSV transcription by 2.5- to 3-fold. Recombinant profilin, expressed in bacteria, stimulated viral transcription as effectively as the native protein and was also inhibited by poly-l-proline. Profilin alone, in the absence of actin, did not activate viral transcription. It is estimated that at optimal levels of transcription, every molecule of viral genomic RNA associates with approximately the following number of protein molecules: 30 molecules of L, 120 molecules of phosphoprotein P, and 60 molecules each of actin and profilin. Together, these results demonstrated for the first time a cardinal role for profilin, an actin-modulatory protein, in the transcription of a paramyxovirus RNA genome. PMID:10623728

Purification of Transcript-Specific mRNP Complexes Formed In Vivo from Saccharomyces cerevisiae.

PubMed

Smith, Jenna E; Baker, Kristian E

2017-01-01

RNA binding proteins play critical roles in shaping the complex life cycle of cellular transcripts. For most RNAs, the association with a distinct complement of proteins serves to orchestrate its unique pattern of maturation, localization, translation, and stability. A key aspect to understanding how transcripts are differentially regulated lies, therefore, in the ability to identify the particular repertoire of protein binding partners associated with an individual transcript. We describe here an optimized experimental procedure for purifying a single mRNA population from yeast cells for the characterization of transcript-specific mRNA-protein complexes (mRNPs) as they exist in vivo. Chemical cross-linking is used to trap native mRNPs and facilitate the co-purification of protein complexes associated with an individual transcript population that is captured under stringent conditions from cell lysates through hybridization to complementary DNA oligonucleotides. The resulting mRNP is highly enriched and largely devoid of non-target transcripts, and can be used for a number of downstream analyses including protein identification by mass spectrometry.

NRF2: Translating the Redox Code

PubMed Central

Tummala, Krishna S.; Kottakis, Filippos; Bardeesy, Nabeel

2016-01-01

Cancer requires mechanisms to mitigate reactive oxygen species (ROS) generated during rapid growth, such as induction of the antioxidant transcription factor, Nrf2. However, the targets of ROS-mediated cytotoxicity are unclear. Recent studies in pancreatic cancer show that redox control by Nrf2 prevents cysteine oxidation of the mRNA translational machinery, thereby supporting efficient protein synthesis. PMID:27555347

Differential regulation of oligodendrocyte markers by glucocorticoids: Post-transcriptional regulation of both proteolipid protein and myelin basic protein and transcriptional regulation of glycerol phosphate dehydrogenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, S.; Cole, R.; Chiappelli, F.

During neonatal development glucocorticoids potentiate oligodendrocyte differentiation and myelinogenesis by regulating the expression of myelin basic protein, proteolipid protein, and glycerol phosphate dehydrogenase. The actual locus at which hydrocortisone exerts its developmental influence on glial physiology is, however, not well understood. Gycerol phosphate dehydrogenase is glucocorticoid-inducible in oligodendrocytes at all stages of development both in vivo and in vitro. In newborn rat cerebral cultures, between 9 and 15 days in vitro, a 2- to 3-fold increase in myelin basic protein and proteolipid protein mRNA levels occurs in oligodendrocytes within 12 hr of hydrocortisone treatment. Immunostaining demonstrates that this increase inmore » mRNAs is followed by a 2- to 3-fold increase in the protein levels within 24 hr. In vitro transcription assays performed with oligodendrocyte nuclei show an 11-fold increase in the transcriptional activity of glycerol phosphate dehydrogenase in response to hydrocortisone but no increase in transcription of myelin basic protein or proteolipid protein. These results indicate that during early myelinogeneis, glucocorticoids influence the expression of key oligodendroglial markers by different processes: The expression of glycerol phosphate dehydrogenase is regulated at the transcriptional level, whereas the expression of myelin basic protein and proteolipid protein is modulated via a different, yet uncharacterized, mechanism involving post-transcriptional regulation.« less

UniPROBE, update 2015: new tools and content for the online database of protein-binding microarray data on protein-DNA interactions.

PubMed

Hume, Maxwell A; Barrera, Luis A; Gisselbrecht, Stephen S; Bulyk, Martha L

2015-01-01

The Universal PBM Resource for Oligonucleotide Binding Evaluation (UniPROBE) serves as a convenient source of information on published data generated using universal protein-binding microarray (PBM) technology, which provides in vitro data about the relative DNA-binding preferences of transcription factors for all possible sequence variants of a length k ('k-mers'). The database displays important information about the proteins and displays their DNA-binding specificity data in terms of k-mers, position weight matrices and graphical sequence logos. This update to the database documents the growth of UniPROBE since the last update 4 years ago, and introduces a variety of new features and tools, including a new streamlined pipeline that facilitates data deposition by universal PBM data generators in the research community, a tool that generates putative nonbinding (i.e. negative control) DNA sequences for one or more proteins and novel motifs obtained by analyzing the PBM data using the BEEML-PBM algorithm for motif inference. The UniPROBE database is available at http://uniprobe.org. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Global Regulatory Functions of the Staphylococcus aureus Endoribonuclease III in Gene Expression

PubMed Central

Lioliou, Efthimia; Sharma, Cynthia M.; Caldelari, Isabelle; Helfer, Anne-Catherine; Fechter, Pierre; Vandenesch, François; Vogel, Jörg; Romby, Pascale

2012-01-01

RNA turnover plays an important role in both virulence and adaptation to stress in the Gram-positive human pathogen Staphylococcus aureus. However, the molecular players and mechanisms involved in these processes are poorly understood. Here, we explored the functions of S. aureus endoribonuclease III (RNase III), a member of the ubiquitous family of double-strand-specific endoribonucleases. To define genomic transcripts that are bound and processed by RNase III, we performed deep sequencing on cDNA libraries generated from RNAs that were co-immunoprecipitated with wild-type RNase III or two different cleavage-defective mutant variants in vivo. Several newly identified RNase III targets were validated by independent experimental methods. We identified various classes of structured RNAs as RNase III substrates and demonstrated that this enzyme is involved in the maturation of rRNAs and tRNAs, regulates the turnover of mRNAs and non-coding RNAs, and autoregulates its synthesis by cleaving within the coding region of its own mRNA. Moreover, we identified a positive effect of RNase III on protein synthesis based on novel mechanisms. RNase III–mediated cleavage in the 5′ untranslated region (5′UTR) enhanced the stability and translation of cspA mRNA, which encodes the major cold-shock protein. Furthermore, RNase III cleaved overlapping 5′UTRs of divergently transcribed genes to generate leaderless mRNAs, which constitutes a novel way to co-regulate neighboring genes. In agreement with recent findings, low abundance antisense RNAs covering 44% of the annotated genes were captured by co-immunoprecipitation with RNase III mutant proteins. Thus, in addition to gene regulation, RNase III is associated with RNA quality control of pervasive transcription. Overall, this study illustrates the complexity of post-transcriptional regulation mediated by RNase III. PMID:22761586

Amyloid Precursor Protein Translation Is Regulated by a 3’UTR Guanine Quadruplex

PubMed Central

Sharoni, Michal; Olson, Kalee; Sebastian, Neeraj P.; Ansaloni, Sara; Schweitzer-Stenner, Reinhard; Akins, Michael R.; Bevilacqua, Philip C.; Saunders, Aleister J.

2015-01-01

A central event in Alzheimer’s disease is the accumulation of amyloid β (Aβ) peptides generated by the proteolytic cleavage of the amyloid precursor protein (APP). APP overexpression leads to increased Aβ generation and Alzheimer’s disease in humans and altered neuronal migration and increased long term depression in mice. Conversely, reduction of APP expression results in decreased Aβ levels in mice as well as impaired learning and memory and decreased numbers of dendritic spines. Together these findings indicate that therapeutic interventions that aim to restore APP and Aβ levels must do so within an ideal range. To better understand the effects of modulating APP levels, we explored the mechanisms regulating APP expression focusing on post-transcriptional regulation. Such regulation can be mediated by RNA regulatory elements such as guanine quadruplexes (G-quadruplexes), non-canonical structured RNA motifs that affect RNA stability and translation. Via a bioinformatics approach, we identified a candidate G-quadruplex within the APP mRNA in its 3’UTR (untranslated region) at residues 3008–3027 (NM_201414.2). This sequence exhibited characteristics of a parallel G-quadruplex structure as revealed by circular dichroism spectrophotometry. Further, as with other G-quadruplexes, the formation of this structure was dependent on the presence of potassium ions. This G-quadruplex has no apparent role in regulating transcription or mRNA stability as wild type and mutant constructs exhibited equivalent mRNA levels as determined by real time PCR. Instead, we demonstrate that this G-quadruplex negatively regulates APP protein expression using dual luciferase reporter and Western blot analysis. Taken together, our studies reveal post-transcriptional regulation by a 3’UTR G-quadruplex as a novel mechanism regulating APP expression. PMID:26618502

Transcriptomic Responses to Salinity Stress in the Pacific Oyster Crassostrea gigas

PubMed Central

Zhao, Xuelin; Yu, Hong; Kong, Lingfeng; Li, Qi

2012-01-01

Background Low salinity is one of the main factors limiting the distribution and survival of marine species. As a euryhaline species, the Pacific oyster Crassostrea gigas is considered to be tolerant to relative low salinity. The genes that regulate C. gigas responses to osmotic stress were monitored using the next-generation sequencing of whole transcriptome with samples taken from gills. By RNAseq technology, transcript catalogs of up- and down-regulated genes were generated from the oysters exposed to low and optimal salinity seawater. Methodology/Principal Findings Through Illumina sequencing, we reported 1665 up-regulated transcripts and 1815 down-regulated transcripts. A total of 45771 protein-coding contigs were identified from two groups based on sequence similarities with known proteins. As determined by GO annotation and KEGG pathway mapping, functional annotation of the genes recovered diverse biological functions and processes. The genes that changed expression significantly were highly represented in cellular process and regulation of biological process, intracellular and cell, binding and protein binding according to GO annotation. The results highlighted genes related to osmoregulation, signaling and interactions of osmotic stress response, anti-apoptotic reactions as well as immune response, cell adhesion and communication, cytoskeleton and cell cycle. Conclusions/Significance Through more than 1.5 million sequence reads and the expression data of the two libraries, the study provided some useful insights into signal transduction pathways in oysters and offered a number of candidate genes as potential markers of tolerance to hypoosmotic stress for oysters. In addition, the characterization of C. gigas transcriptome will not only provide a better understanding of the molecular mechanisms about the response to osmotic stress of the oysters, but also facilitate research into biological processes to find underlying physiological adaptations to hypoosmotic shock for marine invertebrates. PMID:23029449

Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 4: intercellular bridges, mitochondria, nuclear envelope, apoptosis, ubiquitination, membrane/voltage-gated channels, methylation/acetylation, and transcription factors.

PubMed

Hermo, Louis; Pelletier, R-Marc; Cyr, Daniel G; Smith, Charles E

2010-04-01

As germ cells divide and differentiate from spermatogonia to spermatozoa, they share a number of structural and functional features that are common to all generations of germ cells and these features are discussed herein. Germ cells are linked to one another by large intercellular bridges which serve to move molecules and even large organelles from the cytoplasm of one cell to another. Mitochondria take on different shapes and features and topographical arrangements to accommodate their specific needs during spermatogenesis. The nuclear envelope and pore complex also undergo extensive modifications concomitant with the development of germ cell generations. Apoptosis is an event that is normally triggered by germ cells and involves many proteins. It occurs to limit the germ cell pool and acts as a quality control mechanism. The ubiquitin pathway comprises enzymes that ubiquitinate as well as deubiquitinate target proteins and this pathway is present and functional in germ cells. Germ cells express many proteins involved in water balance and pH control as well as voltage-gated ion channel movement. In the nucleus, proteins undergo epigenetic modifications which include methylation, acetylation, and phosphorylation, with each of these modifications signaling changes in chromatin structure. Germ cells contain specialized transcription complexes that coordinate the differentiation program of spermatogenesis, and there are many male germ cell-specific differences in the components of this machinery. All of the above features of germ cells will be discussed along with the specific proteins/genes and abnormalities to fertility related to each topic. Copyright 2009 Wiley-Liss, Inc.

Analysis of persistent changes to γ-aminobutyric acid receptor gene expression in Plutella xylostella subjected to sublethal amounts of spinosad.

PubMed

Yin, X-H; Wu, Q-J; Zhang, Y-J; Long, Y-H; Wu, X-M; Li, R-Y; Wang, M; Tian, X-L; Jiao, X-G

2016-07-25

A multi-generational approach was used to investigate the persistent effects of a sub-lethal dose of spinosad in Plutella xylostella. The susceptibility of various sub-populations of P. xylostella to spinosad and the effects of the insecticide on the gene expression of γ-aminobutyric acid receptor (GABAR) were determined. The results of a leaf dip bioassay showed that the sensitivity of P. xylostella to spinosad decreased across generations. The sub-strains had been previously selected based on a determined LC25 of spinosad. Considering that GABA-gated chloride channels are the primary targets of spinosad, the cDNA of P. xylostella was used to clone GABARα by using reverse transcription-polymerase chain reaction (RT-PCR). The mature peptide cDNA was 1477-bp long and contained a 1449-bp open reading frame encoding a protein of 483 amino acids. The resulting amino acid sequence was used to generate a neighbor-joining dendrogram, and homology search was conducted using NCBI BLAST. The protein had high similarity with the known GABAR sequence from P. xylostella. Subsequent semi-quantitative RT-PCR and real-time PCR analyses indicated that the GABAR transcript levels in the spinosad-resistant strain (RR, 145.82-fold) and in Sub1 strain (selected with LC25 spinosad for one generation) were the highest, followed by those in the spinosad-susceptible strain, the Sub10 strain (selected for ten generations), and the Sub5 strain (selected for five generations). This multi-generational study found significant correlations between spinosad susceptibility and GABAR gene expression, providing insights into the long-term effects of sub-lethal insecticide exposure and its potential to lead to the development of insecticide-resistant insect populations.

Gene regulation network behind drought escape, avoidance and tolerance strategies in black poplar (Populus nigra L.).

PubMed

Yıldırım, Kubilay; Kaya, Zeki

2017-06-01

Drought is the major environmental problem limiting the productivity and survival of plant species. Here, previously identified three black poplar genotypes having contrasting response to drought were subjected to gradual soil water depletion in a pot trial to identify their physiological, morphological and antioxidation related adaptations. We also performed a microarray based transcriptome analyses on the leaves of genotypes by using Affymetrix poplar Genome Array containing 56,000 transcripts. Phenotypic analyses of each genotype confirmed their differential adaptations to drought that could be classified as drought escape, avoidance and tolerance. Comparative transcriptomic analysis indicated highly divergent gene expression patterns among the genotypes in response to drought and post drought re-watering (PDR). We identified 10641, 3824 and 9411 transcripts exclusively regulated in drought escape, avoidance and tolerant genotypes, respectively. The key genes involved in metabolic pathways, such as carbohydrate metabolism, photosynthesis, lipid metabolism, generation of precursor metabolites/energy, protein folding, redox homeostasis, secondary metabolic process and cell wall component biogenesis, were affected by drought stresses in the leaves of these genotypes. Transcript isoforms showed increased expression specificity in the genes coding for bark storage proteins and small heat shock proteins in drought tolerant genotype. On the other hand, drought-avoiding genotype specifically induced the transcripts annotated to the genes functional in secondary metabolite production that linked to enhanced leaf water content and growth performance under drought stress. Transcriptome profiling of drought escape genotype indicated specific regulation of the genes functional in programmed cell death and leaf senescence. Specific upregulation of GTP cyclohydrolase II and transcription factors (WRKY and ERFs) in only this genotype were associated to ROS dependent signalling pathways and gene regulation network responsible in induction of many degrading enzymes acting on cell wall carbohydrates, fatty acids and proteins under drought stress. Our findings provide new insights into the transcriptome dynamics and components of regulatory network associated with drought adaptation strategies. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Global transcriptional repression in C. elegans germline precursors by regulated sequestration of TAF-4.

PubMed

Guven-Ozkan, Tugba; Nishi, Yuichi; Robertson, Scott M; Lin, Rueyling

2008-10-03

In C. elegans, four asymmetric divisions, beginning with the zygote (P0), generate transcriptionally repressed germline blastomeres (P1-P4) and somatic sisters that become transcriptionally active. The protein PIE-1 represses transcription in the later germline blastomeres but not in the earlier germline blastomeres P0 and P1. We show here that OMA-1 and OMA-2, previously shown to regulate oocyte maturation, repress transcription in P0 and P1 by binding to and sequestering in the cytoplasm TAF-4, a component critical for assembly of TFIID and the pol II preinitiation complex. OMA-1/2 binding to TAF-4 is developmentally regulated, requiring phosphorylation by the DYRK kinase MBK-2, which is activated at meiosis II after fertilization. OMA-1/2 are normally degraded after the first mitosis, but ectopic expression of wild-type OMA-1 is sufficient to repress transcription in both somatic and later germline blastomeres. We propose that phosphorylation by MBK-2 serves as a developmental switch, converting OMA-1/2 from oocyte to embryo regulators.

Global transcriptional repression in C. elegans germline precursors by regulated sequestration of TFIID component TAF-4

PubMed Central

Guven-Ozkan, Tugba; Nishi, Yuichi; Robertson, Scott M.; Lin, Rueyling

2008-01-01

In C. elegans, four asymmetric divisions, beginning with the zygote (P0), generate transcriptionally repressed germline blastomeres (P1–P4) and somatic sisters that become transcriptionally active. The protein PIE-1 represses transcription in the later germline blastomeres, but not in the earlier germline blastomeres P0 and P1. We show here that OMA-1 and OMA-2, previously shown to regulate oocyte maturation, repress transcription in P0 and P1 by binding to and sequestering in the cytoplasm TAF-4, a component critical for assembly of TFIID and the pol II preinitiation complex. OMA-1/2 binding to TAF-4 is developmentally regulated, requiring phosphorylation by the DYRK kinase MBK-2, which is activated at meiosis II following fertilization. OMA-1/2 are normally degraded after the first mitosis, but ectopic expression of wildtype OMA-1 is sufficient to repress transcription in both somatic and later germline blastomeres. We propose that phosphorylation by MBK-2 serves as a developmental switch, converting OMA-1/2 from oocyte to embryo regulators. PMID:18854162

Effects of mutations in the Arabidopsis Cold Shock Domain Protein 3 (AtCSP3) gene on leaf cell expansion.

PubMed

Yang, Yongil; Karlson, Dale

2012-08-01

The cold shock domain is among the most evolutionarily conserved nucleic acid binding domains from prokaryotes to higher eukaryotes, including plants. Although eukaryotic cold shock domain proteins have been extensively studied as transcriptional and post-transcriptional regulators during various developmental processes, their functional roles in plants remains poorly understood. In this study, AtCSP3 (At2g17870), which is one of four Arabidopsis thaliana c old s hock domain proteins (AtCSPs), was functionally characterized. Quantitative RT-PCR analysis confirmed high expression of AtCSP3 in reproductive and meristematic tissues. A homozygous atcsp3 loss-of-function mutant exhibits an overall reduced seedling size, stunted and orbicular rosette leaves, reduced petiole length, and curled leaf blades. Palisade mesophyll cells are smaller and more circular in atcsp3 leaves. Cell size analysis indicated that the reduced size of the circular mesophyll cells appears to be generated by a reduction of cell length along the leaf-length axis, resulting in an orbicular leaf shape. It was also determined that leaf cell expansion is impaired for lateral leaf development in the atcsp3 loss-of-function mutant, but leaf cell proliferation is not affected. AtCSP3 loss-of-function resulted in a dramatic reduction of LNG1 transcript, a gene that is involved in two-dimensional leaf polarity regulation. Transient subcellular localization of AtCSP3 in onion epidermal cells confirmed a nucleocytoplasmic localization pattern. Collectively, these data suggest that AtCSP3 is functionally linked to the regulation of leaf length by affecting LNG1 transcript accumulation during leaf development. A putative function of AtCSP3 as an RNA binding protein is also discussed in relation to leaf development.

Neuron-specific specificity protein 4 bigenomically regulates the transcription of all mitochondria- and nucleus-encoded cytochrome c oxidase subunit genes in neurons.

PubMed

Johar, Kaid; Priya, Anusha; Dhar, Shilpa; Liu, Qiuli; Wong-Riley, Margaret T T

2013-11-01

Neurons are highly dependent on oxidative metabolism for their energy supply, and cytochrome c oxidase (COX) is a key energy-generating enzyme in the mitochondria. A unique feature of COX is that it is one of only four proteins in mammalian cells that are bigenomically regulated. Of its thirteen subunits, three are encoded in the mitochondrial genome and ten are nuclear-encoded on nine different chromosomes. The mechanism of regulating this multisubunit, bigenomic enzyme poses a distinct challenge. In recent years, we found that nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) mediate such bigenomic coordination. The latest candidate is the specificity factor (Sp) family of proteins. In N2a cells, we found that Sp1 regulates all 13 COX subunits. However, we discovered recently that in primary neurons, it is Sp4 and not Sp1 that regulates some of the key glutamatergic receptor subunit genes. The question naturally arises as to the role of Sp4 in regulating COX in primary neurons. The present study utilized multiple approaches, including chromatin immunoprecipitation, promoter mutational analysis, knockdown and over-expression of Sp4, as well as functional assays to document that Sp4 indeed functionally regulate all 13 subunits of COX as well as mitochondrial transcription factors A and B. The present study discovered that among the specificity family of transcription factors, it is the less known neuron-specific Sp4 that regulates the expression of all 13 subunits of mitochondrial cytochrome c oxidase (COX) enzyme in primary neurons. Sp4 also regulates the three mitochondrial transcription factors (TFAM, TFB1M, and TFB2M) and a COX assembly protein SURF-1 in primary neurons. © 2013 International Society for Neurochemistry.

Transcriptome and proteomic analysis of mango (Mangifera indica Linn) fruits.

PubMed

Wu, Hong-xia; Jia, Hui-min; Ma, Xiao-wei; Wang, Song-biao; Yao, Quan-sheng; Xu, Wen-tian; Zhou, Yi-gang; Gao, Zhong-shan; Zhan, Ru-lin

2014-06-13

Here we used Illumina RNA-seq technology for transcriptome sequencing of a mixed fruit sample from 'Zill' mango (Mangifera indica Linn) fruit pericarp and pulp during the development and ripening stages. RNA-seq generated 68,419,722 sequence reads that were assembled into 54,207 transcripts with a mean length of 858bp, including 26,413 clusters and 27,794 singletons. A total of 42,515(78.43%) transcripts were annotated using public protein databases, with a cut-off E-value above 10(-5), of which 35,198 and 14,619 transcripts were assigned to gene ontology terms and clusters of orthologous groups respectively. Functional annotation against the Kyoto Encyclopedia of Genes and Genomes database identified 23,741(43.79%) transcripts which were mapped to 128 pathways. These pathways revealed many previously unknown transcripts. We also applied mass spectrometry-based transcriptome data to characterize the proteome of ripe fruit. LC-MS/MS analysis of the mango fruit proteome was using tandem mass spectrometry (MS/MS) in an LTQ Orbitrap Velos (Thermo) coupled online to the HPLC. This approach enabled the identification of 7536 peptides that matched 2754 proteins. Our study provides a comprehensive sequence for a systemic view of transcriptome during mango fruit development and the most comprehensive fruit proteome to date, which are useful for further genomics research and proteomic studies. Our study provides a comprehensive sequence for a systemic view of both the transcriptome and proteome of mango fruit, and a valuable reference for further research on gene expression and protein identification. This article is part of a Special Issue entitled: Proteomics of non-model organisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Critical components of the pluripotency network are targets for the p300/CBP interacting protein (p/CIP) in embryonic stem cells.

PubMed

Chitilian, J M; Thillainadesan, G; Manias, J L; Chang, W Y; Walker, E; Isovic, M; Stanford, W L; Torchia, J

2014-01-01

p/CIP, also known as steroid receptor coactivator 3 (SRC-3)/Nuclear Receptor Coactivator 3 (NCoA3), is a transcriptional coactivator that binds liganded nuclear hormone receptors, as well as other transcription factors, and facilitates transcription through direct recruitment of accessory factors. We have found that p/CIP is highly expressed in undifferentiated mouse embryonic stem cells (mESCs) and is downregulated during differentiation. siRNA-mediated knockdown of p/CIP decreased transcript levels of Nanog, but not Oct4 or Sox2. Microarray expression analysis showed that Klf4, Tbx3, and Dax-1 are significantly downregulated in mESCs when p/CIP is knocked down. Subsequent chromatin immunoprecipitation (ChIP) analysis demonstrated that Tbx3, Klf4, and Dax-1 are direct transcriptional targets of p/CIP. Using the piggyBac transposition system, a mouse ESC line that expresses Flag-p/CIP in a doxycycline-dependent manner was generated. p/CIP overexpression increased the level of target genes and promoted the formation of undifferentiated colonies. Collectively, these results indicate that p/CIP contributes to the maintenance of ESC pluripotency through direct regulation of essential pluripotency genes. To better understand the mechanism by which p/CIP functions in ESC pluripotency, we integrated our ChIP and transcriptome data with published protein-protein interaction and promoter occupancy data to draft a p/CIP gene regulatory network. The p/CIP gene regulatory network identifies various feed-forward modules including one in which p/CIP activates members of the extended pluripotency network, demonstrating that p/CIP is a component of this extended network. © AlphaMed Press.

Integrated omics dissection of proteome dynamics during cardiac remodeling.

PubMed

Lau, Edward; Cao, Quan; Lam, Maggie P Y; Wang, Jie; Ng, Dominic C M; Bleakley, Brian J; Lee, Jessica M; Liem, David A; Wang, Ding; Hermjakob, Henning; Ping, Peipei

2018-01-09

Transcript abundance and protein abundance show modest correlation in many biological models, but how this impacts disease signature discovery in omics experiments is rarely explored. Here we report an integrated omics approach, incorporating measurements of transcript abundance, protein abundance, and protein turnover to map the landscape of proteome remodeling in a mouse model of pathological cardiac hypertrophy. Analyzing the hypertrophy signatures that are reproducibly discovered from each omics data type across six genetic strains of mice, we find that the integration of transcript abundance, protein abundance, and protein turnover data leads to 75% gain in discovered disease gene candidates. Moreover, the inclusion of protein turnover measurements allows discovery of post-transcriptional regulations across diverse pathways, and implicates distinct disease proteins not found in steady-state transcript and protein abundance data. Our results suggest that multi-omics investigations of proteome dynamics provide important insights into disease pathogenesis in vivo.

Phylogenetic and Structural Analysis of the Pluripotency Factor Sex-Determining Region Y box2 Gene of Camelus dromedarius (cSox2).

PubMed

Alawad, Abdullah; Alharbi, Sultan; Alhazzaa, Othman; Alagrafi, Faisal; Alkhrayef, Mohammed; Alhamdan, Ziyad; Alenazi, Abdullah; Al-Johi, Hasan; Alanazi, Ibrahim O; Hammad, Mohamed

2016-01-01

Although the sequencing information of Sox2 cDNA for many mammalian is available, the Sox2 cDNA of Camelus dromedaries has not yet been characterized. The objective of this study was to sequence and characterize Sox2 cDNA from the brain of C. dromedarius (also known as Arabian camel). A full coding sequence of the Sox2 gene from the brain of C. dromedarius was amplified by reverse transcription PCRjmc and then sequenced using the 3730XL series platform Sequencer (Applied Biosystem) for the first time. The cDNA sequence displayed an open reading frame of 822 nucleotides, encoding a protein of 273 amino acids. The molecular weight and the isoelectric point of the translated protein were calculated as 29.825 kDa and 10.11, respectively, using bioinformatics analysis. The predicted cSox2 protein sequence exhibited high identity: 99% for Homo sapiens, Mus musculus, Bos taurus, and Vicugna pacos; 98% for Sus scrofa and 93% for Camelus ferus. A 3D structure was built based on the available crystal structure of the HMG-box domain of human stem cell transcription factor Sox2 (PDB: 2 LE4) with 81 residues and predicting bioinformatics software for 273 amino acid residues. The comparison confirms the presence of the HMG-box domain in the cSox2 protein. The orthologous phylogenetic analysis showed that the Sox2 isoform from C. dromedarius was grouped with humans, alpacas, cattle, and pigs. We believe that this genetic and structural information will be a helpful source for the annotation. Furthermore, Sox2 is one of the transcription factors that contributes to the generation-induced pluripotent stem cells (iPSCs), which in turn will probably help generate camel induced pluripotent stem cells (CiPSCs).

Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis

PubMed Central

Carroll, James A.; Olano, L. Rennee; Sturdevant, Daniel E.; Rosa, Patricia A.

2015-01-01

The saprophyte Leptospira biflexa is an excellent model for studying the physiology of the medically important Leptospira genus, the pathogenic members of which are more recalcitrant to genetic manipulation and have significantly slower in vitro growth. However, relatively little is known regarding the proteome of L. biflexa, limiting its utility as a model for some studies. Therefore, we have generated a proteomic map of both soluble and membrane-associated proteins of L. biflexa during exponential growth and in stationary phase. Using these data, we identified abundantly produced proteins in each cellular fraction and quantified the transcript levels from a subset of these genes using quantitative reverse transcription-PCR (RT-PCR). These proteins should prove useful as cellular markers and as controls for gene expression studies. We also observed a significant number of L. biflexa membrane-associated proteins with multiple isoforms, each having unique isoelectric focusing points. L. biflexa cell lysates were examined for several posttranslational modifications suggested by the protein patterns. Methylation and acetylation of lysine residues were predominately observed in the proteins of the membrane-associated fraction, while phosphorylation was detected mainly among soluble proteins. These three posttranslational modification systems appear to be conserved between the free-living species L. biflexa and the pathogenic species Leptospira interrogans, suggesting an important physiological advantage despite the varied life cycles of the different species. PMID:26655756

Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis.

PubMed

Stewart, Philip E; Carroll, James A; Olano, L Rennee; Sturdevant, Daniel E; Rosa, Patricia A

2016-02-15

The saprophyte Leptospira biflexa is an excellent model for studying the physiology of the medically important Leptospira genus, the pathogenic members of which are more recalcitrant to genetic manipulation and have significantly slower in vitro growth. However, relatively little is known regarding the proteome of L. biflexa, limiting its utility as a model for some studies. Therefore, we have generated a proteomic map of both soluble and membrane-associated proteins of L. biflexa during exponential growth and in stationary phase. Using these data, we identified abundantly produced proteins in each cellular fraction and quantified the transcript levels from a subset of these genes using quantitative reverse transcription-PCR (RT-PCR). These proteins should prove useful as cellular markers and as controls for gene expression studies. We also observed a significant number of L. biflexa membrane-associated proteins with multiple isoforms, each having unique isoelectric focusing points. L. biflexa cell lysates were examined for several posttranslational modifications suggested by the protein patterns. Methylation and acetylation of lysine residues were predominately observed in the proteins of the membrane-associated fraction, while phosphorylation was detected mainly among soluble proteins. These three posttranslational modification systems appear to be conserved between the free-living species L. biflexa and the pathogenic species Leptospira interrogans, suggesting an important physiological advantage despite the varied life cycles of the different species. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Uncovering Hidden Layers of Cell Cycle Regulation through Integrative Multi-omic Analysis

PubMed Central

Aviner, Ranen; Shenoy, Anjana; Elroy-Stein, Orna; Geiger, Tamar

2015-01-01

Studying the complex relationship between transcription, translation and protein degradation is essential to our understanding of biological processes in health and disease. The limited correlations observed between mRNA and protein abundance suggest pervasive regulation of post-transcriptional steps and support the importance of profiling mRNA levels in parallel to protein synthesis and degradation rates. In this work, we applied an integrative multi-omic approach to study gene expression along the mammalian cell cycle through side-by-side analysis of mRNA, translation and protein levels. Our analysis sheds new light on the significant contribution of both protein synthesis and degradation to the variance in protein expression. Furthermore, we find that translation regulation plays an important role at S-phase, while progression through mitosis is predominantly controlled by changes in either mRNA levels or protein stability. Specific molecular functions are found to be co-regulated and share similar patterns of mRNA, translation and protein expression along the cell cycle. Notably, these include genes and entire pathways not previously implicated in cell cycle progression, demonstrating the potential of this approach to identify novel regulatory mechanisms beyond those revealed by traditional expression profiling. Through this three-level analysis, we characterize different mechanisms of gene expression, discover new cycling gene products and highlight the importance and utility of combining datasets generated using different techniques that monitor distinct steps of gene expression. PMID:26439921

RNA-guided transcriptional regulation

DOEpatents

Church, George M.; Mali, Prashant G.; Esvelt, Kevin M.

2016-02-23

Methods of modulating expression of a target nucleic acid in a cell are provided including introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to DNA, wherein the DNA includes the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding a nuclease-null Cas9 protein that binds to the DNA and is guided by the one or more RNAs, introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the one or more RNAs, the nuclease-null Cas9 protein, and the transcriptional regulator protein or domain are expressed, wherein the one or more RNAs, the nuclease-null Cas9 protein and the transcriptional regulator protein or domain co-localize to the DNA and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid.

A transcriptome resource for the koala (Phascolarctos cinereus): insights into koala retrovirus transcription and sequence diversity.

PubMed

Hobbs, Matthew; Pavasovic, Ana; King, Andrew G; Prentis, Peter J; Eldridge, Mark D B; Chen, Zhiliang; Colgan, Donald J; Polkinghorne, Adam; Wilkins, Marc R; Flanagan, Cheyne; Gillett, Amber; Hanger, Jon; Johnson, Rebecca N; Timms, Peter

2014-09-11

The koala, Phascolarctos cinereus, is a biologically unique and evolutionarily distinct Australian arboreal marsupial. The goal of this study was to sequence the transcriptome from several tissues of two geographically separate koalas, and to create the first comprehensive catalog of annotated transcripts for this species, enabling detailed analysis of the unique attributes of this threatened native marsupial, including infection by the koala retrovirus. RNA-Seq data was generated from a range of tissues from one male and one female koala and assembled de novo into transcripts using Velvet-Oases. Transcript abundance in each tissue was estimated. Transcripts were searched for likely protein-coding regions and a non-redundant set of 117,563 putative protein sequences was produced. In similarity searches there were 84,907 (72%) sequences that aligned to at least one sequence in the NCBI nr protein database. The best alignments were to sequences from other marsupials. After applying a reciprocal best hit requirement of koala sequences to those from tammar wallaby, Tasmanian devil and the gray short-tailed opossum, we estimate that our transcriptome dataset represents approximately 15,000 koala genes. The marsupial alignment information was used to look for potential gene duplications and we report evidence for copy number expansion of the alpha amylase gene, and of an aldehyde reductase gene.Koala retrovirus (KoRV) transcripts were detected in the transcriptomes. These were analysed in detail and the structure of the spliced envelope gene transcript was determined. There was appreciable sequence diversity within KoRV, with 233 sites in the KoRV genome showing small insertions/deletions or single nucleotide polymorphisms. Both koalas had sequences from the KoRV-A subtype, but the male koala transcriptome has, in addition, sequences more closely related to the KoRV-B subtype. This is the first report of a KoRV-B-like sequence in a wild population. This transcriptomic dataset is a useful resource for molecular genetic studies of the koala, for evolutionary genetic studies of marsupials, for validation and annotation of the koala genome sequence, and for investigation of koala retrovirus. Annotated transcripts can be browsed and queried at http://koalagenome.org.

Cloning and characterization of mouse extracellular-signal-regulated protein kinase 3 as a unique gene product of 100 kDa.

PubMed

Turgeon, B; Saba-El-Leil, M K; Meloche, S

2000-02-15

MAP (mitogen-activated protein) kinases are a family of serine/threonine kinases that have a pivotal role in signal transduction. Here we report the cloning and characterization of a mouse homologue of extracellular-signal-regulated protein kinase (ERK)3. The mouse Erk3 cDNA encodes a predicted protein of 720 residues, which displays 94% identity with human ERK3. Transcription and translation of this cDNA in vitro generates a 100 kDa protein similar to the human gene product ERK3. Immunoblot analysis with an antibody raised against a unique sequence of ERK3 also recognizes a 100 kDa protein in mouse tissues. A single transcript of Erk3 was detected in every adult mouse tissue examined, with the highest expression being found in the brain. Interestingly, expression of Erk3 mRNA is acutely regulated during mouse development, with a peak of expression observed at embryonic day 11. The mouse Erk3 gene was mapped to a single locus on central mouse chromosome 9, adjacent to the dilute mutation locus and in a region syntenic to human chromosome 15q21. Finally, we provide several lines of evidence to support the existence of a unique Erk3 gene product of 100 kDa in mammalian cells.

An ABRE promoter sequence is involved in osmotic stress-responsive expression of the DREB2A gene, which encodes a transcription factor regulating drought-inducible genes in Arabidopsis.

PubMed

Kim, June-Sik; Mizoi, Junya; Yoshida, Takuya; Fujita, Yasunari; Nakajima, Jun; Ohori, Teppei; Todaka, Daisuke; Nakashima, Kazuo; Hirayama, Takashi; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

2011-12-01

In plants, osmotic stress-responsive transcriptional regulation depends mainly on two major classes of cis-acting elements found in the promoter regions of stress-inducible genes: ABA-responsive elements (ABREs) and dehydration-responsive elements (DREs). ABRE has been shown to perceive ABA-mediated osmotic stress signals, whereas DRE is known to be involved in an ABA-independent pathway. Previously, we reported that the transcription factor DRE-BINDING PROTEIN 2A (DREB2A) regulates DRE-mediated transcription of target genes under osmotic stress conditions in Arabidopsis (Arabidopsis thaliana). However, the transcriptional regulation of DREB2A itself remains largely uncharacterized. To elucidate the transcriptional mechanism associated with the DREB2A gene under osmotic stress conditions, we generated a series of truncated and base-substituted variants of the DREB2A promoter and evaluated their transcriptional activities individually. We found that both ABRE and coupling element 3 (CE3)-like sequences located approximately -100 bp from the transcriptional initiation site are necessary for the dehydration-responsive expression of DREB2A. Coupling our transient expression analyses with yeast one-hybrid and chromatin immunoprecipitation (ChIP) assays indicated that the ABRE-BINDING PROTEIN 1 (AREB1), AREB2 and ABRE-BINDING FACTOR 3 (ABF3) bZIP transcription factors can bind to and activate the DREB2A promoter in an ABRE-dependent manner. Exogenous ABA application induced only a modest accumulation of the DREB2A transcript when compared with the osmotic stress treatment. However, the osmotic stress-induced DREB2A expression was found to be markedly impaired in several ABA-deficient and ABA-insensitive mutants. These results suggest that in addition to an ABA-independent pathway, the ABA-dependent pathway plays a positive role in the osmotic stress-responsive expression of DREB2A.

Identification of unannotated exons of low abundance transcripts in Drosophila melanogaster and cloning of a new serine protease gene upregulated upon injury.

PubMed

Maia, Rafaela M; Valente, Valeria; Cunha, Marco A V; Sousa, Josane F; Araujo, Daniela D; Silva, Wilson A; Zago, Marco A; Dias-Neto, Emmanuel; Souza, Sandro J; Simpson, Andrew J G; Monesi, Nadia; Ramos, Ricardo G P; Espreafico, Enilza M; Paçó-Larson, Maria L

2007-07-24

The sequencing of the D.melanogaster genome revealed an unexpected small number of genes (~ 14,000) indicating that mechanisms acting on generation of transcript diversity must have played a major role in the evolution of complex metazoans. Among the most extensively used mechanisms that accounts for this diversity is alternative splicing. It is estimated that over 40% of Drosophila protein-coding genes contain one or more alternative exons. A recent transcription map of the Drosophila embryogenesis indicates that 30% of the transcribed regions are unannotated, and that 1/3 of this is estimated as missed or alternative exons of previously characterized protein-coding genes. Therefore, the identification of the variety of expressed transcripts depends on experimental data for its final validation and is continuously being performed using different approaches. We applied the Open Reading Frame Expressed Sequence Tags (ORESTES) methodology, which is capable of generating cDNA data from the central portion of rare transcripts, in order to investigate the presence of hitherto unnanotated regions of Drosophila transcriptome. Bioinformatic analysis of 1,303 Drosophila ORESTES clusters identified 68 sequences derived from unannotated regions in the current Drosophila genome version (4.3). Of these, a set of 38 was analysed by polyA+ northern blot hybridization, validating 17 (50%) new exons of low abundance transcripts. For one of these ESTs, we obtained the cDNA encompassing the complete coding sequence of a new serine protease, named SP212. The SP212 gene is part of a serine protease gene cluster located in the chromosome region 88A12-B1. This cluster includes the predicted genes CG9631, CG9649 and CG31326, which were previously identified as up-regulated after immune challenges in genomic-scale microarray analysis. In agreement with the proposal that this locus is co-regulated in response to microorganisms infection, we show here that SP212 is also up-regulated upon injury. Using the ORESTES methodology we identified 17 novel exons from low abundance Drosophila transcripts, and through a PCR approach the complete CDS of one of these transcripts was defined. Our results show that the computational identification and manual inspection are not sufficient to annotate a genome in the absence of experimentally derived data.

Identification of unannotated exons of low abundance transcripts in Drosophila melanogaster and cloning of a new serine protease gene upregulated upon injury

PubMed Central

Maia, Rafaela M; Valente, Valeria; Cunha, Marco AV; Sousa, Josane F; Araujo, Daniela D; Silva, Wilson A; Zago, Marco A; Dias-Neto, Emmanuel; Souza, Sandro J; Simpson, Andrew JG; Monesi, Nadia; Ramos, Ricardo GP; Espreafico, Enilza M; Paçó-Larson, Maria L

2007-01-01

Background The sequencing of the D.melanogaster genome revealed an unexpected small number of genes (~ 14,000) indicating that mechanisms acting on generation of transcript diversity must have played a major role in the evolution of complex metazoans. Among the most extensively used mechanisms that accounts for this diversity is alternative splicing. It is estimated that over 40% of Drosophila protein-coding genes contain one or more alternative exons. A recent transcription map of the Drosophila embryogenesis indicates that 30% of the transcribed regions are unannotated, and that 1/3 of this is estimated as missed or alternative exons of previously characterized protein-coding genes. Therefore, the identification of the variety of expressed transcripts depends on experimental data for its final validation and is continuously being performed using different approaches. We applied the Open Reading Frame Expressed Sequence Tags (ORESTES) methodology, which is capable of generating cDNA data from the central portion of rare transcripts, in order to investigate the presence of hitherto unnanotated regions of Drosophila transcriptome. Results Bioinformatic analysis of 1,303 Drosophila ORESTES clusters identified 68 sequences derived from unannotated regions in the current Drosophila genome version (4.3). Of these, a set of 38 was analysed by polyA+ northern blot hybridization, validating 17 (50%) new exons of low abundance transcripts. For one of these ESTs, we obtained the cDNA encompassing the complete coding sequence of a new serine protease, named SP212. The SP212 gene is part of a serine protease gene cluster located in the chromosome region 88A12-B1. This cluster includes the predicted genes CG9631, CG9649 and CG31326, which were previously identified as up-regulated after immune challenges in genomic-scale microarray analysis. In agreement with the proposal that this locus is co-regulated in response to microorganisms infection, we show here that SP212 is also up-regulated upon injury. Conclusion Using the ORESTES methodology we identified 17 novel exons from low abundance Drosophila transcripts, and through a PCR approach the complete CDS of one of these transcripts was defined. Our results show that the computational identification and manual inspection are not sufficient to annotate a genome in the absence of experimentally derived data. PMID:17650329

The Vigna unguiculata Gene Expression Atlas (VuGEA) from de novo assembly and quantification of RNA-seq data provides insights into seed maturation mechanisms.

PubMed

Yao, Shaolun; Jiang, Chuan; Huang, Ziyue; Torres-Jerez, Ivone; Chang, Junil; Zhang, Heng; Udvardi, Michael; Liu, Renyi; Verdier, Jerome

2016-10-01

Legume research and cultivar development are important for sustainable food production, especially of high-protein seed. Thanks to the development of deep-sequencing technologies, crop species have been taken to the front line, even without completion of their genome sequences. Black-eyed pea (Vigna unguiculata) is a legume species widely grown in semi-arid regions, which has high potential to provide stable seed protein production in a broad range of environments, including drought conditions. The black-eyed pea reference genotype has been used to generate a gene expression atlas of the major plant tissues (i.e. leaf, root, stem, flower, pod and seed), with a developmental time series for pods and seeds. From these various organs, 27 cDNA libraries were generated and sequenced, resulting in more than one billion reads. Following filtering, these reads were de novo assembled into 36 529 transcript sequences that were annotated and quantified across the different tissues. A set of 24 866 unique transcript sequences, called Unigenes, was identified. All the information related to transcript identification, annotation and quantification were stored into a gene expression atlas webserver (http://vugea.noble.org), providing a user-friendly interface and necessary tools to analyse transcript expression in black-eyed pea organs and to compare data with other legume species. Using this gene expression atlas, we inferred details of molecular processes that are active during seed development, and identified key putative regulators of seed maturation. Additionally, we found evidence for conservation of regulatory mechanisms involving miRNA in plant tissues subjected to drought and seeds undergoing desiccation. © 2016 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

Title: Comparative transcriptome profiling of the human and mouse dorsal root ganglia: an RNA-seq-based resource for pain and sensory neuroscience research.

PubMed

Ray, Pradipta; Torck, Andrew; Quigley, Lilyana; Wangzhou, Andi; Neiman, Matthew; Rao, Chandranshu; Lam, Tiffany; Kim, Ji-Young; Kim, Tae Hoon; Zhang, Michael Q; Dussor, Gregory; Price, Theodore J

2018-03-20

Molecular neurobiological insight into human nervous tissues is needed to generate next generation therapeutics for neurological disorders like chronic pain. We obtained human Dorsal Root Ganglia (DRG) samples from organ donors and performed RNA-sequencing (RNA-seq) to study the human DRG (hDRG) transcriptional landscape, systematically comparing it with publicly available data from a variety of human and orthologous mouse tissues, including mouse DRG (mDRG). We characterized the hDRG transcriptional profile in terms of tissue-restricted gene co-expression patterns and putative transcriptional regulators, and formulated an information-theoretic framework to quantify DRG enrichment. Relevant gene families and pathways were also analyzed, including transcription factors (TFs), g-protein coupled receptors (GCPRs) and ion channels. Our analyses reveal a hDRG-enriched protein-coding gene set (∼140), some of which have not been described in the context of DRG or pain signaling. A majority of these show conserved enrichment in mDRG, and were mined for known drug - gene product interactions. Conserved enrichment of the vast majority of TFs suggest that the mDRG is a faithful model system for studying hDRGs, due to evolutionarily conserved regulatory programs. Comparison of hDRG and tibial nerve transcriptomes suggest trafficking of neuronal mRNA to axons in adult hDRG, and are consistent with studies of axonal transport in rodent sensory neurons. We present our work as an online, searchable repository (https://www.utdallas.edu/bbs/painneurosciencelab/sensoryomics/drgtxome), creating a resource for the community. Our analyses provide insight into DRG biology for guiding development of novel therapeutics, and a blueprint for cross-species transcriptomic analyses.

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis.

PubMed

Tsuchiya, Megumi; Karim, M Rezaul; Matsumoto, Taro; Ogawa, Hidesato; Taniguchi, Hiroaki

2017-01-24

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

De novo transcript sequence reconstruction from RNA-Seq: reference generation and analysis with Trinity

PubMed Central

Yassour, Moran; Grabherr, Manfred; Blood, Philip D.; Bowden, Joshua; Couger, Matthew Brian; Eccles, David; Li, Bo; Lieber, Matthias; MacManes, Matthew D.; Ott, Michael; Orvis, Joshua; Pochet, Nathalie; Strozzi, Francesco; Weeks, Nathan; Westerman, Rick; William, Thomas; Dewey, Colin N.; Henschel, Robert; LeDuc, Richard D.; Friedman, Nir; Regev, Aviv

2013-01-01

De novo assembly of RNA-Seq data allows us to study transcriptomes without the need for a genome sequence, such as in non-model organisms of ecological and evolutionary importance, cancer samples, or the microbiome. In this protocol, we describe the use of the Trinity platform for de novo transcriptome assembly from RNA-Seq data in non-model organisms. We also present Trinity’s supported companion utilities for downstream applications, including RSEM for transcript abundance estimation, R/Bioconductor packages for identifying differentially expressed transcripts across samples, and approaches to identify protein coding genes. In an included tutorial we provide a workflow for genome-independent transcriptome analysis leveraging the Trinity platform. The software, documentation and demonstrations are freely available from http://trinityrnaseq.sf.net. PMID:23845962

Robust dynamics in minimal hybrid models of genetic networks

PubMed Central

Perkins, Theodore J.; Wilds, Roy; Glass, Leon

2010-01-01

Many gene-regulatory networks necessarily display robust dynamics that are insensitive to noise and stable under evolution. We propose that a class of hybrid systems can be used to relate the structure of these networks to their dynamics and provide insight into the origin of robustness. In these systems, the genes are represented by logical functions, and the controlling transcription factor protein molecules are real variables, which are produced and destroyed. As the transcription factor concentrations cross thresholds, they control the production of other transcription factors. We discuss mathematical analysis of these systems and show how the concepts of robustness and minimality can be used to generate putative logical organizations based on observed symbolic sequences. We apply the methods to control of the cell cycle in yeast. PMID:20921006

Robust dynamics in minimal hybrid models of genetic networks.

PubMed

Perkins, Theodore J; Wilds, Roy; Glass, Leon

2010-11-13

Many gene-regulatory networks necessarily display robust dynamics that are insensitive to noise and stable under evolution. We propose that a class of hybrid systems can be used to relate the structure of these networks to their dynamics and provide insight into the origin of robustness. In these systems, the genes are represented by logical functions, and the controlling transcription factor protein molecules are real variables, which are produced and destroyed. As the transcription factor concentrations cross thresholds, they control the production of other transcription factors. We discuss mathematical analysis of these systems and show how the concepts of robustness and minimality can be used to generate putative logical organizations based on observed symbolic sequences. We apply the methods to control of the cell cycle in yeast.

Promoter mapping of the mouse Tcp-10bt gene in transgenic mice identifies essential male germ cell regulatory sequences.

PubMed

Ewulonu, U K; Snyder, L; Silver, L M; Schimenti, J C

1996-03-01

Transgenic mice were generated to localize essential promoter elements in the mouse testis-expressed Tcp-10 genes. These genes are expressed exclusively in male germ cells, and exhibit a diffuse range of transcriptional start sites, possibly due to the absence of a TATA box. A series of transgene constructs containing different amounts of 5' flanking DNA revealed that all sequences necessary for appropriate temporal and tissue-specific transcription of Tcp-10 reside between positions -1 to -973. All transgenic animals containing these sequences expressed a chimeric transgene at high levels, in a pattern that paralleled the endogenous genes. These experiments further defined a 227 bp fragment from -746 to -973 that was absolutely essential for expression. In a gel-shift assay, this 227-bp fragment bound nuclear protein from testis, but not other tissues, to yield two retarded bands. Sequence analysis of this fragment revealed a half-site for the AP-2 transcription factor recognition sequence. Gel shift assays using native or mutant oligonucleotides demonstrated that the putative AP-2 recognition sequence was essential for generating the retarded bands. Since the binding activity is testis-specific, but AP-2 expression is not exclusive to male germ cells, it is possible that transcription of Tcp-10 requires interaction between AP-2 and a germ cell-specific transcription factor.

Induction of heat-shock response and alterations of protein phosphorylation by a novel topoisomerase II inhibitor, withangulatin A, in 9L rat brain tumor cells.

PubMed

Lee, W C; Lin, K Y; Chen, C M; Chen, Z T; Liu, H J; Lai, Y K

1991-10-01

Withangulatin A is a newly identified in vitro topoisomerase II inhibitor isolated from the Chinese antitumor herb Physalis angulata. In vivo, it was found to be cytotoxic, capable of suppressing general protein synthesis and of inducing the synthesis of a small set of proteins including those generated by heat-shock treatment. The 70 kDa protein generated by withangulatin A was unequivocally identified as the heat-shock protein 70 (HSP70) since both proteins migrated to the same position on two-dimensional polyacrylamide gels, could be recognized by a monoclonal antibody to human HSP70, and exhibited identical peptide maps. The induction of protein synthesis by withangulatin A was regulated at the transcriptional level since it was aborted in cells pre-treated with actinomycin D. However, the initiation of this process did not require de novo protein synthesis since it was not affected by cycloheximide. Other cellular effect of withangulatin A was alterations of protein phosphorylation including an enhancement of phosphorylation of a 65 kDa protein which was also detected in the heat-shocked cells. Moreover, this process was observed within 7.5 min after the initial heat treatment which is much faster than the onset of HSP synthesis. Therefore, increased phosphorylation of the 65 kDa protein may represent one of the earliest signals generated by both heat-shock and withangluatin A and may be involved in the upstream regulation of heat-shock response in cells.

Expression profiling of Chrysanthemum crassum under salinity stress and the initiation of morphological changes

PubMed Central

Guan, Zhiyong; Feng, Yitong; Song, Aiping; Shi, Xiaomeng; Mao, Yachao; Chen, Sumei; Jiang, Jiafu; Ding, Lian; Chen, Fadi

2017-01-01

Chrysanthemum crassum is a decaploid species of Chrysanthemum with high stress tolerance that allows survival under salinity stress while maintaining a relatively ideal growth rate. We previously recorded morphological changes after salt treatment, such as the expansion of leaf cells. To explore the underlying salinity tolerance mechanisms, we used an Illumina platform and obtained three sequencing libraries from samples collected after 0 h, 12 h and 24 h of salt treatment. Following de novo assembly, 154,944 transcripts were generated, and 97,833 (63.14%) transcripts were annotated, including 55 Gene Ontology (GO) terms and 128 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The expression profile of C. crassum was globally altered after salt treatment. We selected functional genes and pathways that may contribute to salinity tolerance and identified some factors involved in the salinity tolerance strategies of C. crassum, such as signal transduction, transcription factors and plant hormone regulation, enhancement of energy metabolism, functional proteins and osmolyte synthesis, reactive oxygen species (ROS) scavenging, photosystem protection and recovery, and cell wall protein modifications. Forty-six genes were selected for quantitative real-time polymerase chain reaction detection, and their expression patterns were shown to be consistent with the changes in their transcript abundance determined by RNA sequencing. PMID:28437448

TALEN-engineered AR gene rearrangements reveal endocrine uncoupling of androgen receptor in prostate cancer

PubMed Central

Nyquist, Michael D.; Li, Yingming; Hwang, Tae Hyun; Manlove, Luke S.; Vessella, Robert L.; Silverstein, Kevin A. T.; Voytas, Daniel F.; Dehm, Scott M.

2013-01-01

Androgen receptor (AR) target genes direct development and survival of the prostate epithelial lineage, including prostate cancer (PCa). Thus, endocrine therapies that inhibit the AR ligand-binding domain (LBD) are effective in treating PCa. AR transcriptional reactivation is central to resistance, as evidenced by the efficacy of AR retargeting in castration-resistant PCa (CRPC) with next-generation endocrine therapies abiraterone and enzalutamide. However, resistance to abiraterone and enzalutamide limits this efficacy in most men, and PCa remains the second-leading cause of male cancer deaths. Here we show that AR gene rearrangements in CRPC tissues underlie a completely androgen-independent, yet AR-dependent, resistance mechanism. We discovered intragenic AR gene rearrangements in CRPC tissues, which we modeled using transcription activator-like effector nuclease (TALEN)-mediated genome engineering. This modeling revealed that these AR gene rearrangements blocked full-length AR synthesis, but promoted expression of truncated AR variant proteins lacking the AR ligand-binding domain. Furthermore, these AR variant proteins maintained the constitutive activity of the AR transcriptional program and a CRPC growth phenotype independent of full-length AR or androgens. These findings demonstrate that AR gene rearrangements are a unique resistance mechanism by which AR transcriptional activity can be uncoupled from endocrine regulation in CRPC. PMID:24101480

Whole Transcriptome of the Venom Gland from Urodacus yaschenkoi Scorpion

PubMed Central

Juárez-González, Víctor Rivelino; Possani, Lourival D.

2015-01-01

Australian scorpion venoms have been poorly studied, probably because they do not pose an evident threat to humans. In addition, the continent has other medically important venomous animals capable of causing serious health problems. Urodacus yaschenkoi belongs to the most widely distributed family of Australian scorpions (Urodacidae) and it is found all over the continent, making it a useful model system for studying venom composition and evolution. This communication reports the whole set of mRNA transcripts produced by the venom gland. U. yaschenkoi venom is as complex as its overseas counterparts. These transcripts certainly code for several components similar to known scorpion venom components, such as: alpha-KTxs, beta-KTxs, calcins, protease inhibitors, antimicrobial peptides, sodium-channel toxins, toxin-like peptides, allergens, La1-like, hyaluronidases, ribosomal proteins, proteasome components and proteins related to cellular processes. A comparison with the venom gland transcriptome of Centruroides noxius (Buthidae) showed that these two scorpions have similar components related to biological processes, although important differences occur among the venom toxins. In contrast, a comparison with sequences reported for Urodacus manicatus revealed that these two Urodacidae species possess the same subfamily of scorpion toxins. A comparison with sequences of an U. yaschenkoi cDNA library previously reported by our group showed that both techniques are reliable for the description of the venom components, but the whole transcriptome generated with Next Generation Sequencing platform provides sequences of all transcripts expressed. Several of which were identified in the proteome, but many more transcripts were identified including uncommon transcripts. The information reported here constitutes a reference for non-Buthidae scorpion venoms, providing a comprehensive view of genes that are involved in venom production. Further, this work identifies new putative bioactive compounds that could be used to seed research into new pharmacological compounds and increase our understanding of the function of different ion channels. PMID:26020943

Whole Transcriptome of the Venom Gland from Urodacus yaschenkoi Scorpion.

PubMed

Luna-Ramírez, Karen; Quintero-Hernández, Verónica; Juárez-González, Víctor Rivelino; Possani, Lourival D

2015-01-01

Australian scorpion venoms have been poorly studied, probably because they do not pose an evident threat to humans. In addition, the continent has other medically important venomous animals capable of causing serious health problems. Urodacus yaschenkoi belongs to the most widely distributed family of Australian scorpions (Urodacidae) and it is found all over the continent, making it a useful model system for studying venom composition and evolution. This communication reports the whole set of mRNA transcripts produced by the venom gland. U. yaschenkoi venom is as complex as its overseas counterparts. These transcripts certainly code for several components similar to known scorpion venom components, such as: alpha-KTxs, beta-KTxs, calcins, protease inhibitors, antimicrobial peptides, sodium-channel toxins, toxin-like peptides, allergens, La1-like, hyaluronidases, ribosomal proteins, proteasome components and proteins related to cellular processes. A comparison with the venom gland transcriptome of Centruroides noxius (Buthidae) showed that these two scorpions have similar components related to biological processes, although important differences occur among the venom toxins. In contrast, a comparison with sequences reported for Urodacus manicatus revealed that these two Urodacidae species possess the same subfamily of scorpion toxins. A comparison with sequences of an U. yaschenkoi cDNA library previously reported by our group showed that both techniques are reliable for the description of the venom components, but the whole transcriptome generated with Next Generation Sequencing platform provides sequences of all transcripts expressed. Several of which were identified in the proteome, but many more transcripts were identified including uncommon transcripts. The information reported here constitutes a reference for non-Buthidae scorpion venoms, providing a comprehensive view of genes that are involved in venom production. Further, this work identifies new putative bioactive compounds that could be used to seed research into new pharmacological compounds and increase our understanding of the function of different ion channels.

Regulation of RE1 Protein Silencing Transcription Factor (REST) Expression by HIP1 Protein Interactor (HIPPI)*

PubMed Central

Datta, Moumita; Bhattacharyya, Nitai P.

2011-01-01

Earlier we have shown that the proapoptotic protein HIPPI (huntingtin interacting protein 1 (HIP1) protein interactor) along with its molecular partner HIP1 could regulate transcription of the caspase-1 gene. Here we report that RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a new transcriptional target of HIPPI. HIPPI could bind to the promoter of REST and increased its expression in neuronal as well as non-neuronal cells. Such activation of REST down-regulated expression of REST target genes, such as brain-derived neurotrophic factor (BDNF) or proenkephalin (PENK). The ability of HIPPI to activate REST gene transcription was dependent on HIP1, the nuclear transporter of HIPPI. Using a Huntington disease cell model, we have demonstrated that feeble interaction of HIP1 with mutant huntingtin protein resulted in increased nuclear accumulation of HIPPI and HIP1, leading to higher occupancy of HIPPI at the REST promoter, triggering its transcriptional activation and consequent repression of REST target genes. This novel transcription regulatory mechanism of REST by HIPPI may contribute to the deregulation of transcription observed in the cell model of Huntington disease. PMID:21832040

Regulation of RE1 protein silencing transcription factor (REST) expression by HIP1 protein interactor (HIPPI).

PubMed

Datta, Moumita; Bhattacharyya, Nitai P

2011-09-30

Earlier we have shown that the proapoptotic protein HIPPI (huntingtin interacting protein 1 (HIP1) protein interactor) along with its molecular partner HIP1 could regulate transcription of the caspase-1 gene. Here we report that RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a new transcriptional target of HIPPI. HIPPI could bind to the promoter of REST and increased its expression in neuronal as well as non-neuronal cells. Such activation of REST down-regulated expression of REST target genes, such as brain-derived neurotrophic factor (BDNF) or proenkephalin (PENK). The ability of HIPPI to activate REST gene transcription was dependent on HIP1, the nuclear transporter of HIPPI. Using a Huntington disease cell model, we have demonstrated that feeble interaction of HIP1 with mutant huntingtin protein resulted in increased nuclear accumulation of HIPPI and HIP1, leading to higher occupancy of HIPPI at the REST promoter, triggering its transcriptional activation and consequent repression of REST target genes. This novel transcription regulatory mechanism of REST by HIPPI may contribute to the deregulation of transcription observed in the cell model of Huntington disease.

The Zur regulon of Corynebacterium glutamicum ATCC 13032

PubMed Central

2010-01-01

Background Zinc is considered as an essential element for all living organisms, but it can be toxic at large concentrations. Bacteria therefore tightly regulate zinc metabolism. The Cg2502 protein of Corynebacterium glutamicum was a candidate to control zinc metabolism in this species, since it was classified as metalloregulator of the zinc uptake regulator (Zur) subgroup of the ferric uptake regulator (Fur) family of DNA-binding transcription regulators. Results The cg2502 (zur) gene was deleted in the chromosome of C. glutamicum ATCC 13032 by an allelic exchange procedure to generate the zur-deficient mutant C. glutamicum JS2502. Whole-genome DNA microarray hybridizations and real-time RT-PCR assays comparing the gene expression in C. glutamicum JS2502 with that of the wild-type strain detected 18 genes with enhanced expression in the zur mutant. The expression data were combined with results from cross-genome comparisons of shared regulatory sites, revealing the presence of candidate Zur-binding sites in the mapped promoter regions of five transcription units encoding components of potential zinc ABC-type transporters (cg0041-cg0042/cg0043; cg2911-cg2912-cg2913), a putative secreted protein (cg0040), a putative oxidoreductase (cg0795), and a putative P-loop GTPase of the COG0523 protein family (cg0794). Enhanced transcript levels of the respective genes in C. glutamicum JS2502 were verified by real-time RT-PCR, and complementation of the mutant with a wild-type zur gene reversed the effect of differential gene expression. The zinc-dependent expression of the putative cg0042 and cg2911 operons was detected in vivo with a gfp reporter system. Moreover, the zinc-dependent binding of purified Zur protein to double-stranded 40-mer oligonucleotides containing candidate Zur-binding sites was demonstrated in vitro by DNA band shift assays. Conclusion Whole-genome expression profiling and DNA band shift assays demonstrated that Zur directly represses in a zinc-dependent manner the expression of nine genes organized in five transcription units. Accordingly, the Zur (Cg2502) protein is the key transcription regulator for genes involved in zinc homeostasis in C. glutamicum. PMID:20055984

Circadian signaling in Homarus americanus: Region-specific de novo assembled transcriptomes show that both the brain and eyestalk ganglia possess the molecular components of a putative clock system.

PubMed

Christie, Andrew E; Yu, Andy; Pascual, Micah G; Roncalli, Vittoria; Cieslak, Matthew C; Warner, Amanda N; Lameyer, Tess J; Stanhope, Meredith E; Dickinson, Patsy S; Joe Hull, J

2018-04-11

Essentially all organisms exhibit recurring patterns of physiology/behavior that oscillate with a period of ~24-h and are synchronized to the solar day. Crustaceans are no exception, with robust circadian rhythms having been documented in many members of this arthropod subphylum. However, little is known about the molecular underpinnings of their circadian rhythmicity. Moreover, the location of the crustacean central clock has not been firmly established, although both the brain and eyestalk ganglia have been hypothesized as loci. The American lobster, Homarus americanus, is known to exhibit multiple circadian rhythms, and immunodetection data suggest that its central clock is located within the eyestalk ganglia rather than in the brain. Here, brain- and eyestalk ganglia-specific transcriptomes were generated and used to assess the presence/absence of transcripts encoding the commonly recognized protein components of arthropod circadian signaling systems in these two regions of the lobster central nervous system. Transcripts encoding putative homologs of the core clock proteins clock, cryptochrome 2, cycle, period and timeless were found in both the brain and eyestalk ganglia assemblies, as were transcripts encoding similar complements of putative clock-associated, clock input pathway and clock output pathway proteins. The presence and identity of transcripts encoding core clock proteins in both regions were confirmed using PCR. These findings suggest that both the brain and eyestalk ganglia possess all of the molecular components needed for the establishment of a circadian signaling system. Whether the brain and eyestalk clocks are independent of one another or represent a single timekeeping system remains to be determined. Interestingly, while most of the proteins deduced from the identified transcripts are shared by both the brain and eyestalk ganglia, assembly-specific isoforms were also identified, e.g., several period variants, suggesting the possibility of region-specific variation in clock function, especially if the brain and eyestalk clocks represent independent oscillators. Copyright © 2018 Elsevier B.V. All rights reserved.

Simulation-Based Validation of the p53 Transcriptional Activity with Hybrid Functional Petri Net.

PubMed

Doi, Atsushi; Nagasaki, Masao; Matsuno, Hiroshi; Miyano, Satoru

2011-01-01

MDM2 and p19ARF are essential proteins in cancer pathways forming a complex with protein p53 to control the transcriptional activity of protein p53. It is confirmed that protein p53 loses its transcriptional activity by forming the functional dimer with protein MDM2. However, it is still unclear that protein p53 keeps its transcriptional activity when it forms the trimer with proteins MDM2 and p19ARF. We have observed mutual behaviors among genes p53, MDM2, p19ARF and their products on a computational model with hybrid functional Petri net (HFPN) which is constructed based on information described in the literature. The simulation results suggested that protein p53 should have the transcriptional activity in the forms of the trimer of proteins p53, MDM2, and p19ARF. This paper also discusses the advantages of HFPN based modeling method in terms of pathway description for simulations.

Novel mechanism of gene regulation: the protein Rv1222 of Mycobacterium tuberculosis inhibits transcription by anchoring the RNA polymerase onto DNA.

PubMed

Rudra, Paulami; Prajapati, Ranjit Kumar; Banerjee, Rajdeep; Sengupta, Shreya; Mukhopadhyay, Jayanta

2015-07-13

We propose a novel mechanism of gene regulation in Mycobacterium tuberculosis where the protein Rv1222 inhibits transcription by anchoring RNA polymerase (RNAP) onto DNA. In contrast to our existing knowledge that transcriptional repressors function either by binding to DNA at specific sequences or by binding to RNAP, we show that Rv1222-mediated transcription inhibition requires simultaneous binding of the protein to both RNAP and DNA. We demonstrate that the positively charged C-terminus tail of Rv1222 is responsible for anchoring RNAP on DNA, hence the protein slows down the movement of RNAP along the DNA during transcription elongation. The interaction between Rv1222 and DNA is electrostatic, thus the protein could inhibit transcription from any gene. As Rv1222 slows down the RNA synthesis, upon expression of the protein in Mycobacterium smegmatis or Escherichia coli, the growth rate of the bacteria is severely impaired. The protein does not possess any significant affinity for DNA polymerase, thus, is unable to inhibit DNA synthesis. The proposed mechanism by which Rv1222 inhibits transcription reveals a new repertoire of prokaryotic gene regulation. © Crown copyright 2015.

Conserved sequence-specific lincRNA-steroid receptor interactions drive transcriptional repression and direct cell fate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hudson, William H.; Pickard, Mark R.; de Vera, Ian Mitchelle S.

2014-12-23

The majority of the eukaryotic genome is transcribed, generating a significant number of long intergenic noncoding RNAs (lincRNAs). Although lincRNAs represent the most poorly understood product of transcription, recent work has shown lincRNAs fulfill important cellular functions. In addition to low sequence conservation, poor understanding of structural mechanisms driving lincRNA biology hinders systematic prediction of their function. Here we report the molecular requirements for the recognition of steroid receptors (SRs) by the lincRNA growth arrest-specific 5 (Gas5), which regulates steroid-mediated transcriptional regulation, growth arrest and apoptosis. We identify the functional Gas5-SR interface and generate point mutations that ablate the SR-Gas5more » lincRNA interaction, altering Gas5-driven apoptosis in cancer cell lines. Further, we find that the Gas5 SR-recognition sequence is conserved among haplorhines, with its evolutionary origin as a splice acceptor site. This study demonstrates that lincRNAs can recognize protein targets in a conserved, sequence-specific manner in order to affect critical cell functions.« less

Single molecule real-time sequencing of Xanthomonas oryzae genomes reveals a dynamic structure and complex TAL (transcription activator-like) effector gene relationships

PubMed Central

Booher, Nicholas J.; Carpenter, Sara C. D.; Sebra, Robert P.; Wang, Li; Salzberg, Steven L.; Leach, Jan E.

2015-01-01

Pathogen-injected, direct transcriptional activators of host genes, TAL (transcription activator-like) effectors play determinative roles in plant diseases caused by Xanthomonas spp. A large domain of nearly identical, 33–35 aa repeats in each protein mediates DNA recognition. This modularity makes TAL effectors customizable and thus important also in biotechnology. However, the repeats render TAL effector (tal) genes nearly impossible to assemble using next-generation, short reads. Here, we demonstrate that long-read, single molecule real-time (SMRT) sequencing solves this problem. Taking an ensemble approach to first generate local, tal gene contigs, we correctly assembled de novo the genomes of two strains of the rice pathogen X. oryzae completed previously using the Sanger method and even identified errors in those references. Sequencing two more strains revealed a dynamic genome structure and a striking plasticity in tal gene content. Our results pave the way for population-level studies to inform resistance breeding, improve biotechnology and probe TAL effector evolution. PMID:27148456

FoxO proteins restrain osteoclastogenesis and bone resorption by attenuating H2O2 accumulation

PubMed Central

Bartell, Shoshana M.; Kim, Ha-Neui; Ambrogini, Elena; Han, Li; Iyer, Srividhya; Serra Ucer, S.; Rabinovitch, Peter; Jilka, Robert L.; Weinstein, Robert S.; Zhao, Haibo; O’Brien, Charles A.; Manolagas, Stavros C.; Almeida, Maria

2014-01-01

Besides their cell-damaging effects in the setting of oxidative stress, reactive oxygen species (ROS) play an important role in physiological intracellular signalling by triggering proliferation and survival. FoxO transcription factors counteract ROS generation by upregulating antioxidant enzymes. Here we show that intracellular H2O2 accumulation is a critical and purposeful adaptation for the differentiation and survival of osteoclasts, the bone cells responsible for the resorption of mineralized bone matrix. Using mice with conditional loss or gain of FoxO transcription factor function, or mitochondria-targeted catalase in osteoclasts, we demonstrate this is achieved, at least in part, by downregulating the H2O2-inactivating enzyme catalase. Catalase downregulation results from the repression of the transcriptional activity of FoxO1, 3 and 4 by RANKL, the indispensable signal for the generation of osteoclasts, via an Akt-mediated mechanism. Notably, mitochondria-targeted catalase prevented the loss of bone caused by loss of oestrogens, suggesting that decreasing H2O2 production in mitochondria may represent a rational pharmacotherapeutic approach to diseases with increased bone resorption. PMID:24781012

Multi‐omic profiling ­of EPO‐producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production

PubMed Central

Ley, Daniel; Seresht, Ali Kazemi; Engmark, Mikael; Magdenoska, Olivera; Nielsen, Kristian Fog; Kildegaard, Helene Faustrup

2015-01-01

ABSTRACT Chinese hamster ovary (CHO) cells are the preferred production host for many therapeutic proteins. The production of heterologous proteins in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi‐omics approach was applied to study the production of erythropoietin (EPO) in a panel of CHO‐K1 cells under growth‐limited and unlimited conditions in batch and chemostat cultures. Physiological characterization of the EPO‐producing cells included global transcriptome analysis, targeted metabolome analysis, including intracellular pools of glycolytic intermediates, NAD(P)H/NAD(P)+, adenine nucleotide phosphates (ANP), and extracellular concentrations of sugars, organic acids, and amino acids. Potential impact of EPO expression on the protein secretory pathway was assessed at multiple stages using quantitative PCR (qPCR), reverse transcription PCR (qRT‐PCR), Western blots (WB), and global gene expression analysis to assess EPO gene copy numbers, EPO gene expression, intracellular EPO retention, and differentially expressed genes functionally related to secretory protein processing, respectively. We found no evidence supporting the existence of production bottlenecks in energy metabolism (i.e., glycolytic metabolites, NAD(P)H/NAD(P)+ and ANPs) in batch culture or in the secretory protein production pathway (i.e., gene dosage, transcription and post‐translational processing of EPO) in chemostat culture at specific productivities up to 5 pg/cell/day. Time‐course analysis of high‐ and low‐producing clones in chemostat culture revealed rapid adaptation of transcription levels of amino acid catabolic genes in favor of EPO production within nine generations. Interestingly, the adaptation was followed by an increase in specific EPO productivity. Biotechnol. Bioeng. 2015;112: 2373–2387. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. PMID:25995028

Brain transcriptome perturbations in the Hfe(-/-) mouse model of genetic iron loading.

PubMed

Johnstone, Daniel; Graham, Ross M; Trinder, Debbie; Delima, Roheeth D; Riveros, Carlos; Olynyk, John K; Scott, Rodney J; Moscato, Pablo; Milward, Elizabeth A

2012-04-11

Severe disruption of brain iron homeostasis can cause fatal neurodegenerative disease, however debate surrounds the neurologic effects of milder, more common iron loading disorders such as hereditary hemochromatosis, which is usually caused by loss-of-function polymorphisms in the HFE gene. There is evidence from both human and animal studies that HFE gene variants may affect brain function and modify risks of brain disease. To investigate how disruption of HFE influences brain transcript levels, we used microarray and real-time reverse transcription polymerase chain reaction to assess the brain transcriptome in Hfe(-/-) mice relative to wildtype AKR controls (age 10 weeks, n≥4/group). The Hfe(-/-) mouse brain showed numerous significant changes in transcript levels (p<0.05) although few of these related to proteins directly involved in iron homeostasis. There were robust changes of at least 2-fold in levels of transcripts for prominent genes relating to transcriptional regulation (FBJ osteosarcoma oncogene Fos, early growth response genes), neurotransmission (glutamate NMDA receptor Grin1, GABA receptor Gabbr1) and synaptic plasticity and memory (calcium/calmodulin-dependent protein kinase IIα Camk2a). As previously reported for dietary iron-supplemented mice, there were altered levels of transcripts for genes linked to neuronal ceroid lipofuscinosis, a disease characterized by excessive lipofuscin deposition. Labile iron is known to enhance lipofuscin generation which may accelerate brain aging. The findings provide evidence that iron loading disorders can considerably perturb levels of transcripts for genes essential for normal brain function and may help explain some of the neurologic signs and symptoms reported in hemochromatosis patients. Copyright © 2012 Elsevier B.V. All rights reserved.

Transcriptome Analysis of the Chrysanthemum Foliar Nematode, Aphelenchoides ritzemabosi (Aphelenchida: Aphelenchoididae)

PubMed Central

Li, Jun-Yi; Xie, Hui; Xu, Chun-Ling; Li, Yu

2016-01-01

The chrysanthemum foliar nematode (CFN), Aphelenchoides ritzemabosi, is a plant parasitic nematode that attacks many plants. In this study, a transcriptomes of mixed-stage population of CFN was sequenced on the Illumina HiSeq 2000 platform. 68.10 million Illumina high quality paired end reads were obtained which generated 26,817 transcripts with a mean length of 1,032 bp and an N50 of 1,672 bp, of which 16,467 transcripts were annotated against six databases. In total, 20,311 coding region sequences (CDS), 495 simple sequence repeats (SSRs) and 8,353 single-nucleotide polymorphisms (SNPs) were predicted, respectively. The CFN with the most shared sequences was B. xylophilus with 16,846 (62.82%) common transcripts and 10,543 (39.31%) CFN transcripts matched sequences of all of four plant parasitic nematodes compared. A total of 111 CFN transcripts were predicted as homologues of 7 types of carbohydrate-active enzymes (CAZymes) with plant/fungal cell wall-degrading activities, fewer transcripts were predicted as homologues of plant cell wall-degrading enzymes than fungal cell wall-degrading enzymes. The phylogenetic analysis of GH5, GH16, GH43 and GH45 proteins between CFN and other organisms showed CFN and other nematodes have a closer phylogenetic relationship. In the CFN transcriptome, sixteen types of genes orthologues with seven classes of protein families involved in the RNAi pathway in C. elegans were predicted. This research provides comprehensive gene expression information at the transcriptional level, which will facilitate the elucidation of the molecular mechanisms of CFN and the distribution of gene functions at the macro level, potentially revealing improved methods for controlling CFN. PMID:27875578

Mechanism of short-term ERK activation by electromagnetic fields at mobile phone frequencies

PubMed Central

Friedman, Joseph; Kraus, Sarah; Hauptman, Yirmi; Schiff, Yoni; Seger, Rony

2007-01-01

The exposure to non-thermal microwave electromagnetic fields generated by mobile phones affects the expression of many proteins. This effect on transcription and protein stability can be mediated by the MAPK (mitogen-activated protein kinase) cascades, which serve as central signalling pathways and govern essentially all stimulated cellular processes. Indeed, long-term exposure of cells to mobile phone irradiation results in the activation of p38 as well as the ERK (extracellular-signal-regulated kinase) MAPKs. In the present study, we have studied the immediate effect of irradiation on the MAPK cascades, and found that ERKs, but not stress-related MAPKs, are rapidly activated in response to various frequencies and intensities. Using signalling inhibitors, we delineated the mechanism that is involved in this activation. We found that the first step is mediated in the plasma membrane by NADH oxidase, which rapidly generates ROS (reactive oxygen species). These ROS then directly stimulate MMPs (matrix metalloproteinases) and allow them to cleave and release Hb-EGF [heparin-binding EGF (epidermal growth factor)]. This secreted factor activates the EGF receptor, which in turn further activates the ERK cascade. Thus this study demonstrates for the first time a detailed molecular mechanism by which electromagnetic irradiation from mobile phones induces the activation of the ERK cascade and thereby induces transcription and other cellular processes. PMID:17456048

Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms

PubMed Central

Casewell, Nicholas R.; Wagstaff, Simon C.; Wüster, Wolfgang; Cook, Darren A. N.; Bolton, Fiona M. S.; King, Sarah I.; Pla, Davinia; Sanz, Libia; Calvete, Juan J.; Harrison, Robert A.

2014-01-01

Variation in venom composition is a ubiquitous phenomenon in snakes and occurs both interspecifically and intraspecifically. Venom variation can have severe outcomes for snakebite victims by rendering the specific antibodies found in antivenoms ineffective against heterologous toxins found in different venoms. The rapid evolutionary expansion of different toxin-encoding gene families in different snake lineages is widely perceived as the main cause of venom variation. However, this view is simplistic and disregards the understudied influence that processes acting on gene transcription and translation may have on the production of the venom proteome. Here, we assess the venom composition of six related viperid snakes and compare interspecific changes in the number of toxin genes, their transcription in the venom gland, and their translation into proteins secreted in venom. Our results reveal that multiple levels of regulation are responsible for generating variation in venom composition between related snake species. We demonstrate that differential levels of toxin transcription, translation, and their posttranslational modification have a substantial impact upon the resulting venom protein mixture. Notably, these processes act to varying extents on different toxin paralogs found in different snakes and are therefore likely to be as important as ancestral gene duplication events for generating compositionally distinct venom proteomes. Our results suggest that these processes may also contribute to altering the toxicity of snake venoms, and we demonstrate how this variability can undermine the treatment of a neglected tropical disease, snakebite. PMID:24927555

Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms.

PubMed

Casewell, Nicholas R; Wagstaff, Simon C; Wüster, Wolfgang; Cook, Darren A N; Bolton, Fiona M S; King, Sarah I; Pla, Davinia; Sanz, Libia; Calvete, Juan J; Harrison, Robert A

2014-06-24

Variation in venom composition is a ubiquitous phenomenon in snakes and occurs both interspecifically and intraspecifically. Venom variation can have severe outcomes for snakebite victims by rendering the specific antibodies found in antivenoms ineffective against heterologous toxins found in different venoms. The rapid evolutionary expansion of different toxin-encoding gene families in different snake lineages is widely perceived as the main cause of venom variation. However, this view is simplistic and disregards the understudied influence that processes acting on gene transcription and translation may have on the production of the venom proteome. Here, we assess the venom composition of six related viperid snakes and compare interspecific changes in the number of toxin genes, their transcription in the venom gland, and their translation into proteins secreted in venom. Our results reveal that multiple levels of regulation are responsible for generating variation in venom composition between related snake species. We demonstrate that differential levels of toxin transcription, translation, and their posttranslational modification have a substantial impact upon the resulting venom protein mixture. Notably, these processes act to varying extents on different toxin paralogs found in different snakes and are therefore likely to be as important as ancestral gene duplication events for generating compositionally distinct venom proteomes. Our results suggest that these processes may also contribute to altering the toxicity of snake venoms, and we demonstrate how this variability can undermine the treatment of a neglected tropical disease, snakebite.

The C-Module-Binding Factor Supports Amplification of TRE5-A Retrotransposons in the Dictyostelium discoideum Genome ▿

PubMed Central

Bilzer, Annika; Dölz, Heike; Reinhardt, Alexander; Schmith, Anika; Siol, Oliver; Winckler, Thomas

2011-01-01

Retrotransposable elements are molecular parasites that have invaded the genomes of virtually all organisms. Although retrotransposons encode essential proteins to mediate their amplification, they also require assistance by host cell-encoded machineries that perform functions such as DNA transcription and repair. The retrotransposon TRE5-A of the social amoeba Dictyostelium discoideum generates a notable amount of both sense and antisense RNAs, which are generated from element-internal promoters, located in the A module and the C module, respectively. We observed that TRE5-A retrotransposons depend on the C-module-binding factor (CbfA) to maintain high steady-state levels of TRE5-A transcripts and that CbfA supports the retrotransposition activity of TRE5-A elements. The carboxy-terminal domain of CbfA was found to be required and sufficient to mediate the accumulation of TRE5-A transcripts, but it did not support productive retrotransposition of TRE5-A. This result suggests different roles for CbfA protein domains in the regulation of TRE5-A retrotransposition frequency in D. discoideum cells. Although CbfA binds to the C module in vitro, the factor regulates neither C-module nor A-module promoter activity in vivo. We speculate that CbfA supports the amplification of TRE5-A retrotransposons by suppressing the expression of an as yet unidentified component of the cellular posttranscriptional gene silencing machinery. PMID:21076008

Extensive alternative splicing and dual promoter usage generate Tcf-1 protein isoforms with differential transcription control properties.

PubMed Central

Van de Wetering, M; Castrop, J; Korinek, V; Clevers, H

1996-01-01

Previously, we reported the isolation of cDNA clones representing four alternative splice forms of TCF-1, a T-cell-specific transcription factor. In the present study, Western blotting (immunoblotting) yielded a multitude of TCF-1 proteins ranging from 25-55 kDa, a pattern not simply explained from the known splice alternatives. Subsequent cDNA cloning, PCR amplification, and analysis by rapid amplification of 5' cDNA ends revealed (i) the presence of an alternative upstream promoter, which extended the known N terminus by 116 amino acids, (ii) the presence of four alternative exons, and (iii) the existence of a second reading frame in the last exon encoding an extended C terminus. Inclusion of the extended N terminus into the originally reported protein resulted in a striking similarity to the lymphoid factor Lef-1. Several of the TCF-1 isoforms, although less potent, mimicked Lef-1 in transactivating transcription through the T-cell receptor alpha-chain (TCR-alpha) enhancer. These data provide a molecular basis for the complexity of the expressed TCF-1 proteins and establish the existence of functional differences between these isoforms. Furthermore, the functional redundancy between Tcf-1 and Lef-1 explains the apparently normal TCR-alpha expression in single Tcf-1 or Lef-1 knockout mice despite the firm in vitro evidence for the importance of the Tcf/Lef site in the TCR-alpha enhancer. PMID:8622675

Can the 'neuron theory' be complemented by a universal mechanism for generic neuronal differentiation.

PubMed

Ernsberger, Uwe

2015-01-01

With the establishment of the 'neuron theory' at the turn of the twentieth century, this remarkably powerful term was introduced to name a breathtaking diversity of cells unified by a characteristic structural compartmentalization and unique information processing and propagating features. At the beginning of the twenty-first century, developmental, stem cell and reprogramming studies converged to suggest a common mechanism involved in the generation of possibly all vertebrate, and at least a significant number of invertebrate, neurons. Sox and, in particular, SoxB and SoxC proteins as well as basic helix-loop-helix proteins play major roles, even though their precise contributions to progenitor programming, proliferation and differentiation are not fully resolved. In addition to neuronal development, these transcription factors also regulate sensory receptor and endocrine cell development, thus specifying a range of cells with regulatory and communicative functions. To what extent microRNAs contribute to the diversification of these cell types is an upcoming question. Understanding the transcriptional and post-transcriptional regulation of genes coding for cell type-specific cytoskeletal and motor proteins as well as synaptic and ion channel proteins, which mark differences but also similarities between the three communicator cell types, will provide a key to the comprehension of their diversification and the signature of 'generic neuronal' differentiation. Apart from the general scientific significance of a putative universal core instruction for neuronal development, the impact of this line of research for cell replacement therapy and brain tumor treatment will be of considerable interest.

Differential gene expression and alternative splicing between diploid and tetraploid watermelon

PubMed Central

Saminathan, Thangasamy; Nimmakayala, Padma; Manohar, Sumanth; Malkaram, Sridhar; Almeida, Aldo; Cantrell, Robert; Tomason, Yan; Abburi, Lavanya; Rahman, Mohammad A.; Vajja, Venkata G.; Khachane, Amit; Kumar, Brajendra; Rajasimha, Harsha K.; Levi, Amnon; Wehner, Todd; Reddy, Umesh K.

2015-01-01

The exploitation of synthetic polyploids for producing seedless fruits is well known in watermelon. Tetraploid progenitors of triploid watermelon plants, compared with their diploid counterparts, exhibit wide phenotypic differences. Although many factors modulate alternative splicing (AS) in plants, the effects of autopolyploidization on AS are still unknown. In this study, we used tissues of leaf, stem, and fruit of diploid and tetraploid sweet watermelon to understand changes in gene expression and the occurrence of AS. RNA-sequencing analysis was performed along with reverse transcription quantitative PCR and rapid amplification of cDNA ends (RACE)-PCR to demonstrate changes in expression and splicing. All vegetative tissues except fruit showed an increased level of AS in the tetraploid watermelon throughout the growth period. The ploidy levels of diploids and the tetraploid were confirmed using a ploidy analyser. We identified 5362 and 1288 genes that were up- and downregulated, respectively, in tetraploid as compared with diploid plants. We further confirmed that 22 genes underwent AS events across tissues, indicating possibilities of generating different protein isoforms with altered functions of important transcription factors and transporters. Arginine biosynthesis, chlorophyllide synthesis, GDP mannose biosynthesis, trehalose biosynthesis, and starch and sucrose degradation pathways were upregulated in autotetraploids. Phloem protein 2, chloroplastic PGR5-like protein, zinc-finger protein, fructokinase-like 2, MYB transcription factor, and nodulin MtN21 showed AS in fruit tissues. These results should help in developing high-quality seedless watermelon and provide additional transcriptomic information related to other cucurbits. PMID:25520388

Enzymes Involved in Post-transcriptional RNA Metabolism in Gram-negative bacteria

PubMed Central

Mohanty, Bijoy K.

2018-01-01

Gene expression in Gram-negative bacteria is regulated at many levels, including transcription initiation, RNA processing, RNA/RNA interactions, mRNA decay, and translational controls involving enzymes that alter translational efficiency. In this chapter we discuss the various enzymes that control transcription, translation and RNA stability through RNA processing and degradation. RNA processing is essential to generate functional RNAs, while degradation helps control the steady-state level of each individual transcript. For example, all the pre-tRNAs are transcribed with extra nucleotides at both their 5′ and 3′ termini, which are subsequently processed to produce mature tRNAs that can be aminoacylated. Similarly, rRNAs that are transcribed as part of a 30S polycistronic transcript, are matured to individual 16S, 23S and 5S rRNAs. Decay of mRNAs plays a key role in gene regulation through controlling the steady-state level of each transcript, which is essential for maintaining appropriate protein levels. In addition, degradation of both translated and non-translated RNAs recycles nucleotides to facilitate new RNA synthesis. To carry out all these reactions Gram-negative bacteria employ a large number of endonucleases, exonucleases, RNA helicases, and poly(A) polymerase as well as proteins that regulate the catalytic activity of particular ribonucleases. Under certain stress conditions an additional group of specialized endonucleases facilitate the cell’s ability to adapt and survive. Many of the enzymes, such as RNase E, RNase III, polynucleotide phosphorylase, RNase R, and poly(A) polymerase I participate in multiple RNA processing and decay pathways. PMID:29676246

Chronic effects of clofibric acid in zebrafish (Danio rerio): a multigenerational study.

PubMed

Coimbra, Ana M; Peixoto, Maria João; Coelho, Inês; Lacerda, Ricardo; Carvalho, António Paulo; Gesto, Manuel; Lyssimachou, Angeliki; Lima, Daniela; Soares, Joana; André, Ana; Capitão, Ana; Castro, Luís Filipe C; Santos, Miguel M

2015-03-01

Clofibric acid (CA) is an active metabolite of the blood lipid lowering agent clofibrate, a pharmaceutical designed to work as agonist of peroxisome proliferator-activated receptor alpha (PPARa). It is the most commonly reported fibrate in aquatic environments with low degradation rate and potential environmental persistence. Previous fish exposures showed that CA may impact spermatogenesis, growth and the expression of fat binding protein genes. However, there are limited data on the effects of chronic multigenerational CA exposures. Here, we assessed chronic multigenerational effects of CA exposure using zebrafish (Danio rerio) as a teleost model. Zebrafish were exposed through the diet to CA (1 and 10mg/g) during their whole lifetime. Growth, reproduction-related parameters and embryonic development were assessed in the exposed fish (F1 generation) and their offspring (F2 generation), together with muscle triglyceride content and gonad histology. In order to study the potential underlying mechanisms, the transcription levels of genes coding for enzymes involved in lipid metabolism pathways were determined. The results show that chronic life-cycle exposure to CA induced a significant reduction in growth of F1 generation and lowered triglyceride muscle content (10mg/g group). Also, an impact in male gonad development was observed together with a decrease in the fecundity (10mg/g group) and higher frequency of embryo abnormalities in the offspring of fish exposed to the lowest CA dose. The profile of the target genes was sex- and tissue-dependent. In F1 an up-regulation of male hepatic pparaa, pparb and acox transcript levels was observed, suggesting an activation of the fatty acid metabolism (provided that transcript level change indicates also a protein level change). Interestingly, the F2 generation, raised with control diet, displayed a response pattern different from that observed in F1, showing an increase in weight in the descendants of CA exposed fish, in comparison with control animals, which points to a multigenerational effect. Copyright © 2015 Elsevier B.V. All rights reserved.

Probing transcription-specific outputs of β-catenin in vivo

PubMed Central

Valenta, Tomas; Gay, Max; Steiner, Sarah; Draganova, Kalina; Zemke, Martina; Hoffmans, Raymond; Cinelli, Paolo; Aguet, Michel; Sommer, Lukas; Basler, Konrad

2011-01-01

β-Catenin, apart from playing a cell-adhesive role, is a key nuclear effector of Wnt signaling. Based on activity assays in Drosophila, we generated mouse strains where the endogenous β-catenin protein is replaced by mutant forms, which retain the cell adhesion function but lack either or both of the N- and the C-terminal transcriptional outputs. The C-terminal activity is essential for mesoderm formation and proper gastrulation, whereas N-terminal outputs are required later during embryonic development. By combining the double-mutant β-catenin with a conditional null allele and a Wnt1-Cre driver, we probed the role of Wnt/β-catenin signaling in dorsal neural tube development. While loss of β-catenin protein in the neural tube results in severe cell adhesion defects, the morphology of cells and tissues expressing the double-mutant form is normal. Surprisingly, Wnt/β-catenin signaling activity only moderately regulates cell proliferation, but is crucial for maintaining neural progenitor identity and for neuronal differentiation in the dorsal spinal cord. Our model animals thus allow dissecting signaling and structural functions of β-catenin in vivo and provide the first genetic tool to generate cells and tissues that entirely and exclusively lack canonical Wnt pathway activity. PMID:22190459

Sequence and immunogenicity of a clinically approved novel measles virus vaccine vector

PubMed Central

Zuniga, Amando; Liniger, Mathias; Morin, Teldja Neige Azzouz; Marty, René R.; Wiegand, Marian; Ilter, Orhan; Weibel, Sara; Billeter, Martin A.; Knuchel, Marlyse C.; Naim, Hussein Y.

2013-01-01

The measles virus vaccine (MVbv) is a clinically certified and well-tolerated vaccine strain that has been given both parenterally and mucosally. It has been extensively used in children and has proven to be safe and effective in eliciting protective immunity. This specific strain was therefore chosen to generate a measles viral vector. The genome of the commercial MVbv vaccine strain was isolated, sequenced and a plasmid, p(+)MVb, enabling transcription of the viral antigenome and rescue of MVb, was constructed. Phylogenic and phenotypic analysis revealed that MVbv and the rescued MVb constitute another evolutionary branch within the hitherto classified measles vaccines. Plasmid p(+)MVb was modified by insertion of artificial MV-type transcription units (ATUs) for the generation of recombinant viruses (rMVb) expressing additional proteins. Replication characteristics and immunogenicity of rMVb vectors were similar to the parental MVbv and to other vaccine strains. The expression of the additional proteins was stable over 10 serial virus transfers, which corresponds to an amplification greater than 1020. The excellent safety record and its efficient application as aerosol may add to the usefulness of the derived vectors. PMID:23324616

Towards decrypting cryptobiosis--analyzing anhydrobiosis in the tardigrade Milnesium tardigradum using transcriptome sequencing.

PubMed

Wang, Chong; Grohme, Markus A; Mali, Brahim; Schill, Ralph O; Frohme, Marcus

2014-01-01

Many tardigrade species are capable of anhydrobiosis; however, mechanisms underlying their extreme desiccation resistance remain elusive. This study attempts to quantify the anhydrobiotic transcriptome of the limno-terrestrial tardigrade Milnesium tardigradum. A prerequisite for differential gene expression analysis was the generation of a reference hybrid transcriptome atlas by assembly of Sanger, 454 and Illumina sequence data. The final assembly yielded 79,064 contigs (>100 bp) after removal of ribosomal RNAs. Around 50% of them could be annotated by SwissProt and NCBI non-redundant protein sequences. Analysis using CEGMA predicted 232 (93.5%) out of the 248 highly conserved eukaryotic genes in the assembly. We used this reference transcriptome for mapping and quantifying the expression of transcripts regulated under anhdydrobiosis in a time-series during dehydration and rehydration. 834 of the transcripts were found to be differentially expressed in a single stage (dehydration/inactive tun/rehydration) and 184 were overlapping in two stages while 74 were differentially expressed in all three stages. We have found interesting patterns of differentially expressed transcripts that are in concordance with a common hypothesis of metabolic shutdown during anhydrobiosis. This included down-regulation of several proteins of the DNA replication and translational machinery and protein degradation. Among others, heat shock proteins Hsp27 and Hsp30c were up-regulated in response to dehydration and rehydration. In addition, we observed up-regulation of ployubiquitin-B upon rehydration together with a higher expression level of several DNA repair proteins during rehydration than in the dehydration stage. Most of the transcripts identified to be differentially expressed had distinct cellular function. Our data suggest a concerted molecular adaptation in M. tardigradum that permits extreme forms of ametabolic states such as anhydrobiosis. It is temping to surmise that the desiccation tolerance of tradigrades can be achieved by a constitutive cellular protection system, probably in conjunction with other mechanisms such as rehydration-induced cellular repair.

Towards Decrypting Cryptobiosis—Analyzing Anhydrobiosis in the Tardigrade Milnesium tardigradum Using Transcriptome Sequencing

PubMed Central

Wang, Chong; Grohme, Markus A.; Mali, Brahim; Schill, Ralph O.; Frohme, Marcus

2014-01-01

Background Many tardigrade species are capable of anhydrobiosis; however, mechanisms underlying their extreme desiccation resistance remain elusive. This study attempts to quantify the anhydrobiotic transcriptome of the limno-terrestrial tardigrade Milnesium tardigradum. Results A prerequisite for differential gene expression analysis was the generation of a reference hybrid transcriptome atlas by assembly of Sanger, 454 and Illumina sequence data. The final assembly yielded 79,064 contigs (>100 bp) after removal of ribosomal RNAs. Around 50% of them could be annotated by SwissProt and NCBI non-redundant protein sequences. Analysis using CEGMA predicted 232 (93.5%) out of the 248 highly conserved eukaryotic genes in the assembly. We used this reference transcriptome for mapping and quantifying the expression of transcripts regulated under anhdydrobiosis in a time-series during dehydration and rehydration. 834 of the transcripts were found to be differentially expressed in a single stage (dehydration/inactive tun/rehydration) and 184 were overlapping in two stages while 74 were differentially expressed in all three stages. We have found interesting patterns of differentially expressed transcripts that are in concordance with a common hypothesis of metabolic shutdown during anhydrobiosis. This included down-regulation of several proteins of the DNA replication and translational machinery and protein degradation. Among others, heat shock proteins Hsp27 and Hsp30c were up-regulated in response to dehydration and rehydration. In addition, we observed up-regulation of ployubiquitin-B upon rehydration together with a higher expression level of several DNA repair proteins during rehydration than in the dehydration stage. Conclusions Most of the transcripts identified to be differentially expressed had distinct cellular function. Our data suggest a concerted molecular adaptation in M. tardigradum that permits extreme forms of ametabolic states such as anhydrobiosis. It is temping to surmise that the desiccation tolerance of tradigrades can be achieved by a constitutive cellular protection system, probably in conjunction with other mechanisms such as rehydration-induced cellular repair. PMID:24651535

Natural Genetic Variation Influences Protein Abundances in C. elegans Developmental Signalling Pathways

PubMed Central

Singh, Kapil Dev; Roschitzki, Bernd; Snoek, L. Basten; Grossmann, Jonas; Zheng, Xue; Elvin, Mark; Kamkina, Polina; Schrimpf, Sabine P.; Poulin, Gino B.; Kammenga, Jan E.; Hengartner, Michael O.

2016-01-01

Complex traits, including common disease-related traits, are affected by many different genes that function in multiple pathways and networks. The apoptosis, MAPK, Notch, and Wnt signalling pathways play important roles in development and disease progression. At the moment we have a poor understanding of how allelic variation affects gene expression in these pathways at the level of translation. Here we report the effect of natural genetic variation on transcript and protein abundance involved in developmental signalling pathways in Caenorhabditis elegans. We used selected reaction monitoring to analyse proteins from the abovementioned four pathways in a set of recombinant inbred lines (RILs) generated from the wild-type strains N2 (Bristol) and CB4856 (Hawaii) to enable quantitative trait locus (QTL) mapping. About half of the cases from the 44 genes tested showed a statistically significant change in protein abundance between various strains, most of these were however very weak (below 1.3-fold change). We detected a distant QTL on the left arm of chromosome II that affected protein abundance of the phosphatidylserine receptor protein PSR-1, and two separate QTLs that influenced embryonic and ionizing radiation-induced apoptosis on chromosome IV. Our results demonstrate that natural variation in C. elegans is sufficient to cause significant changes in signalling pathways both at the gene expression (transcript and protein abundance) and phenotypic levels. PMID:26985669

Antitumorigenic Effects of ZAKβ, an Alternative Splicing Isoform of ZAK.

PubMed

Lee, Jin-Sun; Lin, Yuh-Yih; Wang, Tsu-Shing; Liu, Jer-Yuh; Lin, Wei-Wen; Yang, Jaw-Ji

2018-02-28

Sterile alpha motif (SAM)- and leucine-zipper-containing kinase (ZAK) plays a role in the regulation of cell cycle progression and oncogenic transformation. The ZAK gene generates two transcript variants, ZAKα and ZAKβ, through alternative splicing. In this study, we identified that ZAKα proteins were upregulated in tumor tissues, whereas ZAKβ proteins were mostly expressed in corresponding normal tissues. The ectopically expressed ZAKβ proteins in cancer cells inhibited cancer cell proliferation as well as anchorage-independent growth. The ZAKβ:ZAKα protein ratio played a role in the regulation of the cyclic adenosine monophosphate (cAMP) signaling pathway, whereas high ZAKβ protein levels led to the activation of cAMP response element binding protein 1 (CREB1) and exerted antitumor properties. Overexpression of ZAKβ or CREB1 cDNAs in cancer cells inhibited anchorage-independent growth and also reduced the levels of cyclooxygenase 2 (Cox2) and β-catenin proteins. Cancer cells treated with doxorubicin (Doxo) resulted in the switching from the expression of ZAKα to ZAKβ and also inhibited cancer cell growth in soft agar, demonstrating that pharmacological drugs could be used to manipulate endogenous reprogramming splicing events and resulting in the activation of endogenous antitumorigenic properties. We showed that the two ZAK transcript variants, ZAKα and ZAKβ, had opposite biological functions in the regulation of tumor cell proliferation in that ZAKβ had powerful antitumor properties and that ZAKα could promote tumor growth.

Specific binding of a HeLa cell nuclear protein to RNA sequences in the human immunodeficiency virus transactivating region.

PubMed Central

Gaynor, R; Soultanakis, E; Kuwabara, M; Garcia, J; Sigman, D S

1989-01-01

The transactivator protein, tat, encoded by the human immunodeficiency virus is a key regulator of viral transcription. Activation by the tat protein requires sequences downstream of the transcription initiation site called the transactivating region (TAR). RNA derived from the TAR is capable of forming a stable stem-loop structure and the maintenance of both the stem structure and the loop sequences located between +19 and +44 is required for complete in vivo activation by tat. Gel retardation assays with RNA from both wild-type and mutant TAR constructs generated in vitro with SP6 polymerase indicated specific binding of HeLa nuclear proteins to the TAR. To characterize this RNA-protein interaction, a method of chemical "imprinting" has been developed using photoactivated uranyl acetate as the nucleolytic agent. This reagent nicks RNA under physiological conditions at all four nucleotides in a reaction that is independent of sequence and secondary structure. Specific interaction of cellular proteins with TAR RNA could be detected by enhanced cleavages or imprints surrounding the loop region. Mutations that either disrupted stem base-pairing or extensively changed the primary sequence resulted in alterations in the cleavage pattern of the TAR RNA. Structural features of the TAR RNA stem-loop essential for tat activation are also required for specific binding of the HeLa cell nuclear protein. Images PMID:2544877

Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks.

PubMed

Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

2016-05-10

RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks.

Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks

NASA Astrophysics Data System (ADS)

Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

2016-05-01

RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks.

iTAK: A program for genome-wide prediction and classification of plant transcription factors, transcriptional regulators and protein kinases

USDA-ARS?s Scientific Manuscript database

Transcription factors (TFs) are proteins that regulate the expression of target genes by binding to specific elements in their regulatory regions. Transcriptional regulators (TRs) also regulate the expression of target genes; however, they operate indirectly via interaction with the basal transcript...

Rapid evolution of cis-regulatory sequences via local point mutations

NASA Technical Reports Server (NTRS)

Stone, J. R.; Wray, G. A.

2001-01-01

Although the evolution of protein-coding sequences within genomes is well understood, the same cannot be said of the cis-regulatory regions that control transcription. Yet, changes in gene expression are likely to constitute an important component of phenotypic evolution. We simulated the evolution of new transcription factor binding sites via local point mutations. The results indicate that new binding sites appear and become fixed within populations on microevolutionary timescales under an assumption of neutral evolution. Even combinations of two new binding sites evolve very quickly. We predict that local point mutations continually generate considerable genetic variation that is capable of altering gene expression.

Revealing stable processing products from ribosome-associated small RNAs by deep-sequencing data analysis.

PubMed

Zywicki, Marek; Bakowska-Zywicka, Kamilla; Polacek, Norbert

2012-05-01

The exploration of the non-protein-coding RNA (ncRNA) transcriptome is currently focused on profiling of microRNA expression and detection of novel ncRNA transcription units. However, recent studies suggest that RNA processing can be a multi-layer process leading to the generation of ncRNAs of diverse functions from a single primary transcript. Up to date no methodology has been presented to distinguish stable functional RNA species from rapidly degraded side products of nucleases. Thus the correct assessment of widespread RNA processing events is one of the major obstacles in transcriptome research. Here, we present a novel automated computational pipeline, named APART, providing a complete workflow for the reliable detection of RNA processing products from next-generation-sequencing data. The major features include efficient handling of non-unique reads, detection of novel stable ncRNA transcripts and processing products and annotation of known transcripts based on multiple sources of information. To disclose the potential of APART, we have analyzed a cDNA library derived from small ribosome-associated RNAs in Saccharomyces cerevisiae. By employing the APART pipeline, we were able to detect and confirm by independent experimental methods multiple novel stable RNA molecules differentially processed from well known ncRNAs, like rRNAs, tRNAs or snoRNAs, in a stress-dependent manner.

Identification and Characterization of Alternative Promoters, Transcripts and Protein Isoforms of Zebrafish R2 Gene

PubMed Central

Shang, Hanqiao; Li, Qing; Feng, Guohui; Cui, Zongbin

2011-01-01

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleoside triphosphates. Expression of RNR subunits is closely associated with DNA replication and repair. Mammalian RNR M2 subunit (R2) functions exclusively in DNA replication of normal cells due to its S phase-specific expression and late mitotic degradation. Herein, we demonstrate the control of R2 expression through alternative promoters, splicing and polyadenylation sites in zebrafish. Three functional R2 promoters were identified to generate six transcript variants with distinct 5′ termini. The proximal promoter contains a conserved E2F binding site and two CCAAT boxes, which are crucial for the transcription of R2 gene during cell cycle. Activity of the distal promoter can be induced by DNA damage to generate four transcript variants through alternative splicing. In addition, two novel splice variants were found to encode distinct N-truncated R2 isoforms containing residues for enzymatic activity but no KEN box essential for its proteolysis. These two N-truncated R2 isoforms remained in the cytoplasm and were able to interact with RNR M1 subunit (R1). Thus, our results suggest that multilayered mechanisms control the differential expression and function of zebrafish R2 gene during cell cycle and under genotoxic stress. PMID:21887375

Identification and characterization of alternative promoters, transcripts and protein isoforms of zebrafish R2 gene.

PubMed

Shang, Hanqiao; Li, Qing; Feng, Guohui; Cui, Zongbin

2011-01-01

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleoside triphosphates. Expression of RNR subunits is closely associated with DNA replication and repair. Mammalian RNR M2 subunit (R2) functions exclusively in DNA replication of normal cells due to its S phase-specific expression and late mitotic degradation. Herein, we demonstrate the control of R2 expression through alternative promoters, splicing and polyadenylation sites in zebrafish. Three functional R2 promoters were identified to generate six transcript variants with distinct 5' termini. The proximal promoter contains a conserved E2F binding site and two CCAAT boxes, which are crucial for the transcription of R2 gene during cell cycle. Activity of the distal promoter can be induced by DNA damage to generate four transcript variants through alternative splicing. In addition, two novel splice variants were found to encode distinct N-truncated R2 isoforms containing residues for enzymatic activity but no KEN box essential for its proteolysis. These two N-truncated R2 isoforms remained in the cytoplasm and were able to interact with RNR M1 subunit (R1). Thus, our results suggest that multilayered mechanisms control the differential expression and function of zebrafish R2 gene during cell cycle and under genotoxic stress.

RNA sequencing of esophageal adenocarcinomas identifies novel fusion transcripts, including NPC1-MELK, arising from a complex chromosomal rearrangement.

PubMed

Wang, Zhixiong; Cheng, Yulan; Abraham, John M; Yan, Rong; Liu, Xi; Chen, Wei; Ibrahim, Sariat; Schroth, Gary P; Ke, Xiquan; He, Yulong; Meltzer, Stephen J

2017-10-15

Studies of chromosomal rearrangements and fusion transcripts have elucidated mechanisms of tumorigenesis and led to targeted cancer therapies. This study was aimed at identifying novel fusion transcripts in esophageal adenocarcinoma (EAC). To identify new fusion transcripts associated with EAC, targeted RNA sequencing and polymerase chain reaction (PCR) verification were performed in 40 EACs and matched nonmalignant specimens from the same patients. Genomic PCR and Sanger sequencing were performed to find the breakpoint of fusion genes. Five novel in-frame fusion transcripts were identified and verified in 40 EACs and in a validation cohort of 15 additional EACs (55 patients in all): fibroblast growth factor receptor 2 (FGFR2)-GRB2-associated binding protein 2 (GAB2) in 2 of 55 or 3.6%, Niemann-Pick C1 (NPC1)-maternal embryonic leucine zipper kinase (MELK) in 2 of 55 or 3.6%, ubiquitin-specific peptidase 54 (USP54)-calcium/calmodulin dependent protein kinase II γ (CAMK2G) in 2 of 55 or 3.6%, megakaryoblastic leukemia (translocation) 1 (MKL1)-fibulin 1 (FBLN1) in 1 of 55 or 1.8%, and CCR4-NOT transcription complex subunit 2 (CNOT2)-chromosome 12 open reading frame 49 (C12orf49) in 1 of 55 or 1.8%. A genomic analysis indicated that NPC1-MELK arose from a complex interchromosomal translocation event involving chromosomes 18, 3, and 9 with 3 rearrangement points, and this was consistent with chromoplexy. These data indicate that fusion transcripts occur at a stable frequency in EAC. Furthermore, our results indicate that chromoplexy is an underlying mechanism that generates fusion transcripts in EAC. These and other fusion transcripts merit further study as diagnostic markers and potential therapeutic targets in EAC. Cancer 2017;123:3916-24. © 2017 American Cancer Society. © 2017 American Cancer Society.

Mutations on the DNA Binding Surface of TBP Discriminate between Yeast TATA and TATA-Less Gene Transcription

PubMed Central

Kamenova, Ivanka; Warfield, Linda

2014-01-01

Most RNA polymerase (Pol) II promoters lack a TATA element, yet nearly all Pol II transcription requires TATA binding protein (TBP). While the TBP-TATA interaction is critical for transcription at TATA-containing promoters, it has been unclear whether TBP sequence-specific DNA contacts are required for transcription at TATA-less genes. Transcription factor IID (TFIID), the TBP-containing coactivator that functions at most TATA-less genes, recognizes short sequence-specific promoter elements in metazoans, but analogous promoter elements have not been identified in Saccharomyces cerevisiae. We generated a set of mutations in the yeast TBP DNA binding surface and found that most support growth of yeast. Both in vivo and in vitro, many of these mutations are specifically defective for transcription of two TATA-containing genes with only minor defects in transcription of two TATA-less, TFIID-dependent genes. TBP binds several TATA-less promoters with apparent high affinity, but our results suggest that this binding is not important for transcription activity. Our results are consistent with the model that sequence-specific TBP-DNA contacts are not important at yeast TATA-less genes and suggest that other general transcription factors or coactivator subunits are responsible for recognition of TATA-less promoters. Our results also explain why yeast TBP derivatives defective for TATA binding appear defective in activated transcription. PMID:24865972

Transcription profiling suggests that mitochondrial topoisomerase IB acts as a topological barrier and regulator of mitochondrial DNA transcription.

PubMed

Dalla Rosa, Ilaria; Zhang, Hongliang; Khiati, Salim; Wu, Xiaolin; Pommier, Yves

2017-12-08

Mitochondrial DNA (mtDNA) is essential for cell viability because it encodes subunits of the respiratory chain complexes. Mitochondrial topoisomerase IB (TOP1MT) facilitates mtDNA replication by removing DNA topological tensions produced during mtDNA transcription, but it appears to be dispensable. To test whether cells lacking TOP1MT have aberrant mtDNA transcription, we performed mitochondrial transcriptome profiling. To that end, we designed and implemented a customized tiling array, which enabled genome-wide, strand-specific, and simultaneous detection of all mitochondrial transcripts. Our technique revealed that Top1mt KO mouse cells process the mitochondrial transcripts normally but that protein-coding mitochondrial transcripts are elevated. Moreover, we found discrete long noncoding RNAs produced by H-strand transcription and encompassing the noncoding regulatory region of mtDNA in human and murine cells and tissues. Of note, these noncoding RNAs were strongly up-regulated in the absence of TOP1MT. In contrast, 7S DNA, produced by mtDNA replication, was reduced in the Top1mt KO cells. We propose that the long noncoding RNA species in the D-loop region are generated by the extension of H-strand transcripts beyond their canonical stop site and that TOP1MT acts as a topological barrier and regulator for mtDNA transcription and D-loop formation.

Mutations on the DNA binding surface of TBP discriminate between yeast TATA and TATA-less gene transcription.

PubMed

Kamenova, Ivanka; Warfield, Linda; Hahn, Steven

2014-08-01

Most RNA polymerase (Pol) II promoters lack a TATA element, yet nearly all Pol II transcription requires TATA binding protein (TBP). While the TBP-TATA interaction is critical for transcription at TATA-containing promoters, it has been unclear whether TBP sequence-specific DNA contacts are required for transcription at TATA-less genes. Transcription factor IID (TFIID), the TBP-containing coactivator that functions at most TATA-less genes, recognizes short sequence-specific promoter elements in metazoans, but analogous promoter elements have not been identified in Saccharomyces cerevisiae. We generated a set of mutations in the yeast TBP DNA binding surface and found that most support growth of yeast. Both in vivo and in vitro, many of these mutations are specifically defective for transcription of two TATA-containing genes with only minor defects in transcription of two TATA-less, TFIID-dependent genes. TBP binds several TATA-less promoters with apparent high affinity, but our results suggest that this binding is not important for transcription activity. Our results are consistent with the model that sequence-specific TBP-DNA contacts are not important at yeast TATA-less genes and suggest that other general transcription factors or coactivator subunits are responsible for recognition of TATA-less promoters. Our results also explain why yeast TBP derivatives defective for TATA binding appear defective in activated transcription. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Seawater inhalation induces acute lung injury via ROS generation and the endoplasmic reticulum stress pathway

PubMed Central

Li, Cong-Cong; Lu, Xi; Qian, Wei-Sheng; Li, Yu-Juan; Jin, Fa-Guang; Mu, De-Guang

2018-01-01

Seawater (SW) inhalation can induce acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). In the present study, SW induced apoptosis of rat alveolar epithelial cells and histopathological alterations to lung tissue. Furthermore, SW administration increased generation of reactive oxygen species (ROS), whereas pretreatment with the ROS scavenger, N-acetyl-L-cysteine (NAC), significantly decreased ROS generation, apoptosis and histopathological alterations. In addition, SW exposure upregulated the expression levels of glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP), which are critical proteins in the endoplasmic reticulum (ER) stress response, thus indicating that SW may activate ER stress. Conversely, blocking ER stress with 4-phenylbutyric acid (4-PBA) significantly improved SW-induced apoptosis and histopathological alterations, whereas an ER stress inducer, thapsigargin, had the opposite effect. Furthermore, blocking ROS with NAC inhibited SW-induced ER stress, as evidenced by the downregulation of GRP78, phosphorylated (p)-protein kinase R-like ER kinase (PERK), p-inositol-requiring kinase 1α (IRE1α), p-50 activating transcription factor 6α and CHOP. In addition, blocking ER stress with 4-PBA decreased ROS generation. In conclusion, the present study indicated that ROS and ER stress pathways, which are involved in alveolar epithelial cell apoptosis, are important in the pathogenesis of SW-induced ALI. PMID:29436612

Comparative transcriptional and translational analysis of leptospiral outer membrane protein expression in response to temperature.

PubMed

Lo, Miranda; Cordwell, Stuart J; Bulach, Dieter M; Adler, Ben

2009-12-08

Leptospirosis is a global zoonosis affecting millions of people annually. Transcriptional changes in response to temperature were previously investigated using microarrays to identify genes potentially expressed upon host entry. Past studies found that various leptospiral outer membrane proteins are differentially expressed at different temperatures. However, our microarray studies highlighted a divergence between protein abundance and transcript levels for some proteins. Given the abundance of post-transcriptional expression control mechanisms, this finding highlighted the importance of global protein analysis systems. To complement our previous transcription study, we evaluated differences in the proteins of the leptospiral outer membrane fraction in response to temperature upshift. Outer membrane protein-enriched fractions from Leptospira interrogans grown at 30 degrees C or overnight upshift to 37 degrees C were isolated and the relative abundance of each protein was determined by iTRAQ analysis coupled with two-dimensional liquid chromatography and tandem mass spectrometry (2-DLC/MS-MS). We identified 1026 proteins with 99% confidence; 27 and 66 were present at elevated and reduced abundance respectively. Protein abundance changes were compared with transcriptional differences determined from the microarray studies. While there was some correlation between the microarray and iTRAQ data, a subset of genes that showed no differential expression by microarray was found to encode temperature-regulated proteins. This set of genes is of particular interest as it is likely that regulation of their expression occurs post-transcriptionally, providing an opportunity to develop hypotheses about the molecular dynamics of the outer membrane of Leptospira in response to changing environments. This is the first study to compare transcriptional and translational responses to temperature shift in L. interrogans. The results thus provide an insight into the mechanisms used by L. interrogans to adapt to conditions encountered in the host and to cause disease. Our results suggest down-regulation of protein expression in response to temperature, and decreased expression of outer membrane proteins may facilitate minimal interaction with host immune mechanisms.

Development of Transcriptomic Resources for Interrogating the Biosynthesis of Monoterpene Indole Alkaloids in Medicinal Plant Species

PubMed Central

Góngora-Castillo, Elsa; Childs, Kevin L.; Fedewa, Greg; Hamilton, John P.; Liscombe, David K.; Magallanes-Lundback, Maria; Mandadi, Kranthi K.; Nims, Ezekiel; Runguphan, Weerawat; Vaillancourt, Brieanne; Varbanova-Herde, Marina; DellaPenna, Dean; McKnight, Thomas D.; O’Connor, Sarah; Buell, C. Robin

2012-01-01

The natural diversity of plant metabolism has long been a source for human medicines. One group of plant-derived compounds, the monoterpene indole alkaloids (MIAs), includes well-documented therapeutic agents used in the treatment of cancer (vinblastine, vincristine, camptothecin), hypertension (reserpine, ajmalicine), malaria (quinine), and as analgesics (7-hydroxymitragynine). Our understanding of the biochemical pathways that synthesize these commercially relevant compounds is incomplete due in part to a lack of molecular, genetic, and genomic resources for the identification of the genes involved in these specialized metabolic pathways. To address these limitations, we generated large-scale transcriptome sequence and expression profiles for three species of Asterids that produce medicinally important MIAs: Camptotheca acuminata, Catharanthus roseus, and Rauvolfia serpentina. Using next generation sequencing technology, we sampled the transcriptomes of these species across a diverse set of developmental tissues, and in the case of C. roseus, in cultured cells and roots following elicitor treatment. Through an iterative assembly process, we generated robust transcriptome assemblies for all three species with a substantial number of the assembled transcripts being full or near-full length. The majority of transcripts had a related sequence in either UniRef100, the Arabidopsis thaliana predicted proteome, or the Pfam protein domain database; however, we also identified transcripts that lacked similarity with entries in either database and thereby lack a known function. Representation of known genes within the MIA biosynthetic pathway was robust. As a diverse set of tissues and treatments were surveyed, expression abundances of transcripts in the three species could be estimated to reveal transcripts associated with development and response to elicitor treatment. Together, these transcriptomes and expression abundance matrices provide a rich resource for understanding plant specialized metabolism, and promotes realization of innovative production systems for plant-derived pharmaceuticals. PMID:23300689

The balance sheet for transcription: an analysis of nuclear RNA metabolism in mammalian cells.

PubMed

Jackson, D A; Pombo, A; Iborra, F

2000-02-01

The control of RNA synthesis from protein-coding genes is fundamental in determining the various cell types of higher eukaryotes. The activation of these genes is driven by promoter complexes, and RNA synthesis is performed by an enzyme mega-complex-the RNA polymerase II holoenzyme. These two complexes are the fundamental components required to initiate gene expression and generate the primary transcripts that, after processing, yield mRNAs that pass to the cytoplasm where protein synthesis occurs. But although this gene expression pathway has been studied intensively, aspects of RNA metabolism remain difficult to comprehend. In particular, it is unclear why >95% of RNA polymerized by polymerase II remains in the nucleus, where it is recycled. To explain this apparent paradox, this review presents a detailed description of nuclear RNA (nRNA) metabolism in mammalian cells. We evaluate the number of active transcription units, discuss the distribution of polymerases on active genes, and assess the efficiency with which the products mature and pass to the cytoplasm. Differences between the behavior of mRNAs on this productive pathway and primary transcripts that never leave the nucleus lead us to propose that these represent distinct populations. We discuss possible roles for nonproductive RNAs and present a model to describe the metabolism of these RNAs in the nuclei of mammalian cells.-Jackson, D. A., Pombo, A., Iborra, F. The balance sheet for transcription: an analysis of nuclear RNA metabolism in mammalian cells.

Genome-wide characterization of differential transcript usage in Arabidopsis thaliana.

PubMed

Vaneechoutte, Dries; Estrada, April R; Lin, Ying-Chen; Loraine, Ann E; Vandepoele, Klaas

2017-12-01

Alternative splicing and the usage of alternate transcription start- or stop sites allows a single gene to produce multiple transcript isoforms. Most plant genes express certain isoforms at a significantly higher level than others, but under specific conditions this expression dominance can change, resulting in a different set of dominant isoforms. These events of differential transcript usage (DTU) have been observed for thousands of Arabidopsis thaliana, Zea mays and Vitis vinifera genes, and have been linked to development and stress response. However, neither the characteristics of these genes, nor the implications of DTU on their protein coding sequences or functions, are currently well understood. Here we present a dataset of isoform dominance and DTU for all genes in the AtRTD2 reference transcriptome based on a protocol that was benchmarked on simulated data and validated through comparison with a published reverse transciptase-polymerase chain reaction panel. We report DTU events for 8148 genes across 206 public RNA-Seq samples, and find that protein sequences are affected in 22% of the cases. The observed DTU events show high consistency across replicates, and reveal reproducible patterns in response to treatment and development. We also demonstrate that genes with different evolutionary ages, expression breadths and functions show large differences in the frequency at which they undergo DTU, and in the effect that these events have on their protein sequences. Finally, we showcase how the generated dataset can be used to explore DTU events for genes of interest or to find genes with specific DTU in samples of interest. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Comparative Proteomics Reveals a Significant Bias Toward Alternative Protein Isoforms with Conserved Structure and Function

PubMed Central

Ezkurdia, Iakes; del Pozo, Angela; Frankish, Adam; Rodriguez, Jose Manuel; Harrow, Jennifer; Ashman, Keith; Valencia, Alfonso; Tress, Michael L.

2012-01-01

Advances in high-throughput mass spectrometry are making proteomics an increasingly important tool in genome annotation projects. Peptides detected in mass spectrometry experiments can be used to validate gene models and verify the translation of putative coding sequences (CDSs). Here, we have identified peptides that cover 35% of the genes annotated by the GENCODE consortium for the human genome as part of a comprehensive analysis of experimental spectra from two large publicly available mass spectrometry databases. We detected the translation to protein of “novel” and “putative” protein-coding transcripts as well as transcripts annotated as pseudogenes and nonsense-mediated decay targets. We provide a detailed overview of the population of alternatively spliced protein isoforms that are detectable by peptide identification methods. We found that 150 genes expressed multiple alternative protein isoforms. This constitutes the largest set of reliably confirmed alternatively spliced proteins yet discovered. Three groups of genes were highly overrepresented. We detected alternative isoforms for 10 of the 25 possible heterogeneous nuclear ribonucleoproteins, proteins with a key role in the splicing process. Alternative isoforms generated from interchangeable homologous exons and from short indels were also significantly enriched, both in human experiments and in parallel analyses of mouse and Drosophila proteomics experiments. Our results show that a surprisingly high proportion (almost 25%) of the detected alternative isoforms are only subtly different from their constitutive counterparts. Many of the alternative splicing events that give rise to these alternative isoforms are conserved in mouse. It was striking that very few of these conserved splicing events broke Pfam functional domains or would damage globular protein structures. This evidence of a strong bias toward subtle differences in CDS and likely conserved cellular function and structure is remarkable and strongly suggests that the translation of alternative transcripts may be subject to selective constraints. PMID:22446687

Mapping of Transcription Termination within the S Segment of SFTS Phlebovirus Facilitated Generation of NSs Deletant Viruses

PubMed Central

Rezelj, Veronica V.; Elliott, Richard M.

2017-01-01

ABSTRACT SFTS phlebovirus (SFTSV) is an emerging tick-borne bunyavirus that was first reported in China in 2009. Here we report the generation of a recombinant SFTSV (rHB29NSsKO) that cannot express the viral nonstructural protein (NSs) upon infection of cells in culture. We show that rHB29NSsKO replication kinetics are greater in interferon (IFN)-incompetent cells and that the virus is unable to suppress IFN induced in response to viral replication. The data confirm for the first time in the context of virus infection that NSs acts as a virally encoded IFN antagonist and that NSs is dispensable for virus replication. Using 3′ rapid amplification of cDNA ends (RACE), we mapped the 3′ end of the N and NSs mRNAs, showing that the mRNAs terminate within the coding region of the opposite open reading frame. We show that the 3′ end of the N mRNA terminates upstream of a 5′-GCCAGCC-3′ motif present in the viral genomic RNA. With this knowledge, and using virus-like particles, we could demonstrate that the last 36 nucleotides of the NSs open reading frame (ORF) were needed to ensure the efficient termination of the N mRNA and were required for recombinant virus rescue. We demonstrate that it is possible to recover viruses lacking NSs (expressing just a 12-amino-acid NSs peptide or encoding enhanced green fluorescent protein [eGFP]) or an NSs-eGFP fusion protein in the NSs locus. This opens the possibility for further studies of NSs and potentially the design of attenuated viruses for vaccination studies. IMPORTANCE SFTS phlebovirus (SFTSV) and related tick-borne viruses have emerged globally since 2009. SFTSV has been shown to cause severe disease in humans. For bunyaviruses, it has been well documented that the nonstructural protein (NSs) enables the virus to counteract the human innate antiviral defenses and that NSs is one of the major determinants of virulence in infection. Therefore, the use of reverse genetics systems to engineer viruses lacking NSs is an attractive strategy to rationally attenuate bunyaviruses. Here we report the generation of several recombinant SFTS viruses that cannot express the NSs protein or have the NSs open reading frame replaced with a reporter gene. These viruses cannot antagonize the mammalian interferon (IFN) response mounted to virus infection. The generation of NSs-lacking viruses was achieved by mapping the transcriptional termination of two S-segment-derived subgenomic mRNAs, which revealed that transcription termination occurs upstream of a 5′-GCCAGCC-3′ motif present in the virus genomic S RNA. PMID:28592543

Mapping of Transcription Termination within the S Segment of SFTS Phlebovirus Facilitated Generation of NSs Deletant Viruses.

PubMed

Brennan, Benjamin; Rezelj, Veronica V; Elliott, Richard M

2017-08-15

SFTS phlebovirus (SFTSV) is an emerging tick-borne bunyavirus that was first reported in China in 2009. Here we report the generation of a recombinant SFTSV (rHB29NSsKO) that cannot express the viral nonstructural protein (NSs) upon infection of cells in culture. We show that rHB29NSsKO replication kinetics are greater in interferon (IFN)-incompetent cells and that the virus is unable to suppress IFN induced in response to viral replication. The data confirm for the first time in the context of virus infection that NSs acts as a virally encoded IFN antagonist and that NSs is dispensable for virus replication. Using 3' rapid amplification of cDNA ends (RACE), we mapped the 3' end of the N and NSs mRNAs, showing that the mRNAs terminate within the coding region of the opposite open reading frame. We show that the 3' end of the N mRNA terminates upstream of a 5'-GCCAGCC-3' motif present in the viral genomic RNA. With this knowledge, and using virus-like particles, we could demonstrate that the last 36 nucleotides of the NSs open reading frame (ORF) were needed to ensure the efficient termination of the N mRNA and were required for recombinant virus rescue. We demonstrate that it is possible to recover viruses lacking NSs (expressing just a 12-amino-acid NSs peptide or encoding enhanced green fluorescent protein [eGFP]) or an NSs-eGFP fusion protein in the NSs locus. This opens the possibility for further studies of NSs and potentially the design of attenuated viruses for vaccination studies. IMPORTANCE SFTS phlebovirus (SFTSV) and related tick-borne viruses have emerged globally since 2009. SFTSV has been shown to cause severe disease in humans. For bunyaviruses, it has been well documented that the nonstructural protein (NSs) enables the virus to counteract the human innate antiviral defenses and that NSs is one of the major determinants of virulence in infection. Therefore, the use of reverse genetics systems to engineer viruses lacking NSs is an attractive strategy to rationally attenuate bunyaviruses. Here we report the generation of several recombinant SFTS viruses that cannot express the NSs protein or have the NSs open reading frame replaced with a reporter gene. These viruses cannot antagonize the mammalian interferon (IFN) response mounted to virus infection. The generation of NSs-lacking viruses was achieved by mapping the transcriptional termination of two S-segment-derived subgenomic mRNAs, which revealed that transcription termination occurs upstream of a 5'-GCCAGCC-3' motif present in the virus genomic S RNA. Copyright © 2017 Brennan et al.

Transcriptional regulatory proteins as biosensing tools.

PubMed

Turner, Kendrick; Joel, Smita; Feliciano, Jessika; Feltus, Agatha; Pasini, Patrizia; Wynn, Daniel; Dau, Peter; Dikici, Emre; Deo, Sapna K; Daunert, Sylvia

2017-06-22

We have developed sensing systems employing different classes of transcriptional regulatory proteins genetically and chemically modified to incorporate a fluorescent reporter molecule for detection of arsenic, hydroxylated polychlorinated biphenyls (OH-PCBs), and cyclic AMP (cAMP). These are the first examples of optical sensing systems based on transcriptional regulatory proteins.

Did Convergent Protein Evolution Enable Phytoplasmas to Generate 'Zombie Plants'?

PubMed

Rümpler, Florian; Gramzow, Lydia; Theißen, Günter; Melzer, Rainer

2015-12-01

Phytoplasmas are pathogenic bacteria that reprogram plant development such that leaf-like structures instead of floral organs develop. Infected plants are sterile and mainly serve to propagate phytoplasmas and thus have been termed 'zombie plants'. The developmental reprogramming relies on specific interactions of the phytoplasma protein SAP54 with a small subset of MADS-domain transcription factors. Here, we propose that SAP54 folds into a structure that is similar to that of the K-domain, a protein-protein interaction domain of MADS-domain proteins. We suggest that undergoing convergent structural and sequence evolution, SAP54 evolved to mimic the K-domain. Given the high specificity of resulting developmental alterations, phytoplasmas might be used to study flower development in genetically intractable plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

Gene expression profiling during asexual development of the late blight pathogen Phytophthora infestans reveals a highly dynamic transcriptome.

PubMed

Judelson, Howard S; Ah-Fong, Audrey M V; Aux, George; Avrova, Anna O; Bruce, Catherine; Cakir, Cahid; da Cunha, Luis; Grenville-Briggs, Laura; Latijnhouwers, Maita; Ligterink, Wilco; Meijer, Harold J G; Roberts, Samuel; Thurber, Carrie S; Whisson, Stephen C; Birch, Paul R J; Govers, Francine; Kamoun, Sophien; van West, Pieter; Windass, John

2008-04-01

Much of the pathogenic success of Phytophthora infestans, the potato and tomato late blight agent, relies on its ability to generate from mycelia large amounts of sporangia, which release zoospores that encyst and form infection structures. To better understand these stages, Affymetrix GeneChips based on 15,650 unigenes were designed and used to profile the life cycle. Approximately half of P. infestans genes were found to exhibit significant differential expression between developmental transitions, with approximately (1)/(10) being stage-specific and most changes occurring during zoosporogenesis. Quantitative reverse-transcription polymerase chain reaction assays confirmed the robustness of the array results and showed that similar patterns of differential expression were obtained regardless of whether hyphae were from laboratory media or infected tomato. Differentially expressed genes encode potential cellular regulators, especially protein kinases; metabolic enzymes such as those involved in glycolysis, gluconeogenesis, or the biosynthesis of amino acids or lipids; regulators of DNA synthesis; structural proteins, including predicted flagellar proteins; and pathogenicity factors, including cell-wall-degrading enzymes, RXLR effector proteins, and enzymes protecting against plant defense responses. Curiously, some stage-specific transcripts do not appear to encode functional proteins. These findings reveal many new aspects of oomycete biology, as well as potential targets for crop protection chemicals.

Efficient exon skipping of SGCG mutations mediated by phosphorodiamidate morpholino oligomers.

PubMed

Wyatt, Eugene J; Demonbreun, Alexis R; Kim, Ellis Y; Puckelwartz, Megan J; Vo, Andy H; Dellefave-Castillo, Lisa M; Gao, Quan Q; Vainzof, Mariz; Pavanello, Rita C M; Zatz, Mayana; McNally, Elizabeth M

2018-05-03

Exon skipping uses chemically modified antisense oligonucleotides to modulate RNA splicing. Therapeutically, exon skipping can bypass mutations and restore reading frame disruption by generating internally truncated, functional proteins to rescue the loss of native gene expression. Limb-girdle muscular dystrophy type 2C is caused by autosomal recessive mutations in the SGCG gene, which encodes the dystrophin-associated protein γ-sarcoglycan. The most common SGCG mutations disrupt the transcript reading frame abrogating γ-sarcoglycan protein expression. In order to treat most SGCG gene mutations, it is necessary to skip 4 exons in order to restore the SGCG transcript reading frame, creating an internally truncated protein referred to as Mini-Gamma. Using direct reprogramming of human cells with MyoD, myogenic cells were tested with 2 antisense oligonucleotide chemistries, 2'-O-methyl phosphorothioate oligonucleotides and vivo-phosphorodiamidate morpholino oligomers, to induce exon skipping. Treatment with vivo-phosphorodiamidate morpholino oligomers demonstrated efficient skipping of the targeted exons and corrected the mutant reading frame, resulting in the expression of a functional Mini-Gamma protein. Antisense-induced exon skipping of SGCG occurred in normal cells and those with multiple distinct SGCG mutations, including the most common 521ΔT mutation. These findings demonstrate a multiexon-skipping strategy applicable to the majority of limb-girdle muscular dystrophy 2C patients.

Tempo and Mode of Gene Duplication in Mammalian Ribosomal Protein Evolution

PubMed Central

Gajdosik, Matthew D.; Simon, Amanda; Nelson, Craig E.

2014-01-01

Gene duplication has been widely recognized as a major driver of evolutionary change and organismal complexity through the generation of multi-gene families. Therefore, understanding the forces that govern the evolution of gene families through the retention or loss of duplicated genes is fundamentally important in our efforts to study genome evolution. Previous work from our lab has shown that ribosomal protein (RP) genes constitute one of the largest classes of conserved duplicated genes in mammals. This result was surprising due to the fact that ribosomal protein genes evolve slowly and transcript levels are very tightly regulated. In our present study, we identified and characterized all RP duplicates in eight mammalian genomes in order to investigate the tempo and mode of ribosomal protein family evolution. We show that a sizable number of duplicates are transcriptionally active and are very highly conserved. Furthermore, we conclude that existing gene duplication models do not readily account for the preservation of a very large number of intact retroduplicated ribosomal protein (RT-RP) genes observed in mammalian genomes. We suggest that selection against dominant-negative mutations may underlie the unexpected retention and conservation of duplicated RP genes, and may shape the fate of newly duplicated genes, regardless of duplication mechanism. PMID:25369106

The molecular topography of silenced chromatin in Saccharomyces cerevisiae

PubMed Central

Thurtle, Deborah M.; Rine, Jasper

2014-01-01

Heterochromatin imparts regional, promoter-independent repression of genes and is epigenetically heritable. Understanding how silencing achieves this regional repression is a fundamental problem in genetics and development. Current models of yeast silencing posit that Sir proteins, recruited by transcription factors bound to the silencers, spread throughout the silenced region. To test this model directly at high resolution, we probed the silenced chromatin architecture by chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-seq) of Sir proteins, histones, and a key histone modification, H4K16-acetyl. These analyses revealed that Sir proteins are strikingly concentrated at and immediately adjacent to the silencers, with lower levels of enrichment over the promoters at HML and HMR, the critical targets for transcriptional repression. The telomeres also showed discrete peaks of Sir enrichment yet a continuous domain of hypoacetylated histone H4K16. Surprisingly, ChIP-seq of cross-linked chromatin revealed a distribution of nucleosomes at silenced loci that was similar to Sir proteins, whereas native nucleosome maps showed a regular distribution throughout silenced loci, indicating that cross-linking captured a specialized chromatin organization imposed by Sir proteins. This specialized chromatin architecture observed in yeast informs the importance of a steric contribution to regional repression in other organisms. PMID:24493645

In Vivo Delivery of Cytoplasmic RNA Virus-derived miRNAs

PubMed Central

Langlois, Ryan A; Shapiro, Jillian S; Pham, Alissa M; tenOever, Benjamin R

2012-01-01

The discovery of microRNAs (miRNAs) revealed an unappreciated level of post-transcriptional control used by the cell to maintain optimal protein levels. This process has represented an attractive strategy for therapeutics that is currently limited by in vivo delivery constraints. Here, we describe the generation of a single-stranded, cytoplasmic virus of negative polarity capable of producing functional miRNAs. Cytoplasmic RNA virus-derived miRNAs accumulated to high levels in vitro, generated significant amounts of miRNA star strand, associated with the RNA-induced silencing complex (RISC), and conferred post transcriptional gene silencing in a sequence-specific manner. Furthermore, we demonstrate that these vectors could deliver miRNAs to a wide range of tissues, and sustain prolonged expression capable of achieving measurable knockdown of physiological targets in vivo. Taken together, these results validate noncanonical processing of cytoplasmic-derived miRNAs and provide a novel platform for small RNA delivery. PMID:22086233

Square cell packing in the Drosophila embryo through spatiotemporally regulated EGF receptor signaling

PubMed Central

Tamada, Masako; Zallen, Jennifer A.

2015-01-01

Summary Cells display dynamic and diverse morphologies during development, but the strategies by which differentiated tissues achieve precise shapes and patterns are not well understood. Here we identify a developmental program that generates a highly ordered square cell grid in the Drosophila embryo through sequential and spatially regulated cell alignment, oriented cell division, and apicobasal cell elongation. The basic leucine zipper transcriptional regulator Cnc is necessary and sufficient to produce a square cell grid in the presence of a midline signal provided by the EGF receptor ligand, Spitz. Spitz orients cell divisions through a Pins/LGN-dependent spindle positioning mechanism and controls cell shape and alignment through a transcriptional pathway that requires the Pointed ETS domain protein. These results identify a strategy for producing ordered square cell packing configurations in epithelia and reveal a molecular mechanism by which organized tissue structure is generated through spatiotemporally regulated responses to EGF receptor activation. PMID:26506305

trans-Resveratrol Protects Ischemic PC12 Cells by Inhibiting the Hypoxia Associated Transcription Factors and Increasing the Levels of Antioxidant Defense Enzymes

PubMed Central

2012-01-01

An in vitro model of ischemic cerebral stroke [oxygen-glucose deprivation (OGD) for 6 h followed by 24 h reoxygenation (R)] with PC12 cells increases Ca2+ influx by upregulating native L-type Ca2+ channels and reactive oxygen species (ROS) generation. This reactive oxygen species generation and increase in intracellular Ca2+ triggers the expression of hypoxic homeostasis transcription factors such as hypoxia induced factor-1 alpha (HIF-1α), Cav-beta 3 (Cav β3), signal transducer and activator of transcription 3 (STAT3), heat shock protein 27 (hsp-27), and cationic channel transient receptor potential melastatin 7 (TRPM7). OGD insulted PC12 cells were subjected to biologically safe doses (5, 10, and 25 μM) of trans-resveratrol in three different treatment groups: 24 h prior to OGD (pre-treatment); 24 h post OGD (post-treatment); and from 24 h before OGD to end of reoxygenation period (whole-treatment). Here, we demonstrated that OGD-R-induced neuronal injury/death is by reactive oxygen species generation, increase in intracellular calcium levels, and decrease in antioxidant defense enzymes. trans-Resveratrol increases the viability of OGD-R insulted PC12 cells, which was assessed by using MTT, NRU, and LDH release assay. In addition, trans-resveratrol significantly decreases reactive oxygen species generation, intracellular Ca2+ levels, and hypoxia associated transcription factors and also increases the level of antioxidant defense enzymes. Our data shows that the whole-treatment group of trans-resveratrol is most efficient in decreasing hypoxia induced cell death through its antioxidant properties. PMID:23421680

Rift valley fever virus nonstructural protein NSs promotes viral RNA replication and transcription in a minigenome system.

PubMed

Ikegami, Tetsuro; Peters, C J; Makino, Shinji

2005-05-01

Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, has a tripartite negative-strand genome (S, M, and L segments) and is an important mosquito-borne pathogen for domestic animals and humans. We established an RVFV T7 RNA polymerase-driven minigenome system in which T7 RNA polymerase from an expression plasmid drove expression of RNA transcripts for viral proteins and minigenome RNA transcripts carrying a reporter gene between both termini of the M RNA segment in 293T cells. Like other viruses of the Bunyaviridae family, replication and transcription of the RVFV minigenome required expression of viral N and L proteins. Unexpectedly, the coexpression of an RVFV nonstructural protein, NSs, with N and L proteins resulted in a significant enhancement of minigenome RNA replication. Coexpression of NSs protein with N and L proteins also enhanced minigenome mRNA transcription in the cells expressing viral-sense minigenome RNA transcripts. NSs protein expression increased the RNA replication of minigenomes that originated from S and L RNA segments. Enhancement of minigenome RNA synthesis by NSs protein occurred in cells lacking alpha/beta interferon (IFN-alpha/beta) genes, indicating that the effect of NSs protein on minigenome RNA replication was unrelated to a putative NSs protein-induced inhibition of IFN-alpha/beta production. Our finding that RVFV NSs protein augmented minigenome RNA synthesis was in sharp contrast to reports that Bunyamwera virus (genus Bunyavirus) NSs protein inhibits viral minigenome RNA synthesis, suggesting that RVFV NSs protein and Bunyamwera virus NSs protein have distinctly different biological roles in viral RNA synthesis.

The drastic reduction of SMN protein in SMA I spinal cord motor neurons is not due to inefficient transcription.

PubMed

Mirabella, M; Servidei, S; Broccolini, A; Gandolfi, N; Ricci, E; Neri, G; Tonali, P; Brahe, C

1999-04-01

Spinal muscular atrophy (SMA) is caused by homozygous absence of the telomeric copy of the survival motor neuron (SMNt) gene. SMNt and its homologous centromeric copy (SMNc) encode the SMN protein, which is markedly reduced in SMA I patients. We have performed SMN transcript and protein studies on spinal cord sections of an SMA I patient using in situ hybridization and immunofluorescence. While the amount of protein was negligible, the level of transcripts was comparable with that of controls. These findings suggest that the reduced protein level is not caused by a deficient transcription of the SMNc gene.

The Homeodomain of PDX-1 Mediates Multiple Protein-Protein Interactions in the Formation of a Transcriptional Activation Complex on the Insulin Promoter

PubMed Central

Ohneda, Kinuko; Mirmira, Raghavendra G.; Wang, Juehu; Johnson, Jeffrey D.; German, Michael S.

2000-01-01

Activation of insulin gene transcription specifically in the pancreatic β cells depends on multiple nuclear proteins that interact with each other and with sequences on the insulin gene promoter to build a transcriptional activation complex. The homeodomain protein PDX-1 exemplifies such interactions by binding to the A3/4 region of the rat insulin I promoter and activating insulin gene transcription by cooperating with the basic-helix-loop-helix (bHLH) protein E47/Pan1, which binds to the adjacent E2 site. The present study provides evidence that the homeodomain of PDX-1 acts as a protein-protein interaction domain to recruit multiple proteins, including E47/Pan1, BETA2/NeuroD1, and high-mobility group protein I(Y), to an activation complex on the E2A3/4 minienhancer. The transcriptional activity of this complex results from the clustering of multiple activation domains capable of interacting with coactivators and the basal transcriptional machinery. These interactions are not common to all homeodomain proteins: the LIM homeodomain protein Lmx1.1 can also activate the E2A3/4 minienhancer in cooperation with E47/Pan1 but does so through different interactions. Cooperation between Lmx1.1 and E47/Pan1 results not only in the aggregation of multiple activation domains but also in the unmasking of a potent activation domain on E47/Pan1 that is normally silent in non-β cells. While more than one activation complex may be capable of activating insulin gene transcription through the E2A3/4 minienhancer, each is dependent on multiple specific interactions among a unique set of nuclear proteins. PMID:10629047

Exploration of Molecular Factors Impairing Superoxide Dismutase Isoforms Activity in Human Senile Cataractous Lenses

PubMed Central

Rajkumar, Sankaranarayanan; Vasavada, Abhay R.; Praveen, Mamidipudi R.; Ananthan, Rajendran; Reddy, Geereddy B.; Tripathi, Harsha; Ganatra, Darshini A.; Arora, Anshul I.; Patel, Alpesh R.

2013-01-01

Purpose. To explore different molecular factors impairing the activities of superoxide dismutase (SOD) isoforms in senile cataractous lenses. Methods. Enzyme activity of SOD isoforms, levels of their corresponding cofactors copper (Cu), manganese (Mn), zinc (Zn), and expression of mRNA transcripts and proteins were determined in the lenses of human subjects with and without cataract. DNA from lens epithelium (LE) and peripheral blood was isolated. Polymerase chain reaction–single strand conformation polymorphism (PCR-SSCP) followed by sequencing was carried out to screen somatic mutations. The impact of intronic insertion/deletion (INDEL) variations on the splicing process and on the resultant transcript was evaluated. Genotyping of IVS4+42delG polymorphism of SOD1 gene was done by PCR–restriction fragment length polymorphism (RFLP). Results. A significant decrease in Cu/Zn- and Mn-SOD activity (P < 0.001) and in Cu/Zn-SOD transcript (P < 0.001) and its protein (P < 0.05) were found in cataractous lenses. No significant change in the level of copper (P = 0.36) and an increase in the level of manganese (P = 0.01) and zinc (P = 0.02) were observed in cataractous lenses. A significant positive correlation between the level of Cu/Zn-SOD activity and the levels of Cu (P = 0.003) and Zn (P = 0.005) was found in the cataractous lenses. DNA sequencing revealed three intronic INDEL variations in exon4 of SOD1 gene. Splice-junction analysis showed the potential of IVS4+42delG in creating a new cryptic acceptor site. If it is involved in alternate splicing, it could result in generation of SOD1 mRNA transcripts lacking exon4 region. Transcript analysis revealed the presence of complete SOD1 mRNA transcripts. Genotyping revealed the presence of IVS4+42delG polymorphism in all subjects. Conclusions. The decrease in the activity of SOD1 isoform in cataractous lenses was associated with the decreased level of mRNA transcripts and their protein expression and was not associated with either modulation in the level of enzyme cofactors or with INDEL variations. PMID:23970468

Optimization of De Novo Short Read Assembly of Seabuckthorn (Hippophae rhamnoides L.) Transcriptome

PubMed Central

Ghangal, Rajesh; Chaudhary, Saurabh; Jain, Mukesh; Purty, Ram Singh; Chand Sharma, Prakash

2013-01-01

Seabuckthorn ( Hippophae rhamnoides L.) is known for its medicinal, nutritional and environmental importance since ancient times. However, very limited efforts have been made to characterize the genome and transcriptome of this wonder plant. Here, we report the use of next generation massive parallel sequencing technology (Illumina platform) and de novo assembly to gain a comprehensive view of the seabuckthorn transcriptome. We assembled 86,253,874 high quality short reads using six assembly tools. At our hand, assembly of non-redundant short reads following a two-step procedure was found to be the best considering various assembly quality parameters. Initially, ABySS tool was used following an additive k-mer approach. The assembled transcripts were subsequently subjected to TGICL suite. Finally, de novo short read assembly yielded 88,297 transcripts (> 100 bp), representing about 53 Mb of seabuckthorn transcriptome. The average length of transcripts was 610 bp, N50 length 1198 BP and 91% of the short reads uniquely mapped back to seabuckthorn transcriptome. A total of 41,340 (46.8%) transcripts showed significant similarity with sequences present in nr protein databases of NCBI (E-value < 1E-06). We also screened the assembled transcripts for the presence of transcription factors and simple sequence repeats. Our strategy involving the use of short read assembler (ABySS) followed by TGICL will be useful for the researchers working with a non-model organism’s transcriptome in terms of saving time and reducing complexity in data management. The seabuckthorn transcriptome data generated here provide a valuable resource for gene discovery and development of functional molecular markers. PMID:23991119

Perigone Lobe Transcriptome Analysis Provides Insights into Rafflesia cantleyi Flower Development.

PubMed

Lee, Xin-Wei; Mat-Isa, Mohd-Noor; Mohd-Elias, Nur-Atiqah; Aizat-Juhari, Mohd Afiq; Goh, Hoe-Han; Dear, Paul H; Chow, Keng-See; Haji Adam, Jumaat; Mohamed, Rahmah; Firdaus-Raih, Mohd; Wan, Kiew-Lian

2016-01-01

Rafflesia is a biologically enigmatic species that is very rare in occurrence and possesses an extraordinary morphology. This parasitic plant produces a gigantic flower up to one metre in diameter with no leaves, stem or roots. However, little is known about the floral biology of this species especially at the molecular level. In an effort to address this issue, we have generated and characterised the transcriptome of the Rafflesia cantleyi flower, and performed a comparison with the transcriptome of its floral bud to predict genes that are expressed and regulated during flower development. Approximately 40 million sequencing reads were generated and assembled de novo into 18,053 transcripts with an average length of 641 bp. Of these, more than 79% of the transcripts had significant matches to annotated sequences in the public protein database. A total of 11,756 and 7,891 transcripts were assigned to Gene Ontology categories and clusters of orthologous groups respectively. In addition, 6,019 transcripts could be mapped to 129 pathways in Kyoto Encyclopaedia of Genes and Genomes Pathway database. Digital abundance analysis identified 52 transcripts with very high expression in the flower transcriptome of R. cantleyi. Subsequently, analysis of differential expression between developing flower and the floral bud revealed a set of 105 transcripts with potential role in flower development. Our work presents a deep transcriptome resource analysis for the developing flower of R. cantleyi. Genes potentially involved in the growth and development of the R. cantleyi flower were identified and provide insights into biological processes that occur during flower development.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Preti, Milena; Guffanti, Elisa; Valitutto, Eleonora

The SNR52 gene, coding for a box C/D snoRNA, is the only snoRNA gene transcribed by RNA polymerase (Pol) III in Saccharomyces cerevisiae. Pol III transcription generates a precisely terminated primary transcript that undergoes extensive 5'-end processing. Here, we show that mutations of the box C/D core motif required for snoRNP assembly compromise 5'-end maturation of the SNR52 snoRNA. Upstream processing was also impaired by specific depletion of either Nop1p or Nop58p snoRNP proteins. We further show that the nuclear exosome is required for 3'-end maturation of SNR52 snoRNA, at variance with all the other known Pol III transcripts. Ourmore » data suggest a functional coupling between snoRNP assembly and 5'-end maturation of independently transcribed box C/D snoRNAs.« less

Small regulatory RNA-induced growth rate heterogeneity of Bacillus subtilis.

PubMed

Mars, Ruben A T; Nicolas, Pierre; Ciccolini, Mariano; Reilman, Ewoud; Reder, Alexander; Schaffer, Marc; Mäder, Ulrike; Völker, Uwe; van Dijl, Jan Maarten; Denham, Emma L

2015-03-01

Isogenic bacterial populations can consist of cells displaying heterogeneous physiological traits. Small regulatory RNAs (sRNAs) could affect this heterogeneity since they act by fine-tuning mRNA or protein levels to coordinate the appropriate cellular behavior. Here we show that the sRNA RnaC/S1022 from the Gram-positive bacterium Bacillus subtilis can suppress exponential growth by modulation of the transcriptional regulator AbrB. Specifically, the post-transcriptional abrB-RnaC/S1022 interaction allows B. subtilis to increase the cell-to-cell variation in AbrB protein levels, despite strong negative autoregulation of the abrB promoter. This behavior is consistent with existing mathematical models of sRNA action, thus suggesting that induction of protein expression noise could be a new general aspect of sRNA regulation. Importantly, we show that the sRNA-induced diversity in AbrB levels generates heterogeneity in growth rates during the exponential growth phase. Based on these findings, we hypothesize that the resulting subpopulations of fast- and slow-growing B. subtilis cells reflect a bet-hedging strategy for enhanced survival of unfavorable conditions.

Identification of the Photoreceptor Transcriptional Co-Repressor SAMD11 as Novel Cause of Autosomal Recessive Retinitis Pigmentosa

PubMed Central

Corton, M.; Avila-Fernández, A.; Campello, L.; Sánchez, M.; Benavides, B.; López-Molina, M. I.; Fernández-Sánchez, L.; Sánchez-Alcudia, R.; da Silva, L. R. J.; Reyes, N.; Martín-Garrido, E.; Zurita, O.; Fernández-San José, P.; Pérez-Carro, R.; García-García, F.; Dopazo, J.; García-Sandoval, B.; Cuenca, N.; Ayuso, C.

2016-01-01

Retinitis pigmentosa (RP), the most frequent form of inherited retinal dystrophy is characterized by progressive photoreceptor degeneration. Many genes have been implicated in RP development, but several others remain to be identified. Using a combination of homozygosity mapping, whole-exome and targeted next-generation sequencing, we found a novel homozygous nonsense mutation in SAMD11 in five individuals diagnosed with adult-onset RP from two unrelated consanguineous Spanish families. SAMD11 is ortholog to the mouse major retinal SAM domain (mr-s) protein that is implicated in CRX-mediated transcriptional regulation in the retina. Accordingly, protein-protein network analysis revealed a significant interaction of SAMD11 with CRX. Immunoblotting analysis confirmed strong expression of SAMD11 in human retina. Immunolocalization studies revealed SAMD11 was detected in the three nuclear layers of the human retina and interestingly differential expression between cone and rod photoreceptors was observed. Our study strongly implicates SAMD11 as novel cause of RP playing an important role in the pathogenesis of human degeneration of photoreceptors. PMID:27734943

Proteomic profiling of white muscle from freshwater catfish Rita rita.

PubMed

Mohanty, Bimal Prasanna; Mitra, Tandrima; Banerjee, Sudeshna; Bhattacharjee, Soma; Mahanty, Arabinda; Ganguly, Satabdi; Purohit, Gopal Krishna; Karunakaran, Dhanasekar; Mohanty, Sasmita

2015-06-01

Muscle tissues contribute 34-48 % of the total body mass in fish. Proteomic analysis enables better understanding of the skeletal muscle physiology and metabolism. A proteome map reflects the general fingerprinting of the fish species and has the potential to identify novel proteins which could serve as biomarkers for many aspects of aquaculture including fish physiology and growth, flesh quality, food safety and aquatic environmental monitoring. The freshwater catfish Rita rita of the family Bagridae inhabiting the tropical rivers and estuaries is an important food fish with high nutritive value and is also considered a species of choice in riverine pollution monitoring. Omics information that could enhance utility of this species in molecular research is meager. Therefore, in the present study, proteomic analysis of Rita rita muscle has been carried out and functional genomics data have been generated. A reference muscle proteome has been developed, and 23 protein spots, representing 18 proteins, have been identified by MALDI-TOF/TOF-MS and LC-MS/MS. Besides, transcript information on a battery of heat shock proteins (Hsps) has been generated. The functional genomics information generated could act as the baseline data for further molecular research on this species.

Expression of Olfactory Signaling Genes in the Eye

PubMed Central

Velmeshev, Dmitry; Faghihi, Mohammad; Shestopalov, Valery I.; Slepak, Vladlen Z.

2014-01-01

Purpose To advance our understanding how the outer eye interacts with its environment, we asked which cellular receptors are expressed in the cornea, focusing on G protein-coupled receptors. Methods Total RNA from the mouse cornea was subjected to next-generation sequencing using the Illumina platform. The data was analyzed with TopHat and CuffLinks software packages. Expression of a representative group of genes detected by RNA-seq was further analyzed by RT-PCR and in situ hybridization using RNAscope technology and fluorescent microscopy. Results We generated more than 46 million pair-end reads from mouse corneal RNA. Bioinformatics analysis revealed that the mouse corneal transcriptome reconstructed from these reads represents over 10,000 gene transcripts. We identified 194 GPCR transcripts, of which 96 were putative olfactory receptors. RT-PCR analysis confirmed the presence of several olfactory receptors and related genes, including olfactory marker protein and the G protein associated with olfaction, Gαolf. In situ hybridization showed that mRNA for olfactory marker protein, Gαolf and possibly some olfactory receptors were found in the corneal epithelial cells. In addition to the corneal epithelium, Gαolf was present in the ganglionic and inner nuclear layers of the retina. One of the olfactory receptors, Olfr558, was present primarily in vessels of the eye co-stained with antibodies against alpha-smooth muscle actin, indicating expression in arterioles. Conclusions Several species of mRNA encoding putative olfactory receptors and related genes are expressed in the mouse cornea and other parts of the eye indicating they may play a role in sensing chemicals in the ocular environment. PMID:24789354

Rapid identification of mutations in the IDS gene of Hunter patients: Analysis of mRNA by the protein truncation test

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hogervorst, F.B.L.; Tuijn, A.C. van der; Ommen, G.J.B. van

Hunter syndrome is an X-linked recessive disorder constituting phenotypes ranging from mild to severe. The gene affected in Hunter syndrome is iduronate-2-sulfatase (IDS). The identification of mutations leading to a defective enzyme could be of benefit for the diagnosis and prognosis of patients. At this moment a variety of mutations have been found, including large deletions and base substitutions. We have previously described a method, designated the protein truncation test (PTT), for the detection of mutations leading to premature translation termination. The method combines reverse transcription and PCR (RT-PCR) with in vitro transcript/translation of the products generated. To facilitate amore » PTT analysis, the forward primer is modified by addition of a T7 promoter sequence and an in-frame protein translation initiation sequence. In our department the method has been successfully applied for DMD and FAP. Here we report on the PTT analysis of 8 Hunter patients, all of them without major gene alterations as determined by Southern analysis. Total RNA was isolated from cultured skin fibroblasts or peripheral blood lymphocytes. PTT analysis revealed 4 novel mutations in the IDS gene: two missense mutations and two frameshift mutations (splice donor site alteration in intron 6 and a 13 bp deletion in exon 9). Furthermore, PTT proved to be a simple method to identify carriers. Currently, we use the generated RT-PCR products of the remaining patients for automated sequence analysis. PTT may be of great value in screening disorders in which affected genes give rise to truncated protein products.« less

A BEN-domain-containing protein associates with heterochromatin and represses transcription.

PubMed

Sathyan, Kizhakke M; Shen, Zhen; Tripathi, Vidisha; Prasanth, Kannanganattu V; Prasanth, Supriya G

2011-09-15

In eukaryotes, higher order chromatin structure governs crucial cellular processes including DNA replication, transcription and post-transcriptional gene regulation. Specific chromatin-interacting proteins play vital roles in the maintenance of chromatin structure. We have identified BEND3, a quadruple BEN domain-containing protein that is highly conserved amongst vertebrates. BEND3 colocalizes with HP1 and H3 trimethylated at K9 at heterochromatic regions in mammalian cells. Using an in vivo gene locus, we have been able to demonstrate that BEND3 associates with the locus only when it is heterochromatic and dissociates upon activation of transcription. Furthermore, tethering BEND3 inhibits transcription from the locus, indicating that BEND3 is involved in transcriptional repression through its interaction with histone deacetylases and Sall4, a transcription repressor. We further demonstrate that BEND3 is SUMOylated and that such modifications are essential for its role in transcriptional repression. Finally, overexpression of BEND3 causes premature chromatin condensation and extensive heterochromatinization, resulting in cell cycle arrest. Taken together, our data demonstrate the role of a novel heterochromatin-associated protein in transcriptional repression.

A BEN-domain-containing protein associates with heterochromatin and represses transcription

PubMed Central

Sathyan, Kizhakke M.; Shen, Zhen; Tripathi, Vidisha; Prasanth, Kannanganattu V.; Prasanth, Supriya G.

2011-01-01

In eukaryotes, higher order chromatin structure governs crucial cellular processes including DNA replication, transcription and post-transcriptional gene regulation. Specific chromatin-interacting proteins play vital roles in the maintenance of chromatin structure. We have identified BEND3, a quadruple BEN domain-containing protein that is highly conserved amongst vertebrates. BEND3 colocalizes with HP1 and H3 trimethylated at K9 at heterochromatic regions in mammalian cells. Using an in vivo gene locus, we have been able to demonstrate that BEND3 associates with the locus only when it is heterochromatic and dissociates upon activation of transcription. Furthermore, tethering BEND3 inhibits transcription from the locus, indicating that BEND3 is involved in transcriptional repression through its interaction with histone deacetylases and Sall4, a transcription repressor. We further demonstrate that BEND3 is SUMOylated and that such modifications are essential for its role in transcriptional repression. Finally, overexpression of BEND3 causes premature chromatin condensation and extensive heterochromatinization, resulting in cell cycle arrest. Taken together, our data demonstrate the role of a novel heterochromatin-associated protein in transcriptional repression. PMID:21914818

Opposing PKA and Hog1 signals control the post-transcriptional response to glucose availability in Cryptococcus neoformans.

PubMed

Banerjee, Dithi; Bloom, Amanda L M; Panepinto, John C

2016-10-01

The pathogenic fungus Cryptococcus neoformans must adapt to glucose-limited conditions in the lung and glucose replete conditions upon dissemination to the brain. We report that glucose controls ribosome biogenesis and translation by modulating mRNA decay through a balance of PKA and Hog1 signalling. Glucose signalling through PKA stabilized ribosomal protein (RP) mRNAs whereas glucose starvation destabilized RP transcripts through Hog1. Glucose starvation-induced oxidative stress response genes, and treatment of glucose-fed cells with reactive oxygen species (ROS) generating compounds repressed RP transcripts, both of which were dependent on Hog1. Stabilization of RP transcripts led to retention of polysomes in a hog1Δ mutant, whereas stabilization of RP transcripts by cyclic AMP did not affect translation repression, suggesting that Hog1 alone signals translation repression. In sum, this work describes a novel antagonism between PKA and Hog1 controlling ribosome biogenesis via mRNA stability in response to glucose availability in this important human pathogen. © 2016 John Wiley & Sons Ltd.

Enhanced stability and polyadenylation of select mRNAs support rapid thermogenesis in the brown fat of a hibernator

PubMed Central

Grabek, Katharine R; Diniz Behn, Cecilia; Barsh, Gregory S; Hesselberth, Jay R; Martin, Sandra L

2015-01-01

During hibernation, animals cycle between torpor and arousal. These cycles involve dramatic but poorly understood mechanisms of dynamic physiological regulation at the level of gene expression. Each cycle, Brown Adipose Tissue (BAT) drives periodic arousal from torpor by generating essential heat. We applied digital transcriptome analysis to precisely timed samples to identify molecular pathways that underlie the intense activity cycles of hibernator BAT. A cohort of transcripts increased during torpor, paradoxical because transcription effectively ceases at these low temperatures. We show that this increase occurs not by elevated transcription but rather by enhanced stabilization associated with maintenance and/or extension of long poly(A) tails. Mathematical modeling further supports a temperature-sensitive mechanism to protect a subset of transcripts from ongoing bulk degradation instead of increased transcription. This subset was enriched in a C-rich motif and genes required for BAT activation, suggesting a model and mechanism to prioritize translation of key proteins for thermogenesis. DOI: http://dx.doi.org/10.7554/eLife.04517.001 PMID:25626169

GABP transcription factor is required for development of chronic myelogenous leukemia via its control of PRKD2.

PubMed

Yang, Zhong-Fa; Zhang, Haojian; Ma, Leyuan; Peng, Cong; Chen, Yaoyu; Wang, Junling; Green, Michael R; Li, Shaoguang; Rosmarin, Alan G

2013-02-05

Hematopoietic stem cells (HSCs) are the source of all blood lineages, and HSCs must balance quiescence, self-renewal, and differentiation to meet lifelong needs for blood cell development. Transformation of HSCs by the breakpoint cluster region-ABL tyrosine kinase (BCR-ABL) oncogene causes chronic myelogenous leukemia (CML). The E-twenty six (ets) transcription factor GA binding protein (GABP) is a tetrameric transcription factor complex that contains GABPα and GABPβ proteins. Deletion in bone marrow of Gabpa, the gene that encodes the DNA-binding component, caused cell cycle arrest in HSCs and profound loss of hematopoietic progenitor cells. Loss of Gabpα prevented development of CML, although mice continued to generate BCR-ABL-expressing Gabpα-null cells for months that were serially transplantable and contributed to all lineages in secondary recipients. A bioinformatic screen identified the serine-threonine kinase protein kinase D2 (PRKD2) as a potential effector of GABP in HSCs. Prkd2 expression was markedly reduced in Gabpα-null HSCs and progenitor cells. Reduced expression of PRKD2 or pharmacologic inhibition decreased cell cycling, and PRKD2 rescued growth of Gabpα-null BCR-ABL-expressing cells. Thus, GABP is required for HSC cell cycle entry and CML development through its control of PRKD2. This offers a potential therapeutic target in leukemia.

Quantitative proteomics unravels that the post-transcriptional regulator Crc modulates the generation of vesicles and secreted virulence determinants of Pseudomonas aeruginosa.

PubMed

Reales-Calderón, Jose Antonio; Corona, Fernando; Monteoliva, Lucía; Gil, Concha; Martínez, Jose Luis

2015-09-08

Recent research indicates that the post-transcriptional regulator Crc modulates susceptibility to antibiotics and virulence in Pseudomonas aeruginosa. Several P. aeruginosa virulence factors are secreted or engulfed in vesicles. To decipher the Crc modulation of P. aeruginosa virulence, we constructed a crc deficient mutant and measure the proteome associated extracellular vesicles and the vesicle-free secretome using iTRAQ. Fifty vesicle-associated proteins were more abundant and 14 less abundant in the crc-defective strain, whereas 37 were more abundant and 17 less abundant in the vesicle-free secretome. Among them, virulence determinants, such as ToxA, protease IV, azurin, chitin-binding protein, PlcB and Hcp1, were less abundant in the crc-defective mutant. Transcriptomic analysis revealed that some of the observed changes were post-transcriptional and, thus, could be attributed to a direct Crc regulatory role; whereas, for other differentially secreted proteins, the regulatory role was likely indirect. We also observed that the crc mutant presented an impaired vesicle-associated secretion of quorum sensing signal molecules and less cytotoxicity than its wild-type strain. Our results offer new insights into the mechanisms by which Crc regulates P. aeruginosa virulence, through the modulation of vesicle formation and secretion of both virulence determinants and quorum sensing signals. This article is part of a Special Issue entitled: HUPO 2014. Published by Elsevier B.V.

Transcriptional profiling reveals the expression of novel genes in response to various stimuli in the human dermatophyte Trichophyton rubrum

PubMed Central

2010-01-01

Background Cutaneous mycoses are common human infections among healthy and immunocompromised hosts, and the anthropophilic fungus Trichophyton rubrum is the most prevalent microorganism isolated from such clinical cases worldwide. The aim of this study was to determine the transcriptional profile of T. rubrum exposed to various stimuli in order to obtain insights into the responses of this pathogen to different environmental challenges. Therefore, we generated an expressed sequence tag (EST) collection by constructing one cDNA library and nine suppression subtractive hybridization libraries. Results The 1388 unigenes identified in this study were functionally classified based on the Munich Information Center for Protein Sequences (MIPS) categories. The identified proteins were involved in transcriptional regulation, cellular defense and stress, protein degradation, signaling, transport, and secretion, among other functions. Analysis of these unigenes revealed 575 T. rubrum sequences that had not been previously deposited in public databases. Conclusion In this study, we identified novel T. rubrum genes that will be useful for ORF prediction in genome sequencing and facilitating functional genome analysis. Annotation of these expressed genes revealed metabolic adaptations of T. rubrum to carbon sources, ambient pH shifts, and various antifungal drugs used in medical practice. Furthermore, challenging T. rubrum with cytotoxic drugs and ambient pH shifts extended our understanding of the molecular events possibly involved in the infectious process and resistance to antifungal drugs. PMID:20144196

The transcriptome of Legionella pneumophila-infected human monocyte-derived macrophages.

PubMed

Price, Christopher T D; Abu Kwaik, Yousef

2014-01-01

Legionella pneumophila is an intracellular bacterial pathogen that invades and replicates within alveolar macrophages through injection of ∼ 300 effector proteins by its Dot/Icm type IV translocation apparatus. The bona fide F-box protein, AnkB, is a nutritional virulence effector that triggers macrophages to generate a surplus of amino acids, which is essential for intravacuolar proliferation. Therefore, the ankB mutant represents a novel genetic tool to determine the transcriptional response of human monocyte-derived macrophages (hMDMs) to actively replicating L. pneumophila. Here, we utilized total human gene microarrays to determine the global transcriptional response of hMDMs to infection by wild type or the ankB mutant of L. pneumophila. The transcriptomes of hMDMs infected with either actively proliferating wild type or non-replicative ankB mutant bacteria were remarkably similar. The transcriptome of infected hMDMs was predominated by up-regulation of inflammatory pathways (IL-10 anti-inflammatory, interferon signaling and amphoterin signaling), anti-apoptosis, and down-regulation of protein synthesis pathways. In addition, L. pneumophila modulated diverse metabolic pathways, particularly those associated with bio-active lipid metabolism, and SLC amino acid transporters expression. Taken together, the hMDM transcriptional response to L. pneumophila is independent of intra-vacuolar replication of the bacteria and primarily involves modulation of the immune response and metabolic as well as nutritional pathways.

An iron-sulfur center essential for transcriptional activation by the redox-sensing SoxR protein.

PubMed Central

Hidalgo, E; Demple, B

1994-01-01

The soxRS oxidative stress regulon of Escherichia coli is triggered by superoxide (O2.-) generating agents or by nitric oxide through two consecutive steps of gene activation. SoxR protein has been proposed as the redox sensing gene activator that triggers this cascade of gene expression. We have now characterized two forms of SoxR: Fe-SoxR contained non-heme iron (up to 1.6 atoms per monomer); apo-SoxR was devoid of Fe or other metals. The spectroscopic properties of Fe-SoxR indicated that it contains a redox active iron-sulfur (FeS) cluster that is oxidized upon extraction from E. coli. Fe-SoxR and apo-SoxR bound the in vivo target, the soxS promoter, with equal affinities and protected the same region from DNase I in vitro. However, only Fe-SoxR stimulated transcription initiation at soxS in vitro > 100-fold, similar to the activation of soxS expression in vivo. This stimulation occurred at a step after the binding of RNAP and indicates a conformational effect of oxidized Fe-SoxR on the soxS promoter. The variable redox state of the SoxR FeS cluster may thus be employed in vivo to modulate the transcriptional activity of this protein in response to specific types of oxidative stress. Images PMID:8306957

Transcriptional Regulation During Zygotic Genome Activation in Zebrafish and Other Anamniote Embryos.

PubMed

Wragg, J; Müller, F

2016-01-01

Embryo development commences with the fusion of two terminally differentiated haploid gametes into the totipotent fertilized egg, which through a series of major cellular and molecular transitions generate a pluripotent cell mass. The activation of the zygotic genome occurs during the so-called maternal to zygotic transition and prepares the embryo for zygotic takeover from maternal factors, in the control of the development of cellular lineages during differentiation. Recent advances in next generation sequencing technologies have allowed the dissection of the genomic and epigenomic processes mediating this transition. These processes include reorganization of the chromatin structure to a transcriptionally permissive state, changes in composition and function of structural and regulatory DNA-binding proteins, and changeover of the transcriptome as it is overhauled from that deposited by the mother in the oocyte to a zygotically transcribed complement. Zygotic genome activation in zebrafish occurs 10 cell cycles after fertilization and provides an ideal experimental platform for elucidating the temporal sequence and dynamics of establishment of a transcriptionally active chromatin state and helps in identifying the determinants of transcription activation at polymerase II transcribed gene promoters. The relatively large number of pluripotent cells generated by the fast cell divisions before zygotic transcription provides sufficient biomass for next generation sequencing technology approaches to establish the temporal dynamics of events and suggest causative relationship between them. However, genomic and genetic technologies need to be improved further to capture the earliest events in development, where cell number is a limiting factor. These technologies need to be complemented with precise, inducible genetic interference studies using the latest genome editing tools to reveal the function of candidate determinants and to confirm the predictions made by classic embryological tools and genome-wide assays. In this review we summarize recent advances in the characterization of epigenetic regulation, transcription control, and gene promoter function during zygotic genome activation and how they fit with old models for the mechanisms of the maternal to zygotic transition. This review will focus on the zebrafish embryo but draw comparisons with other vertebrate model systems and refer to invertebrate models where informative. Copyright © 2016 Elsevier Inc. All rights reserved.

The Conserved Foot Domain of RNA Pol II Associates with Proteins Involved in Transcriptional Initiation and/or Early Elongation

PubMed Central

García-López, M. Carmen; Pelechano, Vicent; Mirón-García, M. Carmen; Garrido-Godino, Ana I.; García, Alicia; Calvo, Olga; Werner, Michel; Pérez-Ortín, José E.; Navarro, Francisco

2011-01-01

RNA polymerase (pol) II establishes many protein–protein interactions with transcriptional regulators to coordinate different steps of transcription. Although some of these interactions have been well described, little is known about the existence of RNA pol II regions involved in contact with transcriptional regulators. We hypothesize that conserved regions on the surface of RNA pol II contact transcriptional regulators. We identified such an RNA pol II conserved region that includes the majority of the “foot” domain and identified interactions of this region with Mvp1, a protein required for sorting proteins to the vacuole, and Spo14, a phospholipase D. Deletion of MVP1 and SPO14 affects the transcription of their target genes and increases phosphorylation of Ser5 in the carboxy-terminal domain (CTD). Genetic, phenotypic, and functional analyses point to a role for these proteins in transcriptional initiation and/or early elongation, consistent with their genetic interactions with CEG1, a guanylyltransferase subunit of the Saccharomyces cerevisiae capping enzyme. PMID:21954159

An MSC2 Promoter-lacZ Fusion Gene Reveals Zinc-Responsive Changes in Sites of Transcription Initiation That Occur across the Yeast Genome

PubMed Central

Wu, Yi-Hsuan; Taggart, Janet; Song, Pamela Xiyao; MacDiarmid, Colin; Eide, David J.

2016-01-01

The Msc2 and Zrg17 proteins of Saccharomyces cerevisiae form a complex to transport zinc into the endoplasmic reticulum. ZRG17 is transcriptionally induced in zinc-limited cells by the Zap1 transcription factor. In this report, we show that MSC2 mRNA also increases (~1.5 fold) in zinc-limited cells. The MSC2 gene has two in-frame ATG codons at its 5’ end, ATG1 and ATG2; ATG2 is the predicted initiation codon. When the MSC2 promoter was fused at ATG2 to the lacZ gene, we found that unlike the chromosomal gene this reporter showed a 4-fold decrease in lacZ mRNA in zinc-limited cells. Surprisingly, β-galactosidase activity generated by this fusion gene increased ~7 fold during zinc deficiency suggesting the influence of post-transcriptional factors. Transcription of MSC2ATG2-lacZ was found to start upstream of ATG1 in zinc-replete cells. In zinc-limited cells, transcription initiation shifted to sites just upstream of ATG2. From the results of mutational and polysome profile analyses, we propose the following explanation for these effects. In zinc-replete cells, MSC2ATG2-lacZ mRNA with long 5’ UTRs fold into secondary structures that inhibit translation. In zinc-limited cells, transcripts with shorter unstructured 5’ UTRs are generated that are more efficiently translated. Surprisingly, chromosomal MSC2 did not show start site shifts in response to zinc status and only shorter 5’ UTRs were observed. However, the shifts that occur in the MSC2ATG2-lacZ construct led us to identify significant transcription start site changes affecting the expression of ~3% of all genes. Therefore, zinc status can profoundly alter transcription initiation across the yeast genome. PMID:27657924

mTOR signaling regulates myotube hypertrophy by modulating protein synthesis, rDNA transcription, and chromatin remodeling.

PubMed

von Walden, Ferdinand; Liu, Chang; Aurigemma, Nicole; Nader, Gustavo A

2016-10-01

Ribosome production is an early event during skeletal muscle hypertrophy and precedes muscle protein accretion. Signaling via mTOR is crucial for ribosome production and hypertrophy; however, the mechanisms by which it regulates these processes remain to be identified. Herein, we investigated the activation of mTOR signaling in hypertrophying myotubes and determined that mTOR coordinates various aspects of gene expression important for ribosome production. First, inhibition of translation with cycloheximide had a more potent effect on protein synthesis than rapamycin indicating that mTOR function during hypertrophy is not on general, but rather on specific protein synthesis. Second, blocking Pol II transcription had a similar effect as Rapamycin and, unexpectedly, revealed the necessity of Pol II transcription for Pol I transcription, suggesting that mTOR may regulate ribosome production also by controlling Class II genes at the transcriptional level. Third, Pol I activity is essential for rDNA transcription and, surprisingly, for protein synthesis as selective Pol I inhibition blunted rDNA transcription, protein synthesis, and the hypertrophic response of myotubes. Finally, mTOR has nuclear localization in muscle, which is not sensitive to rapamycin. Inhibition of mTOR signaling by rapamycin disrupted mTOR-rDNA promoter interaction and resulted in altered histone marks indicative of repressed transcription and formation of higher-order chromatin structure. Thus mTOR signaling appears to regulate muscle hypertrophy by affecting protein synthesis, Class I and II gene expression, and chromatin remodeling. Copyright © 2016 the American Physiological Society.

NRF2: Translating the Redox Code.

PubMed

Tummala, Krishna S; Kottakis, Filippos; Bardeesy, Nabeel

2016-10-01

Cancer requires mechanisms to mitigate reactive oxygen species (ROS) generated during rapid growth, such as induction of the antioxidant transcription factor, Nrf2. However, the targets of ROS-mediated cytotoxicity are unclear. Recent studies in pancreatic cancer show that redox control by Nrf2 prevents cysteine oxidation of the mRNA translational machinery, thereby supporting efficient protein synthesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Transcript levels, alternative splicing and proteolytic cleavage of TFIIIA control 5S rRNA accumulation during Arabidopsis thaliana development.

PubMed

Layat, Elodie; Cotterell, Sylviane; Vaillant, Isabelle; Yukawa, Yasushi; Tutois, Sylvie; Tourmente, Sylvette

2012-07-01

Ribosome biogenesis is critical for eukaryotic cells and requires coordinated synthesis of the protein and rRNA moieties of the ribosome, which are therefore highly regulated. 5S ribosomal RNA, an essential component of the large ribosomal subunit, is transcribed by RNA polymerase III and specifically requires transcription factor IIIA (TFIIIA). To obtain insight into the regulation of 5S rRNA transcription, we have investigated the expression of 5S rRNA and the exon-skipped (ES) and exon-including (EI) TFIIIA transcripts, two transcript isoforms that result from alternative splicing of the TFIIIA gene, and TFIIIA protein amounts with respect to requirements for 5S rRNA during development. We show that 5S rRNA quantities are regulated through distinct but complementary mechanisms operating through transcriptional and post-transcriptional control of TFIIIA transcripts as well as at the post-translational level through proteolytic cleavage of the TFIIIA protein. During the reproductive phase, high expression of the TFIIIA gene together with low proteolytic cleavage contributes to accumulation of functional, full-length TFIIIA protein, and results in 5S rRNA accumulation in the seed. In contrast, just after germination, the levels of TFIIIA-encoding transcripts are low and stable. Full-length TFIIIA protein is undetectable, and the level of 5S rRNA stored in the embryo progressively decreases. After day 4, in correlation with the reorganization of 5S rDNA chromatin to a mature state, full-length TFIIIA protein with transcriptional activity accumulates and permits de novo transcription of 5S rRNA. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

Chromatin immunoprecipitation of mouse embryos.

PubMed

Voss, Anne K; Dixon, Mathew P; McLennan, Tamara; Kueh, Andrew J; Thomas, Tim

2012-01-01

During prenatal development, a large number of different cell types are formed, the vast majority of which contain identical genetic material. The basis of the great variety in cell phenotype and function is the differential expression of the approximately 25,000 genes in the mammalian genome. Transcriptional activity is regulated at many levels by proteins, including members of the basal transcriptional apparatus, DNA-binding transcription factors, and chromatin-binding proteins. Importantly, chromatin structure dictates the availability of a specific genomic locus for transcriptional activation as well as the efficiency, with which transcription can occur. Chromatin immunoprecipitation (ChIP) is a method to assess if chromatin modifications or proteins are present at a specific locus. ChIP involves the cross linking of DNA and associated proteins and immunoprecipitation using specific antibodies to DNA-associated proteins followed by examination of the co-precipitated DNA sequences or proteins. In the last few years, ChIP has become an essential technique for scientists studying transcriptional regulation and chromatin structure. Using ChIP on mouse embryos, we can document the presence or absence of specific proteins and chromatin modifications at genomic loci in vivo during mammalian development. Here, we describe a ChIP technique adapted for mouse embryos.

A homozygous Keap1-knockout human embryonic stem cell line generated using CRISPR/Cas9 mediates gene targeting.

PubMed

Kim, So-Jung; Habib, Omer; Kim, Jin-Soo; Han, Hyo-Won; Koo, Soo Kyung; Kim, Jung-Hyun

2017-03-01

Kelch-like ECH-associated protein 1 (keap1) is a cysteine-rich protein that interacts with transcription factor Nrf2 in a redox-sensitive manner, leading to the degradation of Nrf2 (Kim et al., 2014a). Disruption of Keap1 results in the induction of Nrf2-related signaling pathways involving the expression of a set of anti-oxidant and anti-inflammatory genes. We generated biallelic mutants of the Keap1 gene using a CRISPR-Cas9 genome editing method in the H9 human embryonic stem cell (hESC). The Keap1 homozygous-knockout H9 cell line retained normal morphology, gene expression, and in vivo differentiation potential. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation

DOE PAGES

Xiong, Yi; Coradetti, Samuel T.; Li, Xin; ...

2014-05-29

Improving cellulolytic enzyme production by plant biomass degrading fungi holds great potential in reducing costs associated with production of next-generation biofuels generated from lignocellulose. How fungi sense cellulosic materials and respond by secreting enzymes has mainly been examined by assessing function of transcriptional regulators and via transcriptional profiling. Here, we obtained global proteomic and phosphoproteomic profiles of the plant biomass degrading filamentous fungus Neurospora crassa grown on different carbon sources, i.e. sucrose, no carbon, and cellulose, by performing isobaric tags for relative and absolute quantification (iTRAQ) -based LC-MS/MS analyses. A comparison between proteomes and transcriptomes under identical carbon conditions suggestsmore » that extensive post-transcriptional regulation occurs in N. crassa in response to exposure to cellulosic material. Several hundred amino acid residues with differential phosphorylation levels on crystalline cellulose (Avicel) or carbon-free medium versus sucrose medium were identified, including phosphorylation sites in a major transcriptional activator for cellulase genes, CLR1, as well as a cellobionic acid transporter, CBT1. Finally, we found mutation of phosphorylation sites on CLR1 did not have a major effect on transactivation of cellulase production, while mutation of phosphorylation sites in CBT1 increased its transporting capacity. Our data provides rich information at both the protein and phosphorylation levels of the early cellular responses to carbon starvation and cellulosic induction and aids in a greater understanding of the underlying post-transcriptional regulatory mechanisms in filamentous fungi.« less

Transcriptome and gene expression analysis during flower blooming in Rosa chinensis 'Pallida'.

PubMed

Yan, Huijun; Zhang, Hao; Chen, Min; Jian, Hongying; Baudino, Sylvie; Caissard, Jean-Claude; Bendahmane, Mohammed; Li, Shubin; Zhang, Ting; Zhou, Ningning; Qiu, Xianqin; Wang, Qigang; Tang, Kaixue

2014-04-25

Rosa chinensis 'Pallida' (Rosa L.) is one of the most important ancient rose cultivars originating from China. It contributed the 'tea scent' trait to modern roses. However, little information is available on the gene regulatory networks involved in scent biosynthesis and metabolism in Rosa. In this study, the transcriptome of R. chinensis 'Pallida' petals at different developmental stages, from flower buds to senescent flowers, was investigated using Illumina sequencing technology. De novo assembly generated 89,614 clusters with an average length of 428bp. Based on sequence similarity search with known proteins, 62.9% of total clusters were annotated. Out of these annotated transcripts, 25,705 and 37,159 sequences were assigned to gene ontology and clusters of orthologous groups, respectively. The dataset provides information on transcripts putatively associated with known scent metabolic pathways. Digital gene expression (DGE) was obtained using RNA samples from flower bud, open flower and senescent flower stages. Comparative DGE and quantitative real time PCR permitted the identification of five transcripts encoding proteins putatively associated with scent biosynthesis in roses. The study provides a foundation for scent-related gene discovery in roses. Copyright © 2014. Published by Elsevier B.V.

Stimulation of StAR expression by cAMP is controlled by inhibition of highly inducible SIK1 via CRTC2, a co-activator of CREB.

PubMed

Lee, Jinwoo; Tong, Tiegang; Takemori, Hiroshi; Jefcoate, Colin

2015-06-15

In mouse steroidogenic cells the activation of cholesterol metabolism is mediated by steroidogenic acute regulatory protein (StAR). Here, we visualized a coordinated regulation of StAR transcription, splicing and post-transcriptional processing, which are synchronized by salt inducible kinase (SIK1) and CREB-regulated transcription coactivator (CRTC2). To detect primary RNA (pRNA), spliced primary RNA (Sp-RNA) and mRNA in single cells, we generated probe sets by using fluorescence in situ hybridization (FISH). These methods allowed us to address the nature of StAR gene expression and to visualize protein-nucleic acid interactions through direct detection. We show that SIK1 represses StAR expression in Y1 adrenal and MA10 testis cells through inhibition of processing mediated by CRTC2. Digital image analysis matches qPCR analyses of the total cell culture. Evidence is presented for spatially separate accumulation of StAR pRNA and Sp-RNA at the gene loci in the nucleus. These findings establish that cAMP, SIK and CRTC mediate StAR expression through activation of individual StAR gene loci. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Mapping transcription factor interactome networks using HaloTag protein arrays.

PubMed

Yazaki, Junshi; Galli, Mary; Kim, Alice Y; Nito, Kazumasa; Aleman, Fernando; Chang, Katherine N; Carvunis, Anne-Ruxandra; Quan, Rosa; Nguyen, Hien; Song, Liang; Alvarez, José M; Huang, Shao-Shan Carol; Chen, Huaming; Ramachandran, Niroshan; Altmann, Stefan; Gutiérrez, Rodrigo A; Hill, David E; Schroeder, Julian I; Chory, Joanne; LaBaer, Joshua; Vidal, Marc; Braun, Pascal; Ecker, Joseph R

2016-07-19

Protein microarrays enable investigation of diverse biochemical properties for thousands of proteins in a single experiment, an unparalleled capacity. Using a high-density system called HaloTag nucleic acid programmable protein array (HaloTag-NAPPA), we created high-density protein arrays comprising 12,000 Arabidopsis ORFs. We used these arrays to query protein-protein interactions for a set of 38 transcription factors and transcriptional regulators (TFs) that function in diverse plant hormone regulatory pathways. The resulting transcription factor interactome network, TF-NAPPA, contains thousands of novel interactions. Validation in a benchmarked in vitro pull-down assay revealed that a random subset of TF-NAPPA validated at the same rate of 64% as a positive reference set of literature-curated interactions. Moreover, using a bimolecular fluorescence complementation (BiFC) assay, we confirmed in planta several interactions of biological interest and determined the interaction localizations for seven pairs. The application of HaloTag-NAPPA technology to plant hormone signaling pathways allowed the identification of many novel transcription factor-protein interactions and led to the development of a proteome-wide plant hormone TF interactome network.

RNA polymerase II conserved protein domains as platforms for protein-protein interactions

PubMed Central

García-López, M Carmen

2011-01-01

RNA polymerase II establishes many protein-protein interactions with transcriptional regulators to coordinate gene expression, but little is known about protein domains involved in the contact with them. We use a new approach to look for conserved regions of the RNA pol II of S. cerevisiae located at the surface of the structure of the complex, hypothesizing that they might be involved in the interaction with transcriptional regulators. We defined five different conserved domains and demonstrate that all of them make contact with transcriptional regulators. PMID:21922063

Pharmacological targeting of the transcription factor SOX18 delays breast cancer in mice.

PubMed

Overman, Jeroen; Fontaine, Frank; Moustaqil, Mehdi; Mittal, Deepak; Sierecki, Emma; Sacilotto, Natalia; Zuegg, Johannes; Robertson, Avril Ab; Holmes, Kelly; Salim, Angela A; Mamidyala, Sreeman; Butler, Mark S; Robinson, Ashley S; Lesieur, Emmanuelle; Johnston, Wayne; Alexandrov, Kirill; Black, Brian L; Hogan, Benjamin M; De Val, Sarah; Capon, Robert J; Carroll, Jason S; Bailey, Timothy L; Koopman, Peter; Jauch, Ralf; Smyth, Mark J; Cooper, Matthew A; Gambin, Yann; Francois, Mathias

2017-01-31

Pharmacological targeting of transcription factors holds great promise for the development of new therapeutics, but strategies based on blockade of DNA binding, nuclear shuttling, or individual protein partner recruitment have yielded limited success to date. Transcription factors typically engage in complex interaction networks, likely masking the effects of specifically inhibiting single protein-protein interactions. Here, we used a combination of genomic, proteomic and biophysical methods to discover a suite of protein-protein interactions involving the SOX18 transcription factor, a known regulator of vascular development and disease. We describe a small-molecule that is able to disrupt a discrete subset of SOX18-dependent interactions. This compound selectively suppressed SOX18 transcriptional outputs in vitro and interfered with vascular development in zebrafish larvae. In a mouse pre-clinical model of breast cancer, treatment with this inhibitor significantly improved survival by reducing tumour vascular density and metastatic spread. Our studies validate an interactome-based molecular strategy to interfere with transcription factor activity, for the development of novel disease therapeutics.

Associations between transcriptional changes and protein phenotypes provide insights into immune regulation in corals.

PubMed

Fuess, Lauren E; Pinzόn C, Jorge H; Weil, Ernesto; Mydlarz, Laura D

2016-09-01

Disease outbreaks in marine ecosystems have driven worldwide declines of numerous taxa, including corals. Some corals, such as Orbicella faveolata, are particularly susceptible to disease. To explore the mechanisms contributing to susceptibility, colonies of O. faveolata were exposed to immune challenge with lipopolysaccharides. RNA sequencing and protein activity assays were used to characterize the response of corals to immune challenge. Differential expression analyses identified 17 immune-related transcripts that varied in expression post-immune challenge. Network analyses revealed several groups of transcripts correlated to immune protein activity. Several transcripts, which were annotated as positive regulators of immunity were included in these groups, and some were downregulated following immune challenge. Correlations between expression of these transcripts and protein activity results further supported the role of these transcripts in positive regulation of immunity. The observed pattern of gene expression and protein activity may elucidate the processes contributing to the disease susceptibility of species like O. faveolata. Copyright © 2016 Elsevier Ltd. All rights reserved.

Depletion of the cellular levels of Bag-1 proteins attenuates phorbol ester-induced downregulation of I{kappa}B{alpha} and nuclear accumulation of NF-{kappa}B

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maier, Jana V., E-mail: Jana.maier@kit.edu; Volz, Yvonne; Berger, Caroline

2010-10-22

Research highlights: {yields}Bag-1 depletion only marginally affects the action of the glucocorticoid receptor but strongly regulates the activity of NF-{kappa}B. {yields}Bag-1 depletion attenuates phosphorylation and degradation of I{kappa}B{alpha} and nuclear accumulation of NF-{kappa}B p65 and p50. {yields}Bag-1 interacts with I{kappa}B{alpha} and partially restores I{kappa}B{alpha} and NF-{kappa}B activation in Bag-1 depleted cells. -- Abstract: Bag-1 consists in humans of four isoforms generated from the same RNA by alternative translation. Overexpression of single Bag-1 isoforms has identified Bag-1 as a negative regulator of action of many proteins including the glucocorticoid receptor (GR). Here we have analysed the ability of Bag-1 to regulatemore » the transrepression function of the GR. Silencing Bag-1 expression only marginally affects the transrepression action of the GR but decreased the action of the transcription factor NF-{kappa}B. Furthermore phosphorylation and degradation of the inhibitor protein I{kappa}B{alpha} and nuclear accumulation of p65 and p50 NF-{kappa}B proteins in response to phorbol ester was attenuated following Bag-1 depletion in HeLa cells. Reconstitution of Bag-1 in depleted cells partially restored I{kappa}B{alpha} and NF-{kappa}B activation. Knock-down of Bag-1 expression also did not significantly alter GR-mediated transactivation but affected the basal transcription of some of the target genes. Thus Bag-1 proteins function as regulators of the action of selective transcription factors.« less

Type I human T cell leukemia virus tax protein transforms rat fibroblasts through the cyclic adenosine monophosphate response element binding protein/activating transcription factor pathway.

PubMed Central

Smith, M R; Greene, W C

1991-01-01

The Tax oncoprotein of the type I human T cell leukemia virus (HTLV-I) activates transcription of cellular and viral genes through at least two different transcription factor pathways. Tax activates transcription of the c-fos proto-oncogene by a mechanism that appears to involve members of the cAMP response element binding protein (CREB) and activating transcription factor (ATF) family of DNA-binding proteins. Tax also induces the nuclear expression of the NF-kappa B family of rel oncogene-related enhancer-binding proteins. We have investigated the potential role of these CREB/ATF and NF-kappa B/Rel transcription factors in Tax-mediated transformation by analyzing the oncogenic potential of Tax mutants that functionally segregate these two pathways of transactivation. Rat fibroblasts (Rat2) stably expressing either the wild-type Tax protein or a Tax mutant selectively deficient in the ability to induce NF-kappa B/Rel demonstrated marked changes in morphology and growth characteristics including the ability to form tumors in athymic mice. In contrast, Rat2 cells stably expressing a Tax mutant selectively deficient in the ability to activate transcription through CREB/ATF demonstrated no detectable changes in morphology or growth characteristics. These results suggest that transcriptional activation through the CREB/ATF pathway may play an important role in Tax-mediated cellular transformation. Images PMID:1832173

Genome-wide transcriptional analysis of two soybean genotypes under dehydration and rehydration conditions

PubMed Central

2013-01-01

Background Soybean is an important crop that provides valuable proteins and oils for human use. Because soybean growth and development is extremely sensitive to water deficit, quality and crop yields are severely impacted by drought stress. In the face of limited water resources, drought-responsive genes are therefore of interest. Identification and analysis of dehydration- and rehydration-inducible differentially expressed genes (DEGs) would not only aid elucidation of molecular mechanisms of stress response, but also enable improvement of crop stress tolerance via gene transfer. Using Digital Gene Expression Tag profiling (DGE), a new technique based on Illumina sequencing, we analyzed expression profiles between two soybean genotypes to identify drought-responsive genes. Results Two soybean genotypes—drought-tolerant Jindou21 and drought-sensitive Zhongdou33—were subjected to dehydration and rehydration conditions. For analysis of DEGs under dehydration conditions, 20 cDNA libraries were generated from roots and leaves at two different time points under well-watered and dehydration conditions. We also generated eight libraries for analysis under rehydration conditions. Sequencing of the 28 libraries produced 25,000–33,000 unambiguous tags, which were mapped to reference sequences for annotation of expressed genes. Many genes exhibited significant expression differences among the libraries. DEGs in the drought-tolerant genotype were identified by comparison of DEGs among treatments and genotypes. In Jindou21, 518 and 614 genes were differentially expressed under dehydration in leaves and roots, respectively, with 24 identified both in leaves and roots. The main functional categories enriched in these DEGs were metabolic process, response to stresses, plant hormone signal transduction, protein processing, and plant-pathogen interaction pathway; the associated genes primarily encoded transcription factors, protein kinases, and other regulatory proteins. The seven most significantly expressed (|log2 ratio| ≥ 8) genes— Glyma15g03920, Glyma05g02470, Glyma15g15010, Glyma05g09070, Glyma06g35630, Glyma08g12590, and Glyma11g16000—are more likely to determine drought stress tolerance. The expression patterns of eight randomly-selected genes were confirmed by quantitative RT-PCR; the results of QRT-PCR analysis agreed with transcriptional profile data for 96 out of 128 (75%) data points. Conclusions Many soybean genes were differentially expressed between drought-tolerant and drought-sensitive genotypes. Based on GO functional annotation and pathway enrichment analysis, some of these genes encoded transcription factors, protein kinases, and other regulatory proteins. The seven most significant DEGs are candidates for improving soybean drought tolerance. These findings will be helpful for analysis and elucidation of molecular mechanisms of drought tolerance; they also provide a basis for cultivating new varieties of drought-tolerant soybean. PMID:24093224

GHF-1-promoter-targeted immortalization of a somatotropic progenitor cell results in dwarfism in transgenic mice.

PubMed

Lew, D; Brady, H; Klausing, K; Yaginuma, K; Theill, L E; Stauber, C; Karin, M; Mellon, P L

1993-04-01

During pituitary development, the homeo domain protein GHF-1 is required for generation of somatotropes and lactotropes and for growth hormone (GH) and prolactin (PRL) gene expression. GHF-1 mRNA is detectable several days before the emergence of GH- or PRL-expressing cells, suggesting the existence of a somatotropic progenitor cell in which GHF-1 transcription is first activated. We have immortalized this cell type by using the GHF-1 regulatory region to target SV40 T-antigen (Tag) tumorigenesis in transgenic mice. The GHF-Tag transgene caused developmental entrapment of somatotropic progenitor cells that express GHF-1 but not GH or PRL, resulting in dwarfism. Immortalized cell lines derived from a transgenic pituitary tumor maintain the characteristics of the somato/lactotropic progenitor in that they express GHF-1 mRNA and protein yet fail to activate GH or PRL transcription. Using these cells, we identified an enhancer that activates GHF-1 transcription at this early stage of development yet is inactive in cells representing later developmental stages of the somatotropic lineage or in other cell types. These experiments not only demonstrate the potential for immortalization of developmental progenitor cells using the regulatory regions from cell type-specific transcription factor genes but illustrate the power of such model systems in the study of developmental control.

Expression of human papillomavirus 6 in inverted papilloma arising in a renal transplant recipient.

PubMed

Harris, M O; Beck, J C; Terrell, J E; McClatchey, K D; Carey, T E; Bradford, C R

1998-01-01

A 36-year-old renal transplant recipient taking cyclosporin A presented with bilateral nasal polypoid lesions involving the nasal septum and lateral nasal walls. Pathologic findings from surgical excision demonstrated inverted papilloma (IP) with focal atypia and mild dysplasia. DNA extracted from the tissue was tested with the polymerase chain reaction (PCR) using human papillomavirus (HPV) E6 and L1 consensus primers. This revealed amplification of the expected size fragment consistent with the presence of HPV DNA. Hybridization of PCR products with HPV type-specific oligonucleotide probes revealed a strong signal with only HPV 6. This result was confirmed by PCR amplification with HPV 6 type-specific primers. RNA extracted from the tissue was subjected to reverse transcription PCR (RT-PCR) with a primer pair specific for viral E6/E7 transcripts. The HPV early proteins, E6 and E7, are the transforming proteins implicated as critical for tumorigenesis. RT-PCR experiments generated products representing the E1/E4 spliced transcript originating from the E6/E6 promoter and a smaller unclassified fragment. These results provide evidence for HPV 6 E6/E7 expression in IP, lending credence to the concept that HPV may play a role in the origin of this neoplasm. Histologically normal nasal tissue from the same patient contained HPV DNA and similar transcripts to those described in the IP specimen.

Epigenetic editing of the Dlg4/PSD95 gene improves cognition in aged and Alzheimer's disease mice.

PubMed

Bustos, Fernando J; Ampuero, Estibaliz; Jury, Nur; Aguilar, Rodrigo; Falahi, Fahimeh; Toledo, Jorge; Ahumada, Juan; Lata, Jaclyn; Cubillos, Paula; Henríquez, Berta; Guerra, Miguel V; Stehberg, Jimmy; Neve, Rachael L; Inestrosa, Nibaldo C; Wyneken, Ursula; Fuenzalida, Marco; Härtel, Steffen; Sena-Esteves, Miguel; Varela-Nallar, Lorena; Rots, Marianne G; Montecino, Martin; van Zundert, Brigitte

2017-12-01

The Dlg4 gene encodes for post-synaptic density protein 95 (PSD95), a major synaptic protein that clusters glutamate receptors and is critical for plasticity. PSD95 levels are diminished in ageing and neurodegenerative disorders, including Alzheimer's disease and Huntington's disease. The epigenetic mechanisms that (dys)regulate transcription of Dlg4/PSD95, or other plasticity genes, are largely unknown, limiting the development of targeted epigenome therapy. We analysed the Dlg4/PSD95 epigenetic landscape in hippocampal tissue and designed a Dlg4/PSD95 gene-targeting strategy: a Dlg4/PSD95 zinc finger DNA-binding domain was engineered and fused to effector domains to either repress (G9a, Suvdel76, SKD) or activate (VP64) transcription, generating artificial transcription factors or epigenetic editors (methylating H3K9). These epi-editors altered critical histone marks and subsequently Dlg4/PSD95 expression, which, importantly, impacted several hippocampal neuron plasticity processes. Intriguingly, transduction of the artificial transcription factor PSD95-VP64 rescued memory deficits in aged and Alzheimer's disease mice. Conclusively, this work validates PSD95 as a key player in memory and establishes epigenetic editing as a potential therapy to treat human neurological disorders. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Protein Interaction Profile Sequencing (PIP-seq).

PubMed

Foley, Shawn W; Gregory, Brian D

2016-10-10

Every eukaryotic RNA transcript undergoes extensive post-transcriptional processing from the moment of transcription up through degradation. This regulation is performed by a distinct cohort of RNA-binding proteins which recognize their target transcript by both its primary sequence and secondary structure. Here, we describe protein interaction profile sequencing (PIP-seq), a technique that uses ribonuclease-based footprinting followed by high-throughput sequencing to globally assess both protein-bound RNA sequences and RNA secondary structure. PIP-seq utilizes single- and double-stranded RNA-specific nucleases in the absence of proteins to infer RNA secondary structure. These libraries are also compared to samples that undergo nuclease digestion in the presence of proteins in order to find enriched protein-bound sequences. Combined, these four libraries provide a comprehensive, transcriptome-wide view of RNA secondary structure and RNA protein interaction sites from a single experimental technique. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Exploring the roles of basal transcription factor 3 in eukaryotic growth and development.

PubMed

Jamil, Muhammad; Wang, Wenyi; Xu, Mengyun; Tu, Jumin

2015-01-01

Basal transcription factor 3 (BTF3) has been reported to play a significant part in the transcriptional regulation linking with eukaryotes growth and development. Alteration in the BTF3 gene expression patterns or variation in their activities adds to the explanation of different signaling pathways and regulatory networks. Moreover, BTF3s often respond to numerous stresses, and subsequently they are involved in regulation of various mechanisms. BTF3 proteins also function through protein-protein contact, which can assist us to identify the multifaceted processes of signaling and transcriptional regulation controlled by BTF3 proteins. In this review, we discuss current advances made in starting to explore the roles of BTF3 transcription factors in eukaryotes especially in plant growth and development.

Pioglitazone inhibits LOX-1 expression in human coronary artery endothelial cells by reducing intracellular superoxide radical generation.

PubMed

Mehta, Jawahar L; Hu, Bo; Chen, Jiawei; Li, Dayuan

2003-12-01

LOX-1, a novel lectin-like receptor for oxidized LDL (ox-LDL), is expressed in response to ox-LDL, angiotensin II (Ang II), tumor necrosis factor (TNF)-alpha, and other stress stimuli. It is highly expressed in atherosclerotic tissues. Peroxisome proliferator-activated receptor (PPAR)-gamma ligands, such as pioglitazone, exert antiatherosclerotic effects. This study examined the regulation of LOX-1 expression in human coronary artery endothelial cells (HCAECs) by pioglitazone. Fourth generation HCAECs were treated with ox-LDL, Ang II, or TNF-alpha with or without pioglitazone pretreatment. All 3 stimuli upregulated LOX-1 expression (mRNA and protein). Pioglitazone, in a concentration-dependent manner, reduced LOX-1 expression (P<0.01 versus ox-LDL, Ang II, or TNF-alpha alone). Ox-LDL, Ang II, and TNF-alpha each enhanced intracellular superoxide radical generation, and pioglitazone pretreatment reduced superoxide generation (P<0.01 versus ox-LDL, Ang II, or TNF-alpha). Furthermore, all 3 stimuli upregulated the expression of the transcription factors nuclear factor-kappaB and activator protein-1 (determined by electrophoretic mobility shift assay), and pioglitazone pretreatment reduced this expression (P<0.01 versus ox-LDL, Ang II, or TNF-alpha). To determine the biological significance of pioglitazone-mediated downregulation of LOX-1, we studied monocyte adhesion to ox-LDL-treated HCAECs. Pioglitazone reduced the adhesion of monocytes to activated HCAECs in a fashion similar to that produced by antisense to LOX-1 mRNA. These observations suggest that the PPAR-gamma ligand pioglitazone reduces intracellular superoxide radical generation and subsequently reduces the expression of transcription factors, expression of the LOX-1 gene, and monocyte adhesion to activated endothelium. The salutary effect of PPAR-gamma ligands in atherogenesis may involve the inhibition of LOX-1 and the adhesion of monocytes to endothelium.

Ménage à trois: the complex relationships between mitogen-activated protein kinases, WRKY transcription factors, and VQ-motif-containing proteins.

PubMed

Weyhe, Martin; Eschen-Lippold, Lennart; Pecher, Pascal; Scheel, Dierk; Lee, Justin

2014-01-01

Out of the 34 members of the VQ-motif-containing protein (VQP) family, 10 are phosphorylated by the mitogen-activated protein kinases (MAPKs), MPK3 and MPK6. Most of these MPK3/6-targeted VQPs (MVQs) interacted with specific sub-groups of WRKY transcription factors in a VQ-motif-dependent manner. In some cases, the MAPK appears to phosphorylate either the MVQ or the WRKY, while in other cases, both proteins have been reported to act as MAPK substrates. We propose a network of dynamic interactions between members from the MAPK, MVQ and WRKY families - either as binary or as tripartite interactions. The compositions of the WRKY-MVQ transcriptional protein complexes may change - for instance, through MPK3/6-mediated modulation of protein stability - and therefore control defense gene transcription.

Eaf1p Is Required for Recruitment of NuA4 in Targeting TFIID to the Promoters of the Ribosomal Protein Genes for Transcriptional Initiation In Vivo

PubMed Central

Uprety, Bhawana; Sen, Rwik

2015-01-01

NuA4 (nucleosome acetyltransferase of H4) promotes transcriptional initiation of TFIID (a complex of TBP and TBP-associated factors [TAFs])-dependent ribosomal protein genes involved in ribosome biogenesis. However, it is not clearly understood how NuA4 regulates the transcription of ribosomal protein genes. Here, we show that NuA4 is recruited to the promoters of ribosomal protein genes, such as RPS5, RPL2B, and RPS11B, for TFIID recruitment to initiate transcription, and the recruitment of NuA4 to these promoters is impaired in the absence of its Eaf1p component. Intriguingly, impaired NuA4 recruitment in a Δeaf1 strain depletes recruitment of TFIID (a TAF-dependent form of TBP) but not the TAF-independent form of TBP to the promoters of ribosomal protein genes. However, in the absence of NuA4, SAGA (Spt-Ada-Gcn5-acetyltransferase) is involved in targeting the TAF-independent form of TBP to the promoters of ribosomal protein genes for transcriptional initiation. Thus, NuA4 plays an important role in targeting TFIID to the promoters of ribosomal protein genes for transcriptional initiation in vivo. Such a function is mediated via its targeted histone acetyltransferase activity. In the absence of NuA4, ribosomal protein genes lose TFIID dependency and become SAGA dependent for transcriptional initiation. Collectively, these results provide significant insights into the regulation of ribosomal protein gene expression and, hence, ribosome biogenesis and functions. PMID:26100014

Genome-Wide Cell Type-Specific Mapping of In Vivo Chromatin Protein Binding Using an FLP-Inducible DamID System in Drosophila.

PubMed

Pindyurin, Alexey V

2017-01-01

A thorough study of the genome-wide binding patterns of chromatin proteins is essential for understanding the regulatory mechanisms of genomic processes in eukaryotic nuclei, including DNA replication, transcription, and repair. The DNA adenine methyltransferase identification (DamID) method is a powerful tool to identify genomic binding sites of chromatin proteins. This method does not require fixation of cells and the use of specific antibodies, and has been used to generate genome-wide binding maps of more than a hundred different proteins in Drosophila tissue culture cells. Recent versions of inducible DamID allow performing cell type-specific profiling of chromatin proteins even in small samples of Drosophila tissues that contain heterogeneous cell types. Importantly, with these methods sorting of cells of interest or their nuclei is not necessary as genomic DNA isolated from the whole tissue can be used as an input. Here, I describe in detail an FLP-inducible DamID method, namely generation of suitable transgenic flies, activation of the Dam transgenes by the FLP recombinase, isolation of DNA from small amounts of dissected tissues, and subsequent identification of the DNA binding sites of the chromatin proteins.

Transfer RNA Post-Transcriptional Processing, Turnover, and Subcellular Dynamics in the Yeast Saccharomyces cerevisiae

PubMed Central

Hopper, Anita K.

2013-01-01

Transfer RNAs (tRNAs) are essential for protein synthesis. In eukaryotes, tRNA biosynthesis employs a specialized RNA polymerase that generates initial transcripts that must be subsequently altered via a multitude of post-transcriptional steps before the tRNAs beome mature molecules that function in protein synthesis. Genetic, genomic, biochemical, and cell biological approaches possible in the powerful Saccharomyces cerevisiae system have led to exciting advances in our understandings of tRNA post-transcriptional processing as well as to novel insights into tRNA turnover and tRNA subcellular dynamics. tRNA processing steps include removal of transcribed leader and trailer sequences, addition of CCA to the 3′ mature sequence and, for tRNAHis, addition of a 5′ G. About 20% of yeast tRNAs are encoded by intron-containing genes. The three-step splicing process to remove the introns surprisingly occurs in the cytoplasm in yeast and each of the splicing enzymes appears to moonlight in functions in addition to tRNA splicing. There are 25 different nucleoside modifications that are added post-transcriptionally, creating tRNAs in which ∼15% of the residues are nucleosides other than A, G, U, or C. These modified nucleosides serve numerous important functions including tRNA discrimination, translation fidelity, and tRNA quality control. Mature tRNAs are very stable, but nevertheless yeast cells possess multiple pathways to degrade inappropriately processed or folded tRNAs. Mature tRNAs are also dynamic in cells, moving from the cytoplasm to the nucleus and back again to the cytoplasm; the mechanism and function of this retrograde process is poorly understood. Here, the state of knowledge for tRNA post-transcriptional processing, turnover, and subcellular dynamics is addressed, highlighting the questions that remain. PMID:23633143

Specific Inhibition of the transcription factor Ci by a Cobalt(III)-Schiff base-DNA conjugate

PubMed Central

Hurtado, Ryan R.; Harney, Allison S.; Heffern, Marie C.; Holbrook, Robert J.; Holmgren, Robert A.; Meade, Thomas J.

2012-01-01

We describe the use of Co(III) Schiff base-DNA conjugates, a versatile class of research tools that target C2H2 transcription factors, to inhibit the Hedgehog (Hh) pathway. In developing mammalian embryos, Hh signaling is critical for the formation and development of many tissues and organs. Inappropriate activation of the Hedgehog (Hh) pathway has been implicated in a variety of cancers including medulloblastomas and basal cell carcinomas. It is well known that Hh regulates the activity of the Gli family of C2H2 zinc finger transcription factors in mammals. In Drosophila the function of the Gli proteins is performed by a single transcription factor with an identical DNA binding consensus sequence, Cubitus Interruptus (Ci). We have demonstrated previously that conjugation of a specific 17 base-pair oligonucleotide to a Co(III) Schiff base complex results in a targeted inhibitor of the Snail family C2H2 zinc finger transcription factors. Modification of the oligonucleotide sequence in the Co(III) Schiff base-DNA conjugate to that of Ci’s consensus sequence (Co(III)-Ci) generates an equally selective inhibitor of Ci. Co(III)-Ci irreversibly binds the Ci zinc finger domain and prevents it from binding DNA in vitro. In a Ci responsive tissue culture reporter gene assay, Co(III)-Ci reduces the transcriptional activity of Ci in a concentration dependent manner. In addition, injection of wild-type Drosophila embryos with Co(III)-Ci phenocopies a Ci loss of function phenotype, demonstrating effectiveness in vivo. This study provides evidence that Co(III) Schiff base-DNA conjugates are a versatile class of specific and potent tools for studying zinc finger domain proteins and have potential applications as customizable anti-cancer therapeutics. PMID:22214326

RNAseq versus genome-predicted transcriptomes: a large population of novel transcripts identified in an Illumina-454 Hydra transcriptome.

PubMed

Wenger, Yvan; Galliot, Brigitte

2013-03-25

Evolutionary studies benefit from deep sequencing technologies that generate genomic and transcriptomic sequences from a variety of organisms. Genome sequencing and RNAseq have complementary strengths. In this study, we present the assembly of the most complete Hydra transcriptome to date along with a comparative analysis of the specific features of RNAseq and genome-predicted transcriptomes currently available in the freshwater hydrozoan Hydra vulgaris. To produce an accurate and extensive Hydra transcriptome, we combined Illumina and 454 Titanium reads, giving the primacy to Illumina over 454 reads to correct homopolymer errors. This strategy yielded an RNAseq transcriptome that contains 48'909 unique sequences including splice variants, representing approximately 24'450 distinct genes. Comparative analysis to the available genome-predicted transcriptomes identified 10'597 novel Hydra transcripts that encode 529 evolutionarily-conserved proteins. The annotation of 170 human orthologs points to critical functions in protein biosynthesis, FGF and TOR signaling, vesicle transport, immunity, cell cycle regulation, cell death, mitochondrial metabolism, transcription and chromatin regulation. However, a majority of these novel transcripts encodes short ORFs, at least 767 of them corresponding to pseudogenes. This RNAseq transcriptome also lacks 11'270 predicted transcripts that correspond either to silent genes or to genes expressed below the detection level of this study. We established a simple and powerful strategy to combine Illumina and 454 reads and we produced, with genome assistance, an extensive and accurate Hydra transcriptome. The comparative analysis of the RNAseq transcriptome with genome-predicted transcriptomes lead to the identification of large populations of novel as well as missing transcripts that might reflect Hydra-specific evolutionary events.

RNAseq versus genome-predicted transcriptomes: a large population of novel transcripts identified in an Illumina-454 Hydra transcriptome

PubMed Central

2013-01-01

Background Evolutionary studies benefit from deep sequencing technologies that generate genomic and transcriptomic sequences from a variety of organisms. Genome sequencing and RNAseq have complementary strengths. In this study, we present the assembly of the most complete Hydra transcriptome to date along with a comparative analysis of the specific features of RNAseq and genome-predicted transcriptomes currently available in the freshwater hydrozoan Hydra vulgaris. Results To produce an accurate and extensive Hydra transcriptome, we combined Illumina and 454 Titanium reads, giving the primacy to Illumina over 454 reads to correct homopolymer errors. This strategy yielded an RNAseq transcriptome that contains 48’909 unique sequences including splice variants, representing approximately 24’450 distinct genes. Comparative analysis to the available genome-predicted transcriptomes identified 10’597 novel Hydra transcripts that encode 529 evolutionarily-conserved proteins. The annotation of 170 human orthologs points to critical functions in protein biosynthesis, FGF and TOR signaling, vesicle transport, immunity, cell cycle regulation, cell death, mitochondrial metabolism, transcription and chromatin regulation. However, a majority of these novel transcripts encodes short ORFs, at least 767 of them corresponding to pseudogenes. This RNAseq transcriptome also lacks 11’270 predicted transcripts that correspond either to silent genes or to genes expressed below the detection level of this study. Conclusions We established a simple and powerful strategy to combine Illumina and 454 reads and we produced, with genome assistance, an extensive and accurate Hydra transcriptome. The comparative analysis of the RNAseq transcriptome with genome-predicted transcriptomes lead to the identification of large populations of novel as well as missing transcripts that might reflect Hydra-specific evolutionary events. PMID:23530871

Modulation of Estrogen Response Element-Driven Gene Expressions and Cellular Proliferation with Polar Directions by Designer Transcription Regulators

PubMed Central

Muyan, Mesut; Güpür, Gizem; Yaşar, Pelin; Ayaz, Gamze; User, Sırma Damla; Kazan, Hasan Hüseyin; Huang, Yanfang

2015-01-01

Estrogen receptor α (ERα), as a ligand-dependent transcription factor, mediates 17β-estradiol (E2) effects. ERα is a modular protein containing a DNA binding domain (DBD) and transcription activation domains (AD) located at the amino- and carboxyl-termini. The interaction of the E2-activated ERα dimer with estrogen response elements (EREs) of genes constitutes the initial step in the ERE-dependent signaling pathway necessary for alterations of cellular features. We previously constructed monomeric transcription activators, or monotransactivators, assembled from an engineered ERE-binding module (EBM) using the ERα-DBD and constitutively active ADs from other transcription factors. Monotransactivators modulated cell proliferation by activating and repressing ERE-driven gene expressions that simulate responses observed with E2-ERα. We reasoned here that integration of potent heterologous repression domains (RDs) into EBM could generate monotransrepressors that alter ERE-bearing gene expressions and cellular proliferation in directions opposite to those observed with E2-ERα or monotransactivators. Consistent with this, monotransrepressors suppressed reporter gene expressions that emulate the ERE-dependent signaling pathway. Moreover, a model monotransrepressor regulated DNA synthesis, cell cycle progression and proliferation of recombinant adenovirus infected ER-negative cells through decreasing as well as increasing gene expressions with polar directions compared with E2-ERα or monotransactivator. Our results indicate that an ‘activator’ or a ‘repressor’ possesses both transcription activating/enhancing and repressing/decreasing abilities within a chromatin context. Offering a protein engineering platform to alter signal pathway-specific gene expressions and cell growth, our approach could also be used for the development of tools for epigenetic modifications and for clinical interventions wherein multigenic de-regulations are an issue. PMID:26295471

Comparative Monomethylarginine Proteomics Suggests that Protein Arginine Methyltransferase 1 (PRMT1) is a Significant Contributor to Arginine Monomethylation in Toxoplasma gondii

PubMed Central

Yakubu, Rama R.; Silmon de Monerri, Natalie C.; Nieves, Edward; Kim, Kami; Weiss, Louis M.

2017-01-01

Arginine methylation is a common posttranslational modification found on nuclear and cytoplasmic proteins that has roles in transcriptional regulation, RNA metabolism and DNA repair. The protozoan parasite Toxoplasma gondii has a complex life cycle requiring transcriptional plasticity and has unique transcriptional regulatory pathways. Arginine methylation may play an important part in transcriptional regulation and splicing biology in this organism. The T. gondii genome contains five putative protein arginine methyltransferases (PRMTs), of which PRMT1 is important for cell division and growth. In order to better understand the function(s) of the posttranslational modification monomethyl arginine (MMA) in T. gondii, we performed a proteomic analysis of MMA proteins using affinity purification employing anti-MMA specific antibodies followed by mass spectrometry. The arginine monomethylome of T. gondii contains a large number of RNA binding proteins and multiple ApiAP2 transcription factors, suggesting a role for arginine methylation in RNA biology and transcriptional regulation. Surprisingly, 90% of proteins that are arginine monomethylated were detected as being phosphorylated in a previous phosphoproteomics study which raises the possibility of interplay between MMA and phosphorylation in this organism. Supporting this, a number of kinases are also arginine methylated. Because PRMT1 is thought to be a major PRMT in T. gondii, an organism which lacks a MMA-specific PRMT, we applied comparative proteomics to understand how PRMT1 might contribute to the MMA proteome in T. gondii. We identified numerous putative PRMT1 substrates, which include RNA binding proteins, transcriptional regulators (e.g. AP2 transcription factors), and kinases. Together, these data highlight the importance of MMA and PRMT1 in arginine methylation in T. gondii, as a potential regulator of a large number of processes including RNA biology and transcription. PMID:28143887

Proteopedia: 3D Visualization and Annotation of Transcription Factor-DNA Readout Modes

ERIC Educational Resources Information Center

Dantas Machado, Ana Carolina; Saleebyan, Skyler B.; Holmes, Bailey T.; Karelina, Maria; Tam, Julia; Kim, Sharon Y.; Kim, Keziah H.; Dror, Iris; Hodis, Eran; Martz, Eric; Compeau, Patricia A.; Rohs, Remo

2012-01-01

3D visualization assists in identifying diverse mechanisms of protein-DNA recognition that can be observed for transcription factors and other DNA binding proteins. We used Proteopedia to illustrate transcription factor-DNA readout modes with a focus on DNA shape, which can be a function of either nucleotide sequence (Hox proteins) or base pairing…

Characterization and molecular modeling of Inositol 1,3,4 tris phosphate 5/6 kinase-2 from Glycine max (L) Merr.: comprehending its evolutionary conservancy at functional level.

PubMed

Marathe, Ashish; Krishnan, Veda; Mahajan, Mahesh M; Thimmegowda, Vinutha; Dahuja, Anil; Jolly, Monica; Praveen, Shelly; Sachdev, Archana

2018-01-01

Soybean genome encodes a family of four inositol 1,3,4 trisphosphate 5/6 kinases which belong to the ATP-GRASP group of proteins. Inositol 1,3,4 trisphosphate kinase-2 ( GmItpk2 ), catalyzing the ATP-dependent phosphorylation of Inositol 1,3,4 trisphosphate (IP3) to Inositol 1,3,4,5 tetra phosphate or Inositol 1,3,4,6 tetra phosphate, is a key enzyme diverting the flux of inositol phosphate pool towards phytate biosynthesis. Although considerable research on characterizing genes involved in phytate biosynthesis is accomplished at genomic and transcript level, characterization of the proteins is yet to be explored. In the present study, we report the isolation and expression of single copy Itpk 2 (948 bp) from Glycine max cv Pusa-16 predicted to encode 315 amino acid protein with an isoelectric point of 5.9. Sequence analysis revealed that Gm ITPK2 shared highest similarity (80%) with Phaseolus vulgaris. The predicted 3D model confirmed 12 α helices and 14 β barrel sheets with ATP-binding site close to β sheet present towards the C-terminus of the protein molecule. Spatio-temporal transcript profiling signified GmItpk2 to be seed specific, with higher transcript levels in the early stage of seed development. The present study using various molecular and bio-computational tools could, therefore, help in improving our understanding of this key enzyme and prove to be a potential target towards generating low phytate trait in nutritionally rich crop like soybean.

Temporal regulation of expression of immediate early and second phase transcripts by endothelin-1 in cardiomyocytes

PubMed Central

Cullingford, Timothy E; Markou, Thomais; Fuller, Stephen J; Giraldo, Alejandro; Pikkarainen, Sampsa; Zoumpoulidou, Georgia; Alsafi, Ali; Ekere, Collins; Kemp, Timothy J; Dennis, Jayne L; Game, Laurence; Sugden, Peter H; Clerk, Angela

2008-01-01

Background Endothelin-1 stimulates Gq protein-coupled receptors to promote proliferation in dividing cells or hypertrophy in terminally differentiated cardiomyocytes. In cardiomyocytes, endothelin-1 rapidly (within minutes) stimulates protein kinase signaling, including extracellular-signal regulated kinases 1/2 (ERK1/2; though not ERK5), with phenotypic/physiological changes developing from approximately 12 h. Hypertrophy is associated with changes in mRNA/protein expression, presumably consequent to protein kinase signaling, but the connections between early, transient signaling events and developed hypertrophy are unknown. Results Using microarrays, we defined the early transcriptional responses of neonatal rat cardiomyocytes to endothelin-1 over 4 h, differentiating between immediate early gene (IEG) and second phase RNAs with cycloheximide. IEGs exhibited differential temporal and transient regulation, with expression of second phase RNAs within 1 h. Of transcripts upregulated at 30 minutes encoding established proteins, 28 were inhibited >50% by U0126 (which inhibits ERK1/2/5 signaling), with 9 inhibited 25-50%. Expression of only four transcripts was not inhibited. At 1 h, most RNAs (approximately 67%) were equally changed in total and polysomal RNA with approximately 17% of transcripts increased to a greater extent in polysomes. Thus, changes in expression of most protein-coding RNAs should be reflected in protein synthesis. However, approximately 16% of transcripts were essentially excluded from the polysomes, including some protein-coding mRNAs, presumably inefficiently translated. Conclusion The phasic, temporal regulation of early transcriptional responses induced by endothelin-1 in cardiomyocytes indicates that, even in terminally differentiated cells, signals are propagated beyond the primary signaling pathways through transcriptional networks leading to phenotypic changes (that is, hypertrophy). Furthermore, ERK1/2 signaling plays a major role in this response. PMID:18275597

Decreased expression of ten-eleven translocation 1 protein is associated with some clinicopathological features in gastric cancer.

PubMed

Frycz, Bartosz Adam; Murawa, Dawid; Borejsza-Wysocki, Maciej; Marciniak, Ryszard; Murawa, Paweł; Drews, Michał; Kołodziejczak, Anna; Tomela, Katarzyna; Jagodziński, Paweł Piotr

2014-03-01

A decrease in ten-eleven translocation 1 (TET1) transcript and 5-Hydroxymethylcytosine (5hmC) levels has recently been demonstrated in primary gastric cancer (GC). However, little is known about TET1 protein levels in gastric tumoral and nontumoral tissue. Therefore, using reverse transcription, real-time quantitative polymerase chain reaction and western blotting analysis, we determined the TET1 transcript and protein levels in tumoral and nontumoral tissue from 38 patients with GC. We also assessed the association between the decrease in TET1 transcript and protein levels and some clinicopathological features in primary GC. We found significantly decreased levels of TET1 transcript (P=0.0023) and protein (P=0.00024) in primary tumoral tissues as compared to nontumoral tissues in patients with GC. Moreover, we also observed significantly lower amounts of TET1 transcript (P=0.03) and protein (P=0.00018) in tumoral tissues in patients aged>60. We also found significant lowered TET1 protein levels in male patients (P=0.0014), stomach (P=0.044) and cardia (P=0.013) tumor localization, T3 depth of invasion (P=0.019), N1 (P=0.012) and N3 lymph node metastasis (P=0.013) and G3 histological grade (P=0.0012). There were also significant decreases in TET1 transcript levels in female patients (P=0.042), intestinal histological types (P=0.0079) and T4 depth of invasion (P=0.037). Our results demonstrated that a decrease in TET1 transcript and protein levels is associated with some clinicopathological features in GC. Copyright © 2014. Published by Elsevier Masson SAS.

Dampening DNA binding: a common mechanism of transcriptional repression for both ncRNAs and protein domains.

PubMed

Goodrich, James A; Kugel, Jennifer F

2010-01-01

With eukaryotic non-coding RNAs (ncRNAs) now established as critical regulators of cellular transcription, the true diversity with which they can elicit biological effects is beginning to be appreciated. Two ncRNAs, mouse B2 RNA and human Alu RNA, have been found to repress mRNA transcription in response to heat shock. They do so by binding directly to RNA polymerase II, assembling into complexes on promoter DNA, and disrupting contacts between the polymerase and the DNA. Such a mechanism of repression had not previously been observed for a eukaryotic ncRNA; however, there are examples of eukaryotic protein domains that repress transcription by blocking essential protein-DNA interactions. Comparing the mechanism of transcriptional repression utilized by these protein domains to that used by B2 and Alu RNAs raises intriguing questions regarding transcriptional control, and how B2 and Alu RNAs might themselves be regulated.

Regulation of expression of two LY-6 family genes by intron retention and transcription induced chimerism

PubMed Central

Calvanese, Vincenzo; Mallya, Meera; Campbell, R Duncan; Aguado, Begoña

2008-01-01

Background Regulation of the expression of particular genes can rely on mechanisms that are different from classical transcriptional and translational control. The LY6G5B and LY6G6D genes encode LY-6 domain proteins, whose expression seems to be regulated in an original fashion, consisting of an intron retention event which generates, through an early premature stop codon, a non-coding transcript, preventing expression in most cell lines and tissues. Results The MHC LY-6 non-coding transcripts have shown to be stable and very abundant in the cell, and not subject to Nonsense Mediated Decay (NMD). This retention event appears not to be solely dependent on intron features, because in the case of LY6G5B, when the intron is inserted in the artificial context of a luciferase expression plasmid, it is fully spliced but strongly stabilises the resulting luciferase transcript. In addition, by quantitative PCR we found that the retained and spliced forms are differentially expressed in tissues indicating an active regulation of the non-coding transcript. EST database analysis revealed that these genes have an alternative expression pathway with the formation of Transcription Induced Chimeras (TIC). This data was confirmed by RT-PCR, revealing the presence of different transcripts that would encode the chimeric proteins CSNKβ-LY6G5B and G6F-LY6G6D, in which the LY-6 domain would join to a kinase domain and an Ig-like domain, respectively. Conclusion In conclusion, the LY6G5B and LY6G6D intron-retained transcripts are not subjected to NMD and are more abundant than the properly spliced forms. In addition, these genes form chimeric transcripts with their neighbouring same orientation 5' genes. Of interest is the fact that the 5' genes (CSNKβ or G6F) undergo differential splicing only in the context of the chimera (CSNKβ-LY6G5B or G6F-LY6G6C) and not on their own. PMID:18817541

Targeted repression of AXIN2 and MYC gene expression using designer TALEs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rennoll, Sherri A.; Scott, Samantha A.; Yochum, Gregory S., E-mail: gsy3@psu.edu

Highlights: • We designed TALE–SID fusion proteins to target AXIN2 and MYC. • TALE–SIDs bound the chromosomal AXIN2 and MYC genes and repressed their expression. • TALE–SIDs repress β-catenin{sup S45F}-dependent AXIN2 and MYC transcription. - Abstract: Designer TALEs (dTALEs) are chimeric transcription factors that can be engineered to regulate gene expression in mammalian cells. Whether dTALEs can block gene transcription downstream of signal transduction cascades, however, has yet to be fully explored. Here we tested whether dTALEs can be used to target genes whose expression is controlled by Wnt/β-catenin signaling. TALE DNA binding domains were engineered to recognize sequences adjacentmore » to Wnt responsive enhancer elements (WREs) that control expression of axis inhibition protein 2 (AXIN2) and c-MYC (MYC). These custom DNA binding domains were linked to the mSin3A interaction domain (SID) to generate TALE–SID chimeric repressors. The TALE–SIDs repressed luciferase reporter activity, bound their genomic target sites, and repressed AXIN2 and MYC expression in HEK293 cells. We generated a novel HEK293 cell line to determine whether the TALE–SIDs could function downstream of oncogenic Wnt/β-catenin signaling. Treating these cells with doxycycline and tamoxifen stimulates nuclear accumulation of a stabilized form of β-catenin found in a subset of colorectal cancers. The TALE–SIDs repressed AXIN2 and MYC expression in these cells, which suggests that dTALEs could offer an effective therapeutic strategy for the treatment of colorectal cancer.« less

Long-read sequencing uncovers the adaptive topography of a carnivorous plant genome

PubMed Central

Lan, Tianying; Renner, Tanya; Ibarra-Laclette, Enrique; Farr, Kimberly M.; Chang, Tien-Hao; Cervantes-Pérez, Sergio Alan; Zheng, Chunfang; Sankoff, David; Tang, Haibao; Purbojati, Rikky W.; Putra, Alexander; Drautz-Moses, Daniela I.; Schuster, Stephan C.; Herrera-Estrella, Luis; Albert, Victor A.

2017-01-01

Utricularia gibba, the humped bladderwort, is a carnivorous plant that retains a tiny nuclear genome despite at least two rounds of whole genome duplication (WGD) since common ancestry with grapevine and other species. We used a third-generation genome assembly with several complete chromosomes to reconstruct the two most recent lineage-specific ancestral genomes that led to the modern U. gibba genome structure. Patterns of subgenome dominance in the most recent WGD, both architectural and transcriptional, are suggestive of allopolyploidization, which may have generated genomic novelty and led to instantaneous speciation. Syntenic duplicates retained in polyploid blocks are enriched for transcription factor functions, whereas gene copies derived from ongoing tandem duplication events are enriched in metabolic functions potentially important for a carnivorous plant. Among these are tandem arrays of cysteine protease genes with trap-specific expression that evolved within a protein family known to be useful in the digestion of animal prey. Further enriched functions among tandem duplicates (also with trap-enhanced expression) include peptide transport (intercellular movement of broken-down prey proteins), ATPase activities (bladder-trap acidification and transmembrane nutrient transport), hydrolase and chitinase activities (breakdown of prey polysaccharides), and cell-wall dynamic components possibly associated with active bladder movements. Whereas independently polyploid Arabidopsis syntenic gene duplicates are similarly enriched for transcriptional regulatory activities, Arabidopsis tandems are distinct from those of U. gibba, while still metabolic and likely reflecting unique adaptations of that species. Taken together, these findings highlight the special importance of tandem duplications in the adaptive landscapes of a carnivorous plant genome. PMID:28507139

Using CRISPR-Cas9 to Generate Gene-Corrected Autologous iPSCs for the Treatment of Inherited Retinal Degeneration.

PubMed

Burnight, Erin R; Gupta, Manav; Wiley, Luke A; Anfinson, Kristin R; Tran, Audrey; Triboulet, Robinson; Hoffmann, Jeremy M; Klaahsen, Darcey L; Andorf, Jeaneen L; Jiao, Chunhua; Sohn, Elliott H; Adur, Malavika K; Ross, Jason W; Mullins, Robert F; Daley, George Q; Schlaeger, Thorsten M; Stone, Edwin M; Tucker, Budd A

2017-09-06

Patient-derived induced pluripotent stem cells (iPSCs) hold great promise for autologous cell replacement. However, for many inherited diseases, treatment will likely require genetic repair pre-transplantation. Genome editing technologies are useful for this application. The purpose of this study was to develop CRISPR-Cas9-mediated genome editing strategies to target and correct the three most common types of disease-causing variants in patient-derived iPSCs: (1) exonic, (2) deep intronic, and (3) dominant gain of function. We developed a homology-directed repair strategy targeting a homozygous Alu insertion in exon 9 of male germ cell-associated kinase (MAK) and demonstrated restoration of the retinal transcript and protein in patient cells. We generated a CRISPR-Cas9-mediated non-homologous end joining (NHEJ) approach to excise a major contributor to Leber congenital amaurosis, the IVS26 cryptic-splice mutation in CEP290, and demonstrated correction of the transcript and protein in patient iPSCs. Lastly, we designed allele-specific CRISPR guides that selectively target the mutant Pro23His rhodopsin (RHO) allele, which, following delivery to both patient iPSCs in vitro and pig retina in vivo, created a frameshift and premature stop that would prevent transcription of the disease-causing variant. The strategies developed in this study will prove useful for correcting a wide range of genetic variants in genes that cause inherited retinal degeneration. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

CpG methylation in human papillomavirus (HPV) type 31 long control region (LCR) in cervical infections associated with cytological abnormalities.

PubMed

László, Brigitta; Ferenczi, Annamária; Madar, László; Gyöngyösi, Eszter; Szalmás, Anita; Szakács, Levente; Veress, György; Kónya, József

2016-08-01

The mechanisms that regulate papillomavirus gene expression include DNA methylation. The transcription of papillomavirus oncogenes E6 and E7 is controlled by certain regulatory elements in the LCR, which include binding sites for the E2 protein, a viral regulator of oncogene expression. In HPV-31-infected exfoliated cervical cells, the CpG methylation of the entire LCR was determined by next-generation sequencing after bisulfite modification. Six of the 22 cases had methylated CpG sites in the HPV-31 LCR, including position 7479 and/or 7485, at the promoter distal E2 binding site, thus suggesting a potential regulatory mechanism for papillomavirus transcription.

Induction of Sirt1 by Mechanical Stretch of Skeletal Muscle through the Early Response Factor EGR1 Triggers an Antioxidative Response*

PubMed Central

Pardo, Patricia S.; Mohamed, Junaith S.; Lopez, Michael A.; Boriek, Aladin M.

2011-01-01

Mechanical loading of muscles by intrinsic muscle activity or passive stretch leads to an increase in the production of reactive oxygen species (1, 2). The NAD-dependent protein deacetylase SIRT1 is involved in the protection against oxidative stress by enhancing FOXO-driven Sod2 transcription (3–5). In this report, we unravel a mechanism triggered by mechanical stretch of skeletal muscle cells that leads to an EGR1-dependent transcriptional activation of the Sirt1 gene. The resulting transient increase in SIRT1 expression generates an antioxidative response that contributes to reactive oxygen species scavenging. PMID:20971845

Functional interaction of the DNA-binding transcription factor Sp1 through its DNA-binding domain with the histone chaperone TAF-I.

PubMed

Suzuki, Toru; Muto, Shinsuke; Miyamoto, Saku; Aizawa, Kenichi; Horikoshi, Masami; Nagai, Ryozo

2003-08-01

Transcription involves molecular interactions between general and regulatory transcription factors with further regulation by protein-protein interactions (e.g. transcriptional cofactors). Here we describe functional interaction between DNA-binding transcription factor and histone chaperone. Affinity purification of factors interacting with the DNA-binding domain of the transcription factor Sp1 showed Sp1 to interact with the histone chaperone TAF-I, both alpha and beta isoforms. This interaction was specific as Sp1 did not interact with another histone chaperone CIA nor did other tested DNA-binding regulatory factors (MyoD, NFkappaB, p53) interact with TAF-I. Interaction of Sp1 and TAF-I occurs both in vitro and in vivo. Interaction with TAF-I results in inhibition of DNA-binding, and also likely as a result of such, inhibition of promoter activation by Sp1. Collectively, we describe interaction between DNA-binding transcription factor and histone chaperone which results in negative regulation of the former. This novel regulatory interaction advances our understanding of the mechanisms of eukaryotic transcription through DNA-binding regulatory transcription factors by protein-protein interactions, and also shows the DNA-binding domain to mediate important regulatory interactions.

Systems-wide RNAi analysis of CASP8AP2/FLASH shows transcriptional deregulation of the replication-dependent histone genes and extensive effects on the transcriptome of colorectal cancer cells

PubMed Central

2012-01-01

Background Colorectal carcinomas (CRC) carry massive genetic and transcriptional alterations that influence multiple cellular pathways. The study of proteins whose loss-of-function (LOF) alters the growth of CRC cells can be used to further understand the cellular processes cancer cells depend upon for survival. Results A small-scale RNAi screen of ~400 genes conducted in SW480 CRC cells identified several candidate genes as required for the viability of CRC cells, most prominently CASP8AP2/FLASH. To understand the function of this gene in maintaining the viability of CRC cells in an unbiased manner, we generated gene specific expression profiles following RNAi. Silencing of CASP8AP2/FLASH resulted in altered expression of over 2500 genes enriched for genes associated with cellular growth and proliferation. Loss of CASP8AP2/FLASH function was significantly associated with altered transcription of the genes encoding the replication-dependent histone proteins as a result of the expression of the non-canonical polyA variants of these transcripts. Silencing of CASP8AP2/FLASH also mediated enrichment of changes in the expression of targets of the NFκB and MYC transcription factors. These findings were confirmed by whole transcriptome analysis of CASP8AP2/FLASH silenced cells at multiple time points. Finally, we identified and validated that CASP8AP2/FLASH LOF increases the expression of neurofilament heavy polypeptide (NEFH), a protein recently linked to regulation of the AKT1/ß-catenin pathway. Conclusions We have used unbiased RNAi based approaches to identify and characterize the function of CASP8AP2/FLASH, a protein not previously reported as required for cell survival. This study further defines the role CASP8AP2/FLASH plays in the regulating expression of the replication-dependent histones and shows that its LOF results in broad and reproducible effects on the transcriptome of colorectal cancer cells including the induction of expression of the recently described tumor suppressor gene NEFH. PMID:22216762

Systems-wide RNAi analysis of CASP8AP2/FLASH shows transcriptional deregulation of the replication-dependent histone genes and extensive effects on the transcriptome of colorectal cancer cells.

PubMed

Hummon, Amanda B; Pitt, Jason J; Camps, Jordi; Emons, Georg; Skube, Susan B; Huppi, Konrad; Jones, Tamara L; Beissbarth, Tim; Kramer, Frank; Grade, Marian; Difilippantonio, Michael J; Ried, Thomas; Caplen, Natasha J

2012-01-04

Colorectal carcinomas (CRC) carry massive genetic and transcriptional alterations that influence multiple cellular pathways. The study of proteins whose loss-of-function (LOF) alters the growth of CRC cells can be used to further understand the cellular processes cancer cells depend upon for survival. A small-scale RNAi screen of ~400 genes conducted in SW480 CRC cells identified several candidate genes as required for the viability of CRC cells, most prominently CASP8AP2/FLASH. To understand the function of this gene in maintaining the viability of CRC cells in an unbiased manner, we generated gene specific expression profiles following RNAi. Silencing of CASP8AP2/FLASH resulted in altered expression of over 2500 genes enriched for genes associated with cellular growth and proliferation. Loss of CASP8AP2/FLASH function was significantly associated with altered transcription of the genes encoding the replication-dependent histone proteins as a result of the expression of the non-canonical polyA variants of these transcripts. Silencing of CASP8AP2/FLASH also mediated enrichment of changes in the expression of targets of the NFκB and MYC transcription factors. These findings were confirmed by whole transcriptome analysis of CASP8AP2/FLASH silenced cells at multiple time points. Finally, we identified and validated that CASP8AP2/FLASH LOF increases the expression of neurofilament heavy polypeptide (NEFH), a protein recently linked to regulation of the AKT1/ß-catenin pathway. We have used unbiased RNAi based approaches to identify and characterize the function of CASP8AP2/FLASH, a protein not previously reported as required for cell survival. This study further defines the role CASP8AP2/FLASH plays in the regulating expression of the replication-dependent histones and shows that its LOF results in broad and reproducible effects on the transcriptome of colorectal cancer cells including the induction of expression of the recently described tumor suppressor gene NEFH.

Cloning and expression profiling of the VLDLR gene associated with egg performance in duck (Anas platyrhynchos).

PubMed

Wang, Cui; Li, Shi-Jun; Yu, Wen-Hua; Xin, Qing-Wu; Li, Chuang; Feng, Yan-Ping; Peng, Xiu-Li; Gong, Yan-Zhang

2011-08-05

The very low density lipoprotein receptor gene (VLDLR), a member of the low density lipoprotein receptor (LDLR) gene family, plays a crucial role in the synthesis of yolk protein precursors in oviparous species. Differential splicing of this gene has been reported in human, rabbit and rat. In chicken, studies showed that the VLDLR protein on the oocyte surface mediates the uptake of yolk protein precursors into growing oocytes. However, information on the VLDLR gene in duck is still scarce. Full-length duck VLDLR cDNA was obtained by comparative cloning and rapid amplification of cDNA ends (RACE). Tissue expression patterns were analysed by semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR). Association between the different genotypes and egg performance traits was investigated with the general linear model (GLM) procedure of the SAS® software package. In duck, two VLDLR transcripts were identified, one transcript (variant-a) containing an O-linked sugar domain and the other (variant-b) not containing this sugar domain. These transcripts share ~70 to 90% identity with their counterparts in other species. A phylogenetic tree based on amino acid sequences showed that duck VLDLR proteins were closely related with those of chicken and zebra finch. The two duck VLDLR transcripts are differentially expressed i.e. VLDLR-a is mainly expressed in muscle tissue and VLDLR-b in reproductive organs. We have localized the duck VLDLR gene on chromosome Z. An association analysis using two completely linked SNP sites (T/C at position 2025 bp of the ORF and G/A in intron 13) and records from two generations demonstrated that the duck VLDLR gene was significantly associated with egg production (P < 0.01), age of first egg (P < 0.01) and body weight of first egg (P < 0.05). Duck and chicken VLDLR genes probably perform similar function in the development of growing oocytes and deposition of yolk lipoprotein. Therefore, VLDLR could be a candidate gene for duck egg performance and be used as a genetic marker to improve egg performance in ducks.

Cloning and expression profiling of the VLDLR gene associated with egg performance in duck (Anas platyrhynchos)

PubMed Central

2011-01-01

Background The very low density lipoprotein receptor gene (VLDLR), a member of the low density lipoprotein receptor (LDLR) gene family, plays a crucial role in the synthesis of yolk protein precursors in oviparous species. Differential splicing of this gene has been reported in human, rabbit and rat. In chicken, studies showed that the VLDLR protein on the oocyte surface mediates the uptake of yolk protein precursors into growing oocytes. However, information on the VLDLR gene in duck is still scarce. Methods Full-length duck VLDLR cDNA was obtained by comparative cloning and rapid amplification of cDNA ends (RACE). Tissue expression patterns were analysed by semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR). Association between the different genotypes and egg performance traits was investigated with the general linear model (GLM) procedure of the SAS® software package. Results In duck, two VLDLR transcripts were identified, one transcript (variant-a) containing an O-linked sugar domain and the other (variant-b) not containing this sugar domain. These transcripts share ~70 to 90% identity with their counterparts in other species. A phylogenetic tree based on amino acid sequences showed that duck VLDLR proteins were closely related with those of chicken and zebra finch. The two duck VLDLR transcripts are differentially expressed i.e. VLDLR-a is mainly expressed in muscle tissue and VLDLR-b in reproductive organs. We have localized the duck VLDLR gene on chromosome Z. An association analysis using two completely linked SNP sites (T/C at position 2025 bp of the ORF and G/A in intron 13) and records from two generations demonstrated that the duck VLDLR gene was significantly associated with egg production (P < 0.01), age of first egg (P < 0.01) and body weight of first egg (P < 0.05). Conclusions Duck and chicken VLDLR genes probably perform similar function in the development of growing oocytes and deposition of yolk lipoprotein. Therefore, VLDLR could be a candidate gene for duck egg performance and be used as a genetic marker to improve egg performance in ducks. PMID:21819592

The past and presence of gene targeting: from chemicals and DNA via proteins to RNA.

PubMed

Geel, T M; Ruiters, M H J; Cool, R H; Halby, L; Voshart, D C; Andrade Ruiz, L; Niezen-Koning, K E; Arimondo, P B; Rots, M G

2018-06-05

The ability to target DNA specifically at any given position within the genome allows many intriguing possibilities and has inspired scientists for decades. Early gene-targeting efforts exploited chemicals or DNA oligonucleotides to interfere with the DNA at a given location in order to inactivate a gene or to correct mutations. We here describe an example towards correcting a genetic mutation underlying Pompe's disease using a nucleotide-fused nuclease (TFO-MunI). In addition to the promise of gene correction, scientists soon realized that genes could be inactivated or even re-activated without inducing potentially harmful DNA damage by targeting transcriptional modulators to a particular gene. However, it proved difficult to fuse protein effector domains to the first generation of programmable DNA-binding agents. The engineering of gene-targeting proteins (zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs)) circumvented this problem. The disadvantage of protein-based gene targeting is that a fusion protein needs to be engineered for every locus. The recent introduction of CRISPR/Cas offers a flexible approach to target a (fusion) protein to the locus of interest using cheap designer RNA molecules. Many research groups now exploit this platform and the first human clinical trials have been initiated: CRISPR/Cas has kicked off a new era of gene targeting and is revolutionizing biomedical sciences.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'. © 2018 The Author(s).

Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald, E-mail: gerald.thiel@uks.eu

2015-03-01

Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interruptsmore » the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.« less

S-nitrosylation in the regulation of gene transcription☆

PubMed Central

Sha, Yonggang; Marshall, Harvey E.

2015-01-01

Background Post-translational modification of proteins by S-nitrosylation serves as a major mode of signaling in mammalian cells and a growing body of evidence has shown that transcription factors and their activating pathways are primary targets. S-nitrosylation directly modifies a number of transcription factors, including NF-κB, HIF-1, and AP-1. In addition, S-nitrosylation can indirectly regulate gene transcription by modulating other cell signaling pathways, in particular JNK kinase and ras. Scope of review The evolution of S-nitrosylation as a signaling mechanism in the regulation of gene transcription, physiological advantages of protein S-nitrosylation in the control of gene transcription, and discussion of the many transcriptional proteins modulated by S-nitrosylation is summarized. Major conclusions S-nitrosylation plays a crucial role in the control of mammalian gene transcription with numerous transcription factors regulated by this modification. Many of these proteins serve as immunomodulators, and inducible nitric oxide synthase (iNOS) is regarded as a principal mediatiator of NO-dependent S-nitrosylation. However, additional targets within the nucleus (e.g. histone deacetylases) and alternative mechanisms of S-nitrosylation (e.g. GAPDH-mediated trans-nitrosylation) are thought to play a role in NOS-dependent transcriptional regulation. General significance Derangement of SNO-regulated gene transcription is an important factor in a variety of pathological conditions including neoplasia and sepsis. A better understanding of protein S-nitrosylation as it relates to gene transcription and the physiological mechanisms behind this process is likely to lead to novel therapies for these disorders. This article is part of a Special Issue entitled Regulation of Cellular Processes by S-nitrosylation. PMID:21640163

Deployment of the human immunodeficiency virus type 1 protein arsenal: combating the host to enhance viral transcription and providing targets for therapeutic development

PubMed Central

Dahiya, Satinder; Nonnemacher, Michael R.

2012-01-01

Despite the success of highly active antiretroviral therapy in combating human immunodeficiency virus type 1 (HIV-1) infection, the virus still persists in viral reservoirs, often in a state of transcriptional silence. This review focuses on the HIV-1 protein and regulatory machinery and how expanding knowledge of the function of individual HIV-1-coded proteins has provided valuable insights into understanding HIV transcriptional regulation in selected susceptible cell types. Historically, Tat has been the most studied primary transactivator protein, but emerging knowledge of HIV-1 transcriptional regulation in cells of the monocyte–macrophage lineage has more recently established that a number of the HIV-1 accessory proteins like Vpr may directly or indirectly regulate the transcriptional process. The viral proteins Nef and matrix play important roles in modulating the cellular activation pathways to facilitate viral replication. These observations highlight the cross talk between the HIV-1 transcriptional machinery and cellular activation pathways. The review also discusses the proposed transcriptional regulation mechanisms that intersect with the pathways regulated by microRNAs and how development of the knowledge of chromatin biology has enhanced our understanding of key protein–protein and protein–DNA interactions that form the HIV-1 transcriptome. Finally, we discuss the potential pharmacological approaches to target viral persistence and enhance effective transcription to purge the virus in cellular reservoirs, especially within the central nervous system, and the novel therapeutics that are currently in various stages of development to achieve a much superior prognosis for the HIV-1-infected population. PMID:22422068

Effect of the down-regulation of the high Grain Protein Content (GPC) genes on the wheat transcriptome during monocarpic senescence.

PubMed

Cantu, Dario; Pearce, Stephen P; Distelfeld, Assaf; Christiansen, Michael W; Uauy, Cristobal; Akhunov, Eduard; Fahima, Tzion; Dubcovsky, Jorge

2011-10-07

Increasing the nutrient concentration of wheat grains is important to ameliorate nutritional deficiencies in many parts of the world. Proteins and nutrients in the wheat grain are largely derived from the remobilization of degraded leaf molecules during monocarpic senescence. The down-regulation of the NAC transcription factor Grain Protein Content (GPC) in transgenic wheat plants delays senescence (>3 weeks) and reduces the concentration of protein, Zn and Fe in the grain (>30%), linking senescence and nutrient remobilization.Based on the early and rapid up-regulation of GPC in wheat flag leaves after anthesis, we hypothesized that this transcription factor is an early regulator of monocarpic senescence. To test this hypothesis, we used high-throughput mRNA-seq technologies to characterize the effect of the GPC down-regulation on the wheat flag-leaf transcriptome 12 days after anthesis. At this early stage of senescence GPC transcript levels are significantly lower in transgenic GPC-RNAi plants than in the wild type, but there are still no visible phenotypic differences between genotypes. We generated 1.4 million 454 reads from early senescing flag leaves (average ~350 nt) and assembled 1.2 million into 30,497 contigs that were used as a reference to map 145 million Illumina reads from three wild type and four GPC-RNAi plants. Following normalization and statistical testing, we identified a set of 691 genes differentially regulated by GPC (431 ≥ 2-fold change). Transcript level ratios between transgenic and wild type plants showed a high correlation (R = 0.83) between qRT-PCR and Illumina results, providing independent validation of the mRNA-seq approach. A set of differentially expressed genes were analyzed across an early senescence time-course. Monocarpic senescence is an active process characterized by large-scale changes in gene expression which begins considerably before the appearance of visual symptoms of senescence. The mRNA-seq approach used here was able to detect small differences in transcript levels during the early stages of senescence. This resulted in an extensive list of GPC-regulated genes, which includes transporters, hormone regulated genes, and transcription factors. These GPC-regulated genes, particularly those up-regulated during senescence, provide valuable entry points to dissect the early stages of monocarpic senescence and nutrient remobilization in wheat.

The IRAK-ERK-p67phox-Nox-2 axis mediates TLR4, 2-induced ROS production for IL-1β transcription and processing in monocytes

PubMed Central

Singh, Ankita; Singh, Vishal; Tiwari, Rajiv L.; Chandra, Tulika; Kumar, Ashutosh; Dikshit, Madhu; Barthwal, Manoj K.

2016-01-01

In monocytic cells, Toll-like receptor 4 (TLR4)- and TLR2-induced reactive oxygen species (ROS) cause oxidative stress and inflammatory response; however, the mechanism is not well understood. The present study investigated the role of interleukin-1 receptor-associated kinase (IRAK), extracellular signal-regulated kinase (ERK), p67phox and Nox-2 in TLR4- and TLR2-induced ROS generation during interleukin-1 beta (IL-1β) transcription, processing, and secretion. An IRAK1/4 inhibitor, U0126, PD98059, an NADPH oxidase inhibitor (diphenyleneiodonium (DPI)), and a free radical scavenger (N-acetyl cysteine (NAC))-attenuated TLR4 (lipopolysaccharide (LPS))- and TLR2 (Pam3csk4)-induced ROS generation and IL-1β production in THP-1 and primary human monocytes. An IRAK1/4 inhibitor and siRNA-attenuated LPS- and Pam3csk4-induced ERK-IRAK1 association and ERK phosphorylation and activity. LPS and Pam3csk4 also induced IRAK1/4-, ERK- and ROS-dependent activation of activator protein-1 (AP-1), IL-1β transcription, and IL-1β processing because significant inhibition in AP-1 activity, IL-1β transcription, Pro- and mature IL-β expression, and caspase-1 activity was observed with PD98059, U0126, DPI, NAC, an IRAK1/4 inhibitor, tanshinone IIa, and IRAK1 siRNA treatment. IRAK-dependent ERK-p67phox interaction, p67phox translocation, and p67phox–Nox-2 interaction were observed. Nox-2 siRNA significantly reduced secreted IL-1β, IL-1β transcript, pro- and mature IL-1β expression, and caspase-1 activity indicating a role for Nox-2 in LPS- and Pam3csk4-induced IL-1β production, transcription, and processing. In the present study, we demonstrate that the TLR4- and TLR2-induced IRAK-ERK pathway cross-talks with p67phox-Nox-2 for ROS generation, thus regulating IL-1β transcription and processing in monocytic cells. PMID:26320741

PsRobot: a web-based plant small RNA meta-analysis toolbox.

PubMed

Wu, Hua-Jun; Ma, Ying-Ke; Chen, Tong; Wang, Meng; Wang, Xiu-Jie

2012-07-01

Small RNAs (smRNAs) in plants, mainly microRNAs and small interfering RNAs, play important roles in both transcriptional and post-transcriptional gene regulation. The broad application of high-throughput sequencing technology has made routinely generation of bulk smRNA sequences in laboratories possible, thus has significantly increased the need for batch analysis tools. PsRobot is a web-based easy-to-use tool dedicated to the identification of smRNAs with stem-loop shaped precursors (such as microRNAs and short hairpin RNAs) and their target genes/transcripts. It performs fast analysis to identify smRNAs with stem-loop shaped precursors among batch input data and predicts their targets using a modified Smith-Waterman algorithm. PsRobot integrates the expression data of smRNAs in major plant smRNA biogenesis gene mutants and smRNA-associated protein complexes to give clues to the smRNA generation and functional processes. Besides improved specificity, the reliability of smRNA target prediction results can also be evaluated by mRNA cleavage (degradome) data. The cross species conservation statuses and the multiplicity of smRNA target sites are also provided. PsRobot is freely accessible at http://omicslab.genetics.ac.cn/psRobot/.

Dom34 Links Translation to Protein O-mannosylation

PubMed Central

van Wijlick, Lasse; Geissen, René; Hilbig, Jessica S.; Lagadec, Quentin; Cantero, Pilar D.; Juchimiuk, Mateusz; Kluge, Sven; Wickert, Stephan; Alepuz, Paula; Ernst, Joachim F.

2016-01-01

In eukaryotes, Dom34 upregulates translation by securing levels of activatable ribosomal subunits. We found that in the yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans, Dom34 interacts genetically with Pmt1, a major isoform of protein O-mannosyltransferase. In C. albicans, lack of Dom34 exacerbated defective phenotypes of pmt1 mutants, while they were ameliorated by Dom34 overproduction that enhanced Pmt1 protein but not PMT1 transcript levels. Translational effects of Dom34 required the 5′-UTR of the PMT1 transcript, which bound recombinant Dom34 directly at a CA/AC-rich sequence and regulated in vitro translation. Polysomal profiling revealed that Dom34 stimulates general translation moderately, but that it is especially required for translation of transcripts encoding Pmt isoforms 1, 4 and 6. Because defective protein N- or O-glycosylation upregulates transcription of PMT genes, it appears that Dom34-mediated specific translational upregulation of the PMT transcripts optimizes cellular responses to glycostress. Its translational function as an RNA binding protein acting at the 5′-UTR of specific transcripts adds another facet to the known ribosome-releasing functions of Dom34 at the 3′-UTR of transcripts. PMID:27768707

Dom34 Links Translation to Protein O-mannosylation.

PubMed

van Wijlick, Lasse; Geissen, René; Hilbig, Jessica S; Lagadec, Quentin; Cantero, Pilar D; Pfeifer, Eugen; Juchimiuk, Mateusz; Kluge, Sven; Wickert, Stephan; Alepuz, Paula; Ernst, Joachim F

2016-10-01

In eukaryotes, Dom34 upregulates translation by securing levels of activatable ribosomal subunits. We found that in the yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans, Dom34 interacts genetically with Pmt1, a major isoform of protein O-mannosyltransferase. In C. albicans, lack of Dom34 exacerbated defective phenotypes of pmt1 mutants, while they were ameliorated by Dom34 overproduction that enhanced Pmt1 protein but not PMT1 transcript levels. Translational effects of Dom34 required the 5'-UTR of the PMT1 transcript, which bound recombinant Dom34 directly at a CA/AC-rich sequence and regulated in vitro translation. Polysomal profiling revealed that Dom34 stimulates general translation moderately, but that it is especially required for translation of transcripts encoding Pmt isoforms 1, 4 and 6. Because defective protein N- or O-glycosylation upregulates transcription of PMT genes, it appears that Dom34-mediated specific translational upregulation of the PMT transcripts optimizes cellular responses to glycostress. Its translational function as an RNA binding protein acting at the 5'-UTR of specific transcripts adds another facet to the known ribosome-releasing functions of Dom34 at the 3'-UTR of transcripts.

Selective activation of human heat shock gene transcription by nitrosourea antitumor drugs mediated by isocyanate-induced damage and activation of heat shock transcription factor.

PubMed Central

Kroes, R A; Abravaya, K; Seidenfeld, J; Morimoto, R I

1991-01-01

Treatment of cultured human tumor cells with the chloroethylnitrosourea antitumor drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) selectively induces transcription and protein synthesis of a subset of the human heat shock or stress-induced genes (HSP90 and HSP70) with little effect on other stress genes or on expression of the c-fos, c-myc, or beta-actin genes. The active component of BCNU and related compounds appears to be the isocyanate moiety that causes carbamoylation of proteins and nucleic acids. Transcriptional activation of the human HSP70 gene by BCNU is dependent on the heat shock element and correlates with the level of heat shock transcription factor and its binding to the heat shock element in vivo. Unlike activation by heat or heavy metals, BCNU-mediated activation is strongly dependent upon new protein synthesis. This suggests that BCNU-induced, isocyanate-mediated damage to newly synthesized protein(s) may be responsible for activation of the heat shock transcription factor and increased transcription of the HSP90 and HSP70 genes. Images PMID:2052560

Seawater inhalation induces acute lung injury via ROS generation and the endoplasmic reticulum stress pathway.

PubMed

Li, Peng-Cheng; Wang, Bo-Rong; Li, Cong-Cong; Lu, Xi; Qian, Wei-Sheng; Li, Yu-Juan; Jin, Fa-Guang; Mu, De-Guang

2018-05-01

Seawater (SW) inhalation can induce acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). In the present study, SW induced apoptosis of rat alveolar epithelial cells and histopathological alterations to lung tissue. Furthermore, SW administration increased generation of reactive oxygen species (ROS), whereas pretreatment with the ROS scavenger, N‑acetyl‑L‑cysteine (NAC), significantly decreased ROS generation, apoptosis and histopathological alterations. In addition, SW exposure upregulated the expression levels of glucose‑regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP), which are critical proteins in the endoplasmic reticulum (ER) stress response, thus indicating that SW may activate ER stress. Conversely, blocking ER stress with 4‑phenylbutyric acid (4‑PBA) significantly improved SW‑induced apoptosis and histopathological alterations, whereas an ER stress inducer, thapsigargin, had the opposite effect. Furthermore, blocking ROS with NAC inhibited SW‑induced ER stress, as evidenced by the downregulation of GRP78, phosphorylated (p)‑protein kinase R‑like ER kinase (PERK), p‑inositol‑requiring kinase 1α (IRE1α), p‑50 activating transcription factor 6α and CHOP. In addition, blocking ER stress with 4‑PBA decreased ROS generation. In conclusion, the present study indicated that ROS and ER stress pathways, which are involved in alveolar epithelial cell apoptosis, are important in the pathogenesis of SW‑induced ALI.

Adenovirus E1a prevents the retinoblastoma gene product from repressing the activity of a cellular transcription factor.

PubMed Central

Zamanian, M; La Thangue, N B

1992-01-01

The retinoblastoma (Rb) gene product forms a complex with the cellular transcription factor DRTF1, a property assumed to be important for mediating negative growth control because certain viral oncogenes, such as adenovirus E1a, prevent this interaction and mutant Rb alleles, which have lost the capacity to regulate growth, encode proteins that fail to associate with DRTF1. In this study, we show that the wild-type Rb protein can specifically repress transcription from promoters driven by DRTF1 whereas a naturally occurring mutant Rb protein cannot. Furthermore, Rb-mediated transcriptional repression can be overridden by adenovirus E1a; this requires regions in E1a necessary for cellular transformation. The Rb protein therefore acts in trans to repress the transcriptional activity of DRTF1 whereas adenovirus E1a prevents this interaction and thus maintains DRTF1 in a constitutively active state. The Rb protein and adenovirus E1a therefore have opposite effects on the activity of a common molecular target. Transcriptional repression mediated by the Rb protein and inactivation of repression by the E1a protein are likely to play an important role in mediating their biological effects. Images PMID:1385776

Import routes and nuclear functions of Argonaute and other small RNA-silencing proteins.

PubMed

Schraivogel, Daniel; Meister, Gunter

2014-09-01

Small RNAs are important regulators of gene expression in many different organisms. Nuclear and cytoplasmic biogenesis enzymes generate functional small RNAs from double-stranded (ds) or single-stranded (ss) RNA precursors, and mature small RNAs are loaded into Argonaute proteins. In the cytoplasm, small RNAs guide Argonaute proteins to complementary RNAs leading to cleavage of these targets, translational silencing, or mRNA decay. In the nucleus Argonaute proteins engage in transcriptional silencing processes such as epigenetic silencing of repetitive elements at the chromatin level. During the past few years many novel functions of small RNA-guided gene silencing proteins in the nucleus have been reported. However, their specific import routes are largely unknown. In this review we summarize the current knowledge on nuclear transport routes that Argonaute and other RNA-silencing proteins take to carry out their various functions in the nucleus. Copyright © 2014 Elsevier Ltd. All rights reserved.

Long-term survival of hydrated resting eggs from Brachionus plicatilis.

PubMed

Clark, Melody S; Denekamp, Nadav Y; Thorne, Michael A S; Reinhardt, Richard; Drungowski, Mario; Albrecht, Marcus W; Klages, Sven; Beck, Alfred; Kube, Michael; Lubzens, Esther

2012-01-01

Several organisms display dormancy and developmental arrest at embryonic stages. Long-term survival in the dormant form is usually associated with desiccation, orthodox plant seeds and Artemia cysts being well documented examples. Several aquatic invertebrates display dormancy during embryonic development and survive for tens or even hundreds of years in a hydrated form, raising the question of whether survival in the non-desiccated form of embryonic development depends on pathways similar to those occurring in desiccation tolerant forms. To address this question, Illumina short read sequencing was used to generate transcription profiles from the resting and amictic eggs of an aquatic invertebrate, the rotifer, Brachionus plicatilis. These two types of egg have very different life histories, with the dormant or diapausing resting eggs, the result of the sexual cycle and amictic eggs, the non-dormant products of the asexual cycle. Significant transcriptional differences were found between the two types of egg, with amictic eggs rich in genes involved in the morphological development into a juvenile rotifer. In contrast, representatives of classical "stress" proteins: a small heat shock protein, ferritin and Late Embryogenesis Abundant (LEA) proteins were identified in resting eggs. More importantly however, was the identification of transcripts for messenger ribonucleoprotein particles which stabilise RNA. These inhibit translation and provide a valuable source of useful RNAs which can be rapidly activated on the exit from dormancy. Apoptotic genes were also present. Although apoptosis is inconsistent with maintenance of prolonged dormancy, an altered apoptotic pathway has been proposed for Artemia, and this may be the case with the rotifer. These data represent the first transcriptional profiling of molecular processes associated with dormancy in a non-desiccated form and indicate important similarities in the molecular pathways activated in resting eggs compared with desiccated dormant forms, specifically plant seeds and Artemia.

Methionine sulfoximine-treatment and carbon starvation elicit Snf1-independent phosphorylation of the transcription activator Gln3 in Saccharomyces cerevisiae

PubMed Central

Tate, Jennifer J.; Rai, Rajendra; Cooper, Terrance G.

2008-01-01

SUMMARY Tor proteins are global regulators situated at the top of a signal transduction pathway conserved from yeast to humans. Specific inhibition of the two S. cerevisiae Tor proteins by rapamycin alters many cellular processes and the expression of hundreds of genes. Among the regulated genes are those whose expression is activated by the GATA-family transcription activator, Gln3. The extent of Gln3 phosphorylation has been thought to determine its intracellular localization, with phosphorylated and dephosphorylated forms accumulating in the cytoplasm and nucleus, respectively. Data presented here demonstrate that rapamycin and the glutamine synthetase inhibitor, methionine sulfoximine (MSX), although eliciting the same outcomes with respect to Gln3-Myc13 nuclear accumulation and NCR-sensitive transcription, generate diametrically opposite effects on Gln3-Myc13 phosphorylation. MSX increases Gln3-Myc13 phosphorylation while rapamycin decreases it. Gln3-Myc13 phosphorylation levels are regulated by at least three mechanisms: (i) one, observed during carbon starvation, depends on Snf1 kinase, (ii) another, observed during both carbon-starvation and MSX-treatment, is Snf1-independent, and (iii) the last is rapamycin-induced dephosphorylation. MSX and rapamycin act additively on Gln3-Myc13 phosphorylation, but MSX clearly predominates. These results suggest that MSX- and rapamycin-inhibited proteins are more likely to function in separate regulatory pathways than they are to function tandemly in a single pathway as previously thought. Further, Gln3 phosphorylation/dephosphorylation, that we and others have detected thus far, is not a demonstrably required step in achieving Gln3 nuclear localization and NCR-sensitive transcription in response to MSX- or rapamycin-treatment. PMID:15911613

Comprehensive Transcriptome Analysis of Response to Nickel Stress in White Birch (Betula papyrifera)

PubMed Central

Theriault, Gabriel; Michael, Paul; Nkongolo, Kabwe

2016-01-01

White birch (Betula papyrifera) is a dominant tree species of the Boreal Forest. Recent studies have shown that it is fairly resistant to heavy metal contamination, specifically to nickel. Knowledge of regulation of genes associated with metal resistance in higher plants is very sketchy. Availability and annotation of the dwarf birch (B. nana) enables the use of high throughout sequencing approaches to understanding responses to environmental challenges in other Betula species such as B. papyrifera. The main objectives of this study are to 1) develop and characterize the B. papyrifera transcriptome, 2) assess gene expression dynamics of B. papyrifera in response to nickel stress, and 3) describe gene function based on ontology. Nickel resistant and susceptible genotypes were selected and used for transcriptome analysis. A total of 208,058 trinity genes were identified and were assembled to 275,545 total trinity transcripts. The transcripts were mapped to protein sequences and based on best match; we annotated the B. papyrifera genes and assigned gene ontology. In total, 215,700 transcripts were annotated and were compared to the published B. nana genome. Overall, a genomic match for 61% transcripts with the reference genome was found. Expression profiles were generated and 62,587 genes were found to be significantly differentially expressed among the nickel resistant, susceptible, and untreated libraries. The main nickel resistance mechanism in B. papyrifera is a downregulation of genes associated with translation (in ribosome), binding, and transporter activities. Five candidate genes associated to nickel resistance were identified. They include Glutathione S–transferase, thioredoxin family protein, putative transmembrane protein and two Nramp transporters. These genes could be useful for genetic engineering of birch trees. PMID:27082755

Regulation of matrix metalloproteinases (MMPs) expression and secretion in MDA-MB-231 breast cancer cells by LIM and SH3 protein 1 (LASP1).

PubMed

Endres, Marcel; Kneitz, Susanne; Orth, Martin F; Perera, Ruwan K; Zernecke, Alma; Butt, Elke

2016-09-27

The process of tumor invasion requires degradation of extracellular matrix by proteolytic enzymes. Cancer cells form protrusive invadopodia, which produce and release matrix metalloproteinases (MMPs) to degrade the basement membrane thereby enabling metastasis. We investigated the effect of LASP1, a newly identified protein in invadopodia, on expression, secretion and activation of MMPs in invasive breast tumor cell lines.By analyzing microarray data of in-house generated control and LASP1-depleted MDA-MB-231 breast cancer cells, we observed downregulation of MMP1, -3 and -9 upon LASP1 depletion. This was confirmed by Western blot analysis. Conversely, rescue experiments restored in part MMP expression and secretion. The regulatory effect of LASP1 on MMP expression was also observed in BT-20 breast cancer cells as well as in prostate and bladder cancer cell lines.In line with bioinformatic FunRich analysis of our data, which mapped a high regulation of transcription factors by LASP1, public microarray data analysis detected a correlation between high LASP1 expression and enhanced c-Fos levels, a protein that is part of the transcription factor AP-1 and known to regulate MMP expression. Compatibly, in luciferase reporter assays, AP-1 showed a decreased transcriptional activity after LASP1 knockdown.Zymography assays and Western blot analysis revealed an additional promotion of MMP secretion into the extracellular matrix by LASP1, thus, most likely, altering the microenvironment during cancer progression.The newly identified role of LASP1 in regulating matrix degradation by affecting MMP transcription and secretion elucidated the migratory potential of LASP1 overexpressing aggressive tumor cells in earlier studies.

Novel Nine-Exon AR Transcripts (Exon 1/Exon 1b/Exons 2-8) in Normal and Cancerous Breast and Prostate Cells.

PubMed

Hu, Dong Gui; McKinnon, Ross A; Hulin, Julie-Ann; Mackenzie, Peter I; Meech, Robyn

2016-12-27

Nearly 20 different transcripts of the human androgen receptor (AR) are reported with two currently listed as Refseq isoforms in the NCBI database. Isoform 1 encodes wild-type AR (type 1 AR) and isoform 2 encodes the variant AR45 (type 2 AR). Both variants contain eight exons: they share common exons 2-8 but differ in exon 1 with the canonical exon 1 in isoform 1 and the variant exon 1b in isoform 2. Splicing of exon 1 or exon 1b is reported to be mutually exclusive. In this study, we identified a novel exon 1b (1b/TAG) that contains an additional TAG trinucleotide upstream of exon 1b. Moreover, we identified AR transcripts in both normal and cancerous breast and prostate cells that contained either exon 1b or 1b/TAG spliced between the canonical exon 1 and exon 2, generating nine-exon AR transcripts that we have named isoforms 3a and 3b. The proteins encoded by these new AR variants could regulate androgen-responsive reporters in breast and prostate cancer cells under androgen-depleted conditions. Analysis of type 3 AR-GFP fusion proteins showed partial nuclear localization in PC3 cells under androgen-depleted conditions, supporting androgen-independent activation of the AR. Type 3 AR proteins inhibited androgen-induced growth of LNCaP cells. Microarray analysis identified a small set of type 3a AR target genes in LNCaP cells, including genes known to modulate growth and proliferation of prostate cancer ( PCGEM1 , PEG3 , EPHA3 , and EFNB2 ) or other types of human cancers ( TOX3 , ST8SIA4 , and SLITRK3 ), and genes that are diagnostic/prognostic biomarkers of prostate cancer ( GRINA3 , and BCHE ).

dFOXO Activates Large and Small Heat Shock Protein Genes in Response to Oxidative Stress to Maintain Proteostasis in Drosophila.

PubMed

Donovan, Marissa R; Marr, Michael T

2016-09-02

Maintaining protein homeostasis is critical for survival at the cellular and organismal level (Morimoto, R. I. (2011) Cold Spring Harb. Symp. Quant. Biol. 76, 91-99). Cells express a family of molecular chaperones, the heat shock proteins, during times of oxidative stress to protect against proteotoxicity. We have identified a second stress responsive transcription factor, dFOXO, that works alongside the heat shock transcription factor to activate transcription of both the small heat shock protein and the large heat shock protein genes. This expression likely protects cells from protein misfolding associated with oxidative stress. Here we identify the regions of the Hsp70 promoter essential for FOXO-dependent transcription using in vitro methods and find a physiological role for FOXO-dependent expression of heat shock proteins in vivo. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural and functional aspects of winged-helix domains at the core of transcription initiation complexes.

PubMed

Teichmann, Martin; Dumay-Odelot, Hélène; Fribourg, Sébastien

2012-01-01

The winged helix (WH) domain is found in core components of transcription systems in eukaryotes and prokaryotes. It represents a sub-class of the helix-turn-helix motif. The WH domain participates in establishing protein-DNA and protein-protein-interactions. Here, we discuss possible explanations for the enrichment of this motif in transcription systems.

Mosaic genome structure of the barley powdery mildew pathogen and conservation of transcriptional programs in divergent hosts

PubMed Central

Hacquard, Stéphane; Kracher, Barbara; Maekawa, Takaki; Vernaldi, Saskia; Schulze-Lefert, Paul; Ver Loren van Themaat, Emiel

2013-01-01

Barley powdery mildew, Blumeria graminis f. sp. hordei (Bgh), is an obligate biotrophic ascomycete fungal pathogen that can grow and reproduce only on living cells of wild or domesticated barley (Hordeum sp.). Domestication and deployment of resistant barley cultivars by humans selected for amplification of Bgh isolates with different virulence combinations. We sequenced the genomes of two European Bgh isolates, A6 and K1, for comparative analysis with the reference genome of isolate DH14. This revealed a mosaic genome structure consisting of large isolate-specific DNA blocks with either high or low SNP densities. Some of the highly polymorphic blocks likely accumulated SNPs for over 10,000 years, well before the domestication of barley. These isolate-specific blocks of alternating monomorphic and polymorphic regions imply an exceptionally large standing genetic variation in the Bgh population and might be generated and maintained by rare outbreeding and frequent clonal reproduction. RNA-sequencing experiments with isolates A6 and K1 during four early stages of compatible and incompatible interactions on leaves of partially immunocompromised Arabidopsis mutants revealed a conserved Bgh transcriptional program during pathogenesis compared with the natural host barley despite ∼200 million years of reproductive isolation of these hosts. Transcripts encoding candidate-secreted effector proteins are massively induced in successive waves. A specific decrease in candidate-secreted effector protein transcript abundance in the incompatible interaction follows extensive transcriptional reprogramming of the host transcriptome and coincides with the onset of localized host cell death, suggesting a host-inducible defense mechanism that targets fungal effector secretion or production. PMID:23696672

Analysis of tandem E-box motifs within human Complement receptor 2 (CR2/CD21) promoter reveals cell specific roles for RP58, E2A, USF and localized chromatin accessibility.

PubMed

Cruickshank, Mark N; Dods, James; Taylor, Rhonda L; Karimi, Mahdad; Fenwick, Emily J; Quail, Elizabeth A; Rea, Alexander J; Holers, V Michael; Abraham, Lawrence J; Ulgiati, Daniela

2015-07-01

Complement receptor 2 (CR2/CD21) plays an important role in the generation of normal B cell immune responses. As transcription appears to be the prime mechanism via which surface CR2/CD21 expression is controlled, understanding transcriptional regulation of this gene will have broader implications to B cell biology. Here we report opposing, cell-context specific control of CR2/CD21 promoter activity by tandem E-box elements, spaced 22 bp apart and within 70 bp of the transcription initiation site. We have identified E2A and USF transcription factors as binding to the distal and proximal E-box sites respectively in CR2-positive B-cells, at a site that is hypersensitive to restriction enzyme digestion compared to non-expressing K562 cells. However, additional unidentified proteins have also been found to bind these functionally important elements. By utilizing a proteomics approach we have identified a repressor protein, RP58, binding the distal E-box motif. Co-transfection experiments using RP58 overexpression constructs demonstrated a specific 10-fold repression of CR2/CD21 transcriptional activity mediated through the distal E-box repressor element. Taken together, our results indicate that repression of the CR2/CD21 promoter can occur through one of the E-box motifs via recruitment of RP58 and other factors to bring about a silenced chromatin context within CR2/CD21 non-expressing cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

Serratia marcescens Cyclic AMP Receptor Protein Controls Transcription of EepR, a Novel Regulator of Antimicrobial Secondary Metabolites.

PubMed

Stella, Nicholas A; Lahr, Roni M; Brothers, Kimberly M; Kalivoda, Eric J; Hunt, Kristin M; Kwak, Daniel H; Liu, Xinyu; Shanks, Robert M Q

2015-08-01

Serratia marcescens generates secondary metabolites and secreted enzymes, and it causes hospital infections and community-acquired ocular infections. Previous studies identified cyclic AMP (cAMP) receptor protein (CRP) as an indirect inhibitor of antimicrobial secondary metabolites. Here, we identified a putative two-component regulator that suppressed crp mutant phenotypes. Evidence supports that the putative response regulator eepR was directly transcriptionally inhibited by cAMP-CRP. EepR and the putative sensor kinase EepS were necessary for the biosynthesis of secondary metabolites, including prodigiosin- and serratamolide-dependent phenotypes, swarming motility, and hemolysis. Recombinant EepR bound to the prodigiosin and serratamolide promoters in vitro. Together, these data introduce a novel regulator of secondary metabolites that directly connects the broadly conserved metabolism regulator CRP with biosynthetic genes that may contribute to competition with other microbes. This study identifies a new transcription factor that is directly controlled by a broadly conserved transcription factor, CRP. CRP is well studied in its role to help bacteria respond to the amount of nutrients in their environment. The new transcription factor EepR is essential for the bacterium Serratia marcescens to produce two biologically active compounds, prodigiosin and serratamolide. These two compounds are antimicrobial and may allow S. marcescens to compete for limited nutrients with other microorganisms. Results from this study tie together the CRP environmental nutrient sensor with a new regulator of antimicrobial compounds. Beyond microbial ecology, prodigiosin and serratamolide have therapeutic potential; therefore, understanding their regulation is important for both applied and basic science. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Analysis of early dengue virus infection in mice as modulated by Aedes aegypti probing.

PubMed

McCracken, M K; Christofferson, R C; Chisenhall, D M; Mores, C N

2014-02-01

Dengue virus (DENV), the etiologic agent of dengue fever, is transmitted during probing of human skin by infected-mosquito bite. The expectorated viral inoculum also contains an assortment of mosquito salivary proteins that have been shown to modulate host hemostasis and innate immune responses. To examine the potential role of mosquito probing in DENV establishment within the vertebrate host, we inoculated mice intradermally with DENV serotype 2 strain 1232 at sites where Aedes aegypti had or had not probed immediately prior. We assayed these sites 3 h postinoculation with transcript arrays for the Toll-like receptor (TLR), RIG-I-like receptor, and NOD-like receptor signaling pathways of the innate immune system. We then chose TLR7, transcription factor p65 (RelA), gamma interferon (IFN-γ), and IFN-γ-inducible protein 10 (IP-10) from the arrays for further investigation and assayed these transcripts at 10 min, 3 h, and 6 h postinoculation. The transcripts for TLR7, RelA, IFN-γ, and IP-10 were significantly downregulated between 2- and 3-fold in the group subjected to mosquito probing relative to the virus-only inoculation group at 3 h postinoculation. A reduction in these transcripts could indicate reduced DENV recognition and antigen presentation and diminished inhibition of viral replication and spread. Further, mosquito probing resulted in viremia titers significantly higher than those in mice that did not receive probing. A. aegypti probing has a significant effect on the innate immune response to DENV infection and generates an early immune environment more permissive to the establishment of infection.

The 193-base pair Gsg2 (haspin) promoter region regulates germ cell-specific expression bidirectionally and synchronously.

PubMed

Tokuhiro, Keizo; Miyagawa, Yasushi; Yamada, Shuichi; Hirose, Mika; Ohta, Hiroshi; Nishimune, Yoshitake; Tanaka, Hiromitsu

2007-03-01

Haspin is a unique protein kinase expressed predominantly in haploid male germ cells. The genomic structure of haspin (Gsg2) has revealed it to be intronless, and the entire transcription unit is in an intron of the integrin alphaE (Itgae) gene. Transcription occurs from a bidirectional promoter that also generates an alternatively spliced integrin alphaE-derived mRNA (Aed). In mice, the testis-specific alternative splicing of Aed is expressed bidirectionally downstream from the Gsg2 transcription initiation site, and a segment consisting of 26 bp transcribes both genomic DNA strands between Gsg2 and the Aed transcription initiation sites. To investigate the mechanisms for this unique gene regulation, we cloned and characterized the Gsg2 promoter region. The 193-bp genomic fragment from the 5' end of the Gsg2 and Aed genes, fused with EGFP and DsRed genes, drove the expression of both proteins in haploid germ cells of transgenic mice. This promoter element contained only a GC-rich sequence, and not the previously reported DNA sequences known to bind various transcription factors--with the exception of E2F1, TCFAP2A1 (AP2), and SP1. Here, we show that the 193-bp DNA sequence is sufficient for the specific, bidirectional, and synchronous expression in germ cells in the testis. We also demonstrate the existence of germ cell nuclear factors specifically bound to the promoter sequence. This activity may be regulated by binding to the promoter sequence with germ cell-specific nuclear complex(es) without regulation via DNA methylation.

Nuclear magnetic resonance-based model of a TF1/HmU-DNA complex.

PubMed

Silva, M V; Pasternack, L B; Kearns, D R

1997-12-15

Transcription factor 1 (TF1), a type II DNA-binding protein encoded by the Bacillus subtilis bacteriophage SPO1, has the capacity for sequence-selective DNA binding and a preference for 5-hydroxymethyl-2'-deoxyuridine (HmU)-containing DNA. In NMR studies of the TF1/HmU-DNA complex, intermolecular NOEs indicate that the flexible beta-ribbon and C-terminal alpha-helix are involved in the DNA-binding site of TF1, placing it in the beta-sheet category of DNA-binding proteins proposed to bind by wrapping two beta-ribbon "arms" around the DNA. Intermolecular and intramolecular NOEs were used to generate an energy-minimized model of the protein-DNA complex in which both DNA bending and protein structure changes are evident.

PLETHORA gradient formation mechanism separates auxin responses.

PubMed

Mähönen, Ari Pekka; Ten Tusscher, Kirsten; Siligato, Riccardo; Smetana, Ondřej; Díaz-Triviño, Sara; Salojärvi, Jarkko; Wachsman, Guy; Prasad, Kalika; Heidstra, Renze; Scheres, Ben

2014-11-06

During plant growth, dividing cells in meristems must coordinate transitions from division to expansion and differentiation, thus generating three distinct developmental zones: the meristem, elongation zone and differentiation zone. Simultaneously, plants display tropisms, rapid adjustments of their direction of growth to adapt to environmental conditions. It is unclear how stable zonation is maintained during transient adjustments in growth direction. In Arabidopsis roots, many aspects of zonation are controlled by the phytohormone auxin and auxin-induced PLETHORA (PLT) transcription factors, both of which display a graded distribution with a maximum near the root tip. In addition, auxin is also pivotal for tropic responses. Here, using an iterative experimental and computational approach, we show how an interplay between auxin and PLTs controls zonation and gravitropism. We find that the PLT gradient is not a direct, proportionate readout of the auxin gradient. Rather, prolonged high auxin levels generate a narrow PLT transcription domain from which a gradient of PLT protein is subsequently generated through slow growth dilution and cell-to-cell movement. The resulting PLT levels define the location of developmental zones. In addition to slowly promoting PLT transcription, auxin also rapidly influences division, expansion and differentiation rates. We demonstrate how this specific regulatory design in which auxin cooperates with PLTs through different mechanisms and on different timescales enables both the fast tropic environmental responses and stable zonation dynamics necessary for coordinated cell differentiation.

Intron retention generates ANKRD1 splice variants that are co-regulated with the main transcript in normal and failing myocardium.

PubMed

Torrado, Mario; Iglesias, Raquel; Nespereira, Beatriz; Centeno, Alberto; López, Eduardo; Mikhailov, Alexander T

2009-07-01

The cardiac ankyrin repeat domain 1 protein (ANKRD1, also known as CARP) has been extensively characterized with regard to its proposed functions as a cardio-enriched transcriptional co-factor and stress-inducible myofibrillar protein. The present results show the occurrence of alternative splicing by intron retention events in the pig and human ankrd1 gene. In pig heart, ankrd1 is expressed as four alternatively spliced transcripts, three of which have non-excised introns: ankrd1-contained introns 6, 7 and 8 (i.e., ankrd1-i6,7,8), ankrd1-contained introns 7 and 8 (i.e., ankrd1-i7,8), and ankrd1 retained only intron 8 (i.e., ankrd1-i8). In the human heart, two orthologues of porcine intron-retaining ankrd1 variants (i.e., ankrd1-i8 and ankrd1-i7,8) are detected. We demonstrate that these newly-identified intron-retaining ankrd1 transcripts are functionally intact, efficiently translated into protein in vitro and exported to the cytoplasm in cardiomyocytes in vivo. In the piglet heart, both the intronless and intron-retaining ankrd1 mRNAs are co-expressed in a chamber-dependent manner being more abundant in the left as compared to the right myocardium. Our data further indicate co-upregulation of the ankrd1 spliced variants in myocardium in the porcine model of diastolic heart failure. Most significantly, we demonstrate that in vivo forced expression of recombinant intronless ankrd1 markedly increases the levels of intron-retaining ankrd1 variants (but not of the endogenous main transcript) in piglet myocardium, suggesting that ANKRD1 may positively regulate the expression of its own intron-containing RNAs in response to cardiac stress. Overall, our findings demonstrate that in cardiomyocytes ANKRD1 can exist in multiple isoforms which may contribute to the functional diversity of this factor in heart development and disease.

PcFKH1, a novel regulatory factor from the forkhead family, controls the biosynthesis of penicillin in Penicillium chrysogenum.

PubMed

Domínguez-Santos, Rebeca; García-Estrada, Carlos; Kosalková, Katarina; Prieto, Carlos; Santamarta, Irene; Martín, Juan-Francisco

2015-08-01

Penicillin biosynthesis in Penicillium chrysogenum (re-identified as Penicillium rubens) is a good example of a biological process subjected to complex global regulatory networks and serves as a model to study fungal secondary metabolism. The winged-helix family of transcription factors recently described, which includes the forkhead type of proteins, is a key type of regulatory proteins involved in this process. In yeasts and humans, forkhead transcription factors are involved in different processes (cell cycle regulation, cell death control, pre-mRNA processing and morphogenesis); one member of this family of proteins has been identified in the P. chrysogenum genome (Pc18g00430). In this work, we have characterized this novel transcription factor (named PcFKH1) by generating knock-down mutants and overexpression strains. Results clearly indicate that PcFKH1 positively controls antibiotic biosynthesis through the specific interaction with the promoter region of the penDE gene, thus regulating penDE mRNA levels. PcFKH1 also binds to the pcbC promoter, but with low affinity. In addition, it also controls other ancillary genes of the penicillin biosynthetic process, such as phlA (encoding phenylacetyl CoA ligase) and ppt (encoding phosphopantetheinyl transferase). PcFKH1 also plays a role in conidiation and spore pigmentation, but it does not seem to be involved in hyphal morphology or cell division in the improved laboratory reference strain Wisconsin 54-1255. A genome-wide analysis of processes putatively coregulated by PcFKH1 and PcRFX1 (another winged-helix transcription factor) in P. chrysogenum provided evidence of the global effect of these transcription factors in P. chrysogenum metabolism. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

DNA wrapping and distortion by an oligomeric homeodomain protein.

PubMed

Williams, Hannah; Jayaraman, Padma-Sheela; Gaston, Kevin

2008-10-31

Many transcription factors alter DNA or chromatin structure. Changes in chromatin structure are often brought about by the recruitment of chromatin-binding proteins, chromatin-modifying proteins, or other transcription co-activator or co-repressor proteins. However, some transcription factors form oligomeric assemblies that may themselves induce changes in DNA conformation and chromatin structure. The proline-rich homeodomain (PRH/Hex) protein is a transcription factor that regulates cell differentiation and cell proliferation, and has multiple roles in embryonic development. Earlier, we showed that PRH can repress transcription by multiple mechanisms, including the recruitment of co-repressor proteins belonging to the TLE family of chromatin-binding proteins. Our in vivo crosslinking studies have shown that PRH forms oligomeric complexes in cells and a variety of biophysical techniques suggest that the protein forms octamers. However, as yet we have little knowledge of the role played by PRH oligomerisation in the regulation of promoter activity or of the architecture of promoters that are regulated directly by PRH in cells. Here, we compare the binding of PRH and the isolated PRH homeodomain to DNA fragments with single and multiple PRH sites, using gel retardation assays and DNase I and chemical footprinting. We show that the PRH oligomer binds to multiple sites within the human Goosecoid promoter with high affinity and that the binding of PRH brings about DNA distortion. We suggest that PRH octamers wrap DNA in order to bring about transcriptional repression.

Probing transcription-specific outputs of β-catenin in vivo.

PubMed

Valenta, Tomas; Gay, Max; Steiner, Sarah; Draganova, Kalina; Zemke, Martina; Hoffmans, Raymond; Cinelli, Paolo; Aguet, Michel; Sommer, Lukas; Basler, Konrad

2011-12-15

β-Catenin, apart from playing a cell-adhesive role, is a key nuclear effector of Wnt signaling. Based on activity assays in Drosophila, we generated mouse strains where the endogenous β-catenin protein is replaced by mutant forms, which retain the cell adhesion function but lack either or both of the N- and the C-terminal transcriptional outputs. The C-terminal activity is essential for mesoderm formation and proper gastrulation, whereas N-terminal outputs are required later during embryonic development. By combining the double-mutant β-catenin with a conditional null allele and a Wnt1-Cre driver, we probed the role of Wnt/β-catenin signaling in dorsal neural tube development. While loss of β-catenin protein in the neural tube results in severe cell adhesion defects, the morphology of cells and tissues expressing the double-mutant form is normal. Surprisingly, Wnt/β-catenin signaling activity only moderately regulates cell proliferation, but is crucial for maintaining neural progenitor identity and for neuronal differentiation in the dorsal spinal cord. Our model animals thus allow dissecting signaling and structural functions of β-catenin in vivo and provide the first genetic tool to generate cells and tissues that entirely and exclusively lack canonical Wnt pathway activity. © 2011 by Cold Spring Harbor Laboratory Press

[CNC proteins in physiology and pathology].

PubMed

Gęgotek, Agnieszka; Skrzydlewska, Elżbieta

2015-07-06

CNC proteins consist of Bach1, Bach2 and 4 homologous transcription factors: Nrf1, Nrf2, Nrf3 and p45NF-E2. Transcription factors belonging to this group of proteins play a crucial role in protection of cells against oxidative stress. Under physiological conditions, they remain in the cytoplasm in the inactive form or are degraded. However, in oxidative stress conditions, they are translocated to the nucleus, and bind to DNA in the ARE sequence. Consequently, there is transcription of genes encoding cytoprotective proteins, such as phase II enzymes, or low molecular weight antioxidant proteins (i.e., thioredoxin, ferritin, metallothionein) responsible for protecting cells from reactive oxygen species (ROS) action. The activity of transcriptional proteins depends directly on the redox state of the cell. ROS as second messenger signals, control inhibitors of cytoplasmic CNC proteins or potentiate the activity of kinases (MAPK, PKC, PI3K, PERK), leading to phosphorylation of transcription factors. This is conducive to translocation of these molecules into the nucleus and to formation of complexes that initiate the gene expression. Disorders of regulation of the activity of transcription factors belonging to the CNC proteins caused by gene mutations, epigenetic modifications or increased activity of p62, p21, or k-Ras, B-Raf and c-Myc oncogenes, induce changes in the level of ARE-dependent gene expression, which can lead even to the development of carcinogenesis. On the other hand, Nrf transcription factors, inducing the expression of antioxidants and enzymes responsible for the detoxification of xenobiotics, can be considered as a potential target of the action of chemopreventive factors in anticancer therapy.

Hairless Streaks in Cattle Implicate TSR2 in Early Hair Follicle Formation

PubMed Central

Murgiano, Leonardo; Shirokova, Vera; Welle, Monika Maria; Jagannathan, Vidhya; Plattet, Philippe; Oevermann, Anna; Pienkowska-Schelling, Aldona; Gallo, Daniele; Gentile, Arcangelo; Mikkola, Marja; Drögemüller, Cord

2015-01-01

Four related cows showed hairless streaks on various parts of the body with no correlation to the pigmentation pattern. The stripes occurred in a consistent pattern resembling the lines of Blaschko. The non-syndromic hairlessness phenotype observed occurred across three generations of a single family and was compatible with an X-linked mode of inheritance. Linkage analysis and subsequent whole genome sequencing of one affected female identified two perfectly associated non-synonymous sequence variants in the critical interval on bovine chromosome X. Both variants occurred in complete linkage disequilibrium and were absent in more than 3900 controls. An ERCC6L missense mutation was predicted to cause an amino acid substitution of a non-conserved residue. Analysis in mice showed no specific Ercc6l expression pattern related to hair follicle development and therefore ERCC6L was not considered as causative gene. A point mutation at the 5'-splice junction of exon 5 of the TSR2, 20S rRNA accumulation, homolog (S. cerevisiae), gene led to the production of two mutant transcripts, both of which contain a frameshift and generate a premature stop codon predicted to truncate approximately 25% of the protein. Interestingly, in addition to the presence of both physiological TSR2 transcripts, the two mutant transcripts were predominantly detected in the hairless skin of the affected cows. Immunohistochemistry, using an antibody against the N-terminal part of the bovine protein demonstrated the specific expression of the TSR2 protein in the skin and the hair of the affected and the control cows as well as in bovine fetal skin and hair. The RNA hybridization in situ showed that Tsr2 was expressed in pre- and post-natal phases of hair follicle development in mice. Mammalian TSR2 proteins are highly conserved and are known to be broadly expressed, but their precise in vivo functions are poorly understood. Thus, by dissecting a naturally occurring mutation in a domestic animal species, we identified TSR2 as a regulator of hair follicle development. PMID:26203908

HuR interacts with human immunodeficiency virus type 1 reverse transcriptase, and modulates reverse transcription in infected cells

PubMed Central

Lemay, Julie; Maidou-Peindara, Priscilla; Bader, Thomas; Ennifar, Eric; Rain, Jean-Christophe; Benarous, Richard; Liu, Lang Xia

2008-01-01

Reverse transcription of the genetic material of human immunodeficiency virus type 1 (HIV-1) is a critical step in the replication cycle of this virus. This process, catalyzed by reverse transcriptase (RT), is well characterized at the biochemical level. However, in infected cells, reverse transcription occurs in a multiprotein complex – the reverse transcription complex (RTC) – consisting of viral genomic RNA associated with viral proteins (including RT) and, presumably, as yet uncharacterized cellular proteins. Very little is known about the cellular proteins interacting with the RTC, and with reverse transcriptase in particular. We report here that HIV-1 reverse transcription is affected by the levels of a nucleocytoplasmic shuttling protein – the RNA-binding protein HuR. A direct protein-protein interaction between RT and HuR was observed in a yeast two-hybrid screen and confirmed in vitro by homogenous time-resolved fluorescence (HTRF). We mapped the domain interacting with HuR to the RNAse H domain of RT, and the binding domain for RT to the C-terminus of HuR, partially overlapping the third RRM RNA-binding domain of HuR. HuR silencing with specific siRNAs greatly impaired early and late steps of reverse transcription, significantly inhibiting HIV-1 infection. Moreover, by mutagenesis and immunoprecipitation studies, we could not detect the binding of HuR to the viral RNA. These results suggest that HuR may be involved in and may modulate the reverse transcription reaction of HIV-1, by an as yet unknown mechanism involving a protein-protein interaction with HIV-1 RT. PMID:18544151

Post-Transcriptional Coordination of the Arabidopsis Iron Deficiency Response is Partially Dependent on the E3 Ligases RING DOMAIN LIGASE1 (RGLG1) and RING DOMAIN LIGASE2 (RGLG2)*

PubMed Central

Pan, I-Chun; Tsai, Huei-Hsuan; Cheng, Ya-Tan; Wen, Tuan-Nan; Buckhout, Thomas J.; Schmidt, Wolfgang

2015-01-01

Acclimation to changing environmental conditions is mediated by proteins, the abundance of which is carefully tuned by an elaborate interplay of DNA-templated and post-transcriptional processes. To dissect the mechanisms that control and mediate cellular iron homeostasis, we conducted quantitative high-resolution iTRAQ proteomics and microarray-based transcriptomic profiling of iron-deficient Arabidopsis thaliana plants. A total of 13,706 and 12,124 proteins was identified with a quadrupole-Orbitrap hybrid mass spectrometer in roots and leaves, respectively. This deep proteomic coverage allowed accurate estimates of post-transcriptional regulation in response to iron deficiency. Similarly regulated transcripts were detected in only 13% (roots) and 11% (leaves) of the 886 proteins that differentially accumulated between iron-sufficient and iron-deficient plants, indicating that the majority of the iron-responsive proteins was post-transcriptionally regulated. Mutants harboring defects in the RING DOMAIN LIGASE1 (RGLG1)1 and RING DOMAIN LIGASE2 (RGLG2) showed a pleiotropic phenotype that resembled iron-deficient plants with reduced trichome density and the formation of branched root hairs. Proteomic and transcriptomic profiling of rglg1 rglg2 double mutants revealed that the functional RGLG protein is required for the regulation of a large set of iron-responsive proteins including the coordinated expression of ribosomal proteins. This integrative analysis provides a detailed catalog of post-transcriptionally regulated proteins and allows the concept of a chiefly transcriptionally regulated iron deficiency response to be revisited. Protein data are available via ProteomeXchange with identifier PXD002126. PMID:26253232

Huntingtin interacting protein 1 modulates the transcriptional activity of nuclear hormone receptors.

PubMed

Mills, Ian G; Gaughan, Luke; Robson, Craig; Ross, Theodora; McCracken, Stuart; Kelly, John; Neal, David E

2005-07-18

Internalization of activated receptors regulates signaling, and endocytic adaptor proteins are well-characterized in clathrin-mediated uptake. One of these adaptor proteins, huntingtin interacting protein 1 (HIP1), induces cellular transformation and is overexpressed in some prostate cancers. We have discovered that HIP1 associates with the androgen receptor through a central coiled coil domain and is recruited to DNA response elements upon androgen stimulation. HIP1 is a novel androgen receptor regulator, significantly repressing transcription when knocked down using a silencing RNA approach and activating transcription when overexpressed. We have also identified a functional nuclear localization signal at the COOH terminus of HIP1, which contributes to the nuclear translocation of the protein. In conclusion, we have discovered that HIP1 is a nucleocytoplasmic protein capable of associating with membranes and DNA response elements and regulating transcription.

Evaluating the Autonomy of the Drosophila Circadian Clock in Dissociated Neuronal Culture.

PubMed

Sabado, Virginie; Vienne, Ludovic; Nagoshi, Emi

2017-01-01

Circadian behavioral rhythms offer an excellent model to study intricate interactions between the molecular and neuronal mechanisms of behavior. In mammals, pacemaker neurons in the suprachiasmatic nucleus (SCN) generate rhythms cell-autonomously, which are synchronized by the network interactions within the circadian circuit to drive behavioral rhythms. However, whether this principle is universal to circadian systems in animals remains unanswered. Here, we examined the autonomy of the Drosophila circadian clock by monitoring transcriptional and post-transcriptional rhythms of individual clock neurons in dispersed culture with time-lapse microscopy. Expression patterns of the transcriptional reporter show that CLOCK/CYCLE (CLK/CYC)-mediated transcription is constantly active in dissociated clock neurons. In contrast, the expression profile of the post-transcriptional reporter indicates that PERIOD (PER) protein levels fluctuate and ~10% of cells display rhythms in PER levels with periods in the circadian range. Nevertheless, PER and TIM are enriched in the cytoplasm and no periodic PER nuclear accumulation was observed. These results suggest that repression of CLK/CYC-mediated transcription by nuclear PER is impaired, and thus the negative feedback loop of the molecular clock is incomplete in isolated clock neurons. We further demonstrate that, by pharmacological assays using the non-amidated form of neuropeptide pigment-dispersing factor (PDF), which could be specifically secreted from larval LNvs and adult s-LNvs, downstream events of the PDF signaling are partly impaired in dissociated larval clock neurons. Although non-amidated PDF is likely to be less active than the amidated one, these results point out the possibility that alteration in PDF downstream signaling may play a role in dampening of molecular rhythms in isolated clock neurons. Taken together, our results suggest that Drosophila clocks are weak oscillators that need to be in the intact circadian circuit to generate robust 24-h rhythms.

Pharmacological targeting of the transcription factor SOX18 delays breast cancer in mice

PubMed Central

Overman, Jeroen; Fontaine, Frank; Moustaqil, Mehdi; Mittal, Deepak; Sierecki, Emma; Sacilotto, Natalia; Zuegg, Johannes; Robertson, Avril AB; Holmes, Kelly; Salim, Angela A; Mamidyala, Sreeman; Butler, Mark S; Robinson, Ashley S; Lesieur, Emmanuelle; Johnston, Wayne; Alexandrov, Kirill; Black, Brian L; Hogan, Benjamin M; De Val, Sarah; Capon, Robert J; Carroll, Jason S; Bailey, Timothy L; Koopman, Peter; Jauch, Ralf; Smyth, Mark J; Cooper, Matthew A; Gambin, Yann; Francois, Mathias

2017-01-01

Pharmacological targeting of transcription factors holds great promise for the development of new therapeutics, but strategies based on blockade of DNA binding, nuclear shuttling, or individual protein partner recruitment have yielded limited success to date. Transcription factors typically engage in complex interaction networks, likely masking the effects of specifically inhibiting single protein-protein interactions. Here, we used a combination of genomic, proteomic and biophysical methods to discover a suite of protein-protein interactions involving the SOX18 transcription factor, a known regulator of vascular development and disease. We describe a small-molecule that is able to disrupt a discrete subset of SOX18-dependent interactions. This compound selectively suppressed SOX18 transcriptional outputs in vitro and interfered with vascular development in zebrafish larvae. In a mouse pre-clinical model of breast cancer, treatment with this inhibitor significantly improved survival by reducing tumour vascular density and metastatic spread. Our studies validate an interactome-based molecular strategy to interfere with transcription factor activity, for the development of novel disease therapeutics. DOI: http://dx.doi.org/10.7554/eLife.21221.001 PMID:28137359

Casein Kinase 2-Mediated Phosphorylation of Respiratory Syncytial Virus Phosphoprotein P Is Essential for the Transcription Elongation Activity of the Viral Polymerase; Phosphorylation by Casein Kinase 1 Occurs Mainly at Ser215 and Is without Effect

PubMed Central

Dupuy, Lesley C.; Dobson, Sean; Bitko, Vira; Barik, Sailen

1999-01-01

The major site of in vitro phosphorylation by casein kinase 2 (CK2) was the conserved Ser232 in the P proteins of human, bovine, and ovine strains of respiratory syncytial virus (RSV). Enzymatic removal of this phosphate group from the P protein instantly halted transcription elongation in vitro. Transcription reconstituted in the absence of P protein or in the presence of phosphate-free P protein produced abortive initiation products but no full-length transcripts. A recombinant P protein in which Ser232 was mutated to Asp exhibited about half of the transcriptional activity of the wild-type phosphorylated protein, suggesting that the negative charge of the phosphate groups is an important contributor to P protein function. Use of a temperature-sensitive CK2 mutant yeast revealed that in yeast, phosphorylation of recombinant P by non-CK2 kinase(s) occurs mainly at Ser215. In vitro, P protein could be phosphorylated by purified CK1 at Ser215 but this phosphorylation did not result in transcriptionally active P protein. A triple mutant P protein in which Ser215, Ser232, and Ser237 were all mutated to Ala was completely defective in phosphorylation in vitro as well as ex vivo. The xanthate compound D609 inhibited CK2 but not CK1 in vitro and had a very modest effect on P protein phosphorylation and RSV yield ex vivo. Together, these results suggest a role for CK2-mediated phosphorylation of the P protein in the promoter clearance and elongation properties of the viral RNA-dependent RNA polymerase. PMID:10482589

Molecular pathophysiology of cerebral edema

PubMed Central

Gerzanich, Volodymyr; Simard, J Marc

2015-01-01

Advancements in molecular biology have led to a greater understanding of the individual proteins responsible for generating cerebral edema. In large part, the study of cerebral edema is the study of maladaptive ion transport. Following acute CNS injury, cells of the neurovascular unit, particularly brain endothelial cells and astrocytes, undergo a program of pre- and post-transcriptional changes in the activity of ion channels and transporters. These changes can result in maladaptive ion transport and the generation of abnormal osmotic forces that, ultimately, manifest as cerebral edema. This review discusses past models and current knowledge regarding the molecular and cellular pathophysiology of cerebral edema. PMID:26661240