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Sample records for nuclear transcription factor-kappab

  1. Transcriptional regulation at the yeast nuclear envelope

    PubMed Central

    Steglich, Babett; Sazer, Shelley; Ekwall, Karl

    2013-01-01

    The spatial organization of the genome inside the nucleus affects many nuclear processes, such as DNA replication, DNA repair, and gene transcription. In metazoans, the nuclear periphery harbors mainly repressed genes that associate with the nuclear lamina. This review discusses how peripheral positioning is connected to transcriptional regulation in yeasts. Tethering of reporter genes to the nuclear envelope was found to result in transcriptional silencing. Similarly, repression of the silent mating type loci and subtelomeric genes is influenced by their position close to the nuclear envelope. In contrast, active genes are bound by nucleoporins and inducible genes associate with the nuclear pore complex upon activation. Taken together, these results portray the nuclear envelope as a platform for transcriptional regulation, both through activation at nuclear pores and silencing at the nuclear envelope. PMID:24021962

  2. Nuclear Actin in Development and Transcriptional Reprogramming.

    PubMed

    Misu, Shinji; Takebayashi, Marina; Miyamoto, Kei

    2017-01-01

    Actin is a highly abundant protein in eukaryotic cells and dynamically changes its polymerized states with the help of actin-binding proteins. Its critical function as a constituent of cytoskeleton has been well-documented. Growing evidence demonstrates that actin is also present in nuclei, referred to as nuclear actin, and is involved in a number of nuclear processes, including transcriptional regulation and chromatin remodeling. The contribution of nuclear actin to transcriptional regulation can be explained by its direct interaction with transcription machineries and chromatin remodeling factors and by controlling the activities of transcription factors. In both cases, polymerized states of nuclear actin affect the transcriptional outcome. Nuclear actin also plays an important role in activating strongly silenced genes in somatic cells for transcriptional reprogramming. When these nuclear functions of actin are considered, it is plausible to speculate that nuclear actin is also implicated in embryonic development, in which numerous genes need to be activated in a well-coordinated manner. In this review, we especially focus on nuclear actin's roles in transcriptional activation, reprogramming and development, including stem cell differentiation and we discuss how nuclear actin can be an important player in development and cell differentiation.

  3. Nuclear Actin in Development and Transcriptional Reprogramming

    PubMed Central

    Misu, Shinji; Takebayashi, Marina; Miyamoto, Kei

    2017-01-01

    Actin is a highly abundant protein in eukaryotic cells and dynamically changes its polymerized states with the help of actin-binding proteins. Its critical function as a constituent of cytoskeleton has been well-documented. Growing evidence demonstrates that actin is also present in nuclei, referred to as nuclear actin, and is involved in a number of nuclear processes, including transcriptional regulation and chromatin remodeling. The contribution of nuclear actin to transcriptional regulation can be explained by its direct interaction with transcription machineries and chromatin remodeling factors and by controlling the activities of transcription factors. In both cases, polymerized states of nuclear actin affect the transcriptional outcome. Nuclear actin also plays an important role in activating strongly silenced genes in somatic cells for transcriptional reprogramming. When these nuclear functions of actin are considered, it is plausible to speculate that nuclear actin is also implicated in embryonic development, in which numerous genes need to be activated in a well-coordinated manner. In this review, we especially focus on nuclear actin’s roles in transcriptional activation, reprogramming and development, including stem cell differentiation and we discuss how nuclear actin can be an important player in development and cell differentiation. PMID:28326098

  4. Sumoylation and transcription regulation at nuclear pores.

    PubMed

    Texari, Lorane; Stutz, Françoise

    2015-03-01

    Increasing evidence indicates that besides promoters, enhancers, and epigenetic modifications, nuclear organization is another parameter contributing to optimal control of gene expression. Although differences between species exist, the influence of gene positioning on expression seems to be a conserved feature from yeast to Drosophila and mammals. The nuclear periphery is one of the nuclear compartments implicated in gene regulation. It consists of the nuclear envelope (NE) and the nuclear pore complexes (NPC), which have distinct roles in the control of gene expression. The NPC has recently been shown to tether proteins involved in the sumoylation pathway. Here, we will focus on the importance of gene positioning and NPC-linked sumoylation/desumoylation in transcription regulation. We will mainly discuss observations made in the yeast Saccharomyces cerevisiae model system and highlight potential parallels in metazoan species.

  5. Spliceosome twin introns in fungal nuclear transcripts.

    PubMed

    Flipphi, Michel; Fekete, Erzsébet; Ag, Norbert; Scazzocchio, Claudio; Karaffa, Levente

    2013-08-01

    The spliceosome is an RNA/protein complex, responsible for intron excision from eukaryotic nuclear transcripts. In bacteria, mitochondria and plastids, intron excision does not involve the spliceosome, but occurs through mechanisms dependent on intron RNA secondary and tertiary structure. For group II/III chloroplast introns, "twintrons" (introns within introns) have been described. The excision of the external intron, and thus proper RNA maturation, necessitates prior removal of the internal intron, which interrupts crucial sequences of the former. We have here predicted analogous instances of spliceosomal twintrons ("stwintrons") in filamentous fungi. In two specific cases, where the internal intron interrupts the donor of the external intron after the first or after the second nucleotide, respectively, we show that intermediates with the sequence predicted by the "stwintron" hypothesis, are produced in the splicing process. This implies that two successive rounds of RNA scanning by the spliceosome are necessary to produce the mature mRNA. The phylogenetic distributions of the stwintrons we have identified suggest that they derive from "late" events, subsequent to the appearance of the host intron. They may well not be limited to fungal nuclear transcripts, and their generation and eventual disappearance in the evolutionary process are relevant to hypotheses of intron origin and alternative splicing.

  6. Persistent nuclear actin filaments inhibit transcription by RNA polymerase II.

    PubMed

    Serebryannyy, Leonid A; Parilla, Megan; Annibale, Paolo; Cruz, Christina M; Laster, Kyle; Gratton, Enrico; Kudryashov, Dmitri; Kosak, Steven T; Gottardi, Cara J; de Lanerolle, Primal

    2016-09-15

    Actin is abundant in the nucleus and it is clear that nuclear actin has important functions. However, mystery surrounds the absence of classical actin filaments in the nucleus. To address this question, we investigated how polymerizing nuclear actin into persistent nuclear actin filaments affected transcription by RNA polymerase II. Nuclear filaments impaired nuclear actin dynamics by polymerizing and sequestering nuclear actin. Polymerizing actin into stable nuclear filaments disrupted the interaction of actin with RNA polymerase II and correlated with impaired RNA polymerase II localization, dynamics, gene recruitment, and reduced global transcription and cell proliferation. Polymerizing and crosslinking nuclear actin in vitro similarly disrupted the actin-RNA-polymerase-II interaction and inhibited transcription. These data rationalize the general absence of stable actin filaments in mammalian somatic nuclei. They also suggest a dynamic pool of nuclear actin is required for the proper localization and activity of RNA polymerase II.

  7. Using nuclear run-on transcription assays in RNAi studies.

    PubMed

    Khraiwesh, Basel

    2011-01-01

    RNA interference (RNAi) is a mechanism regulating gene transcript levels either by transcriptional gene silencing or by posttranscriptional gene silencing, which act in the genome maintenance and the regulation of gene expression which is typically inferred from measuring transcript abundance. Nuclear "run-on" (or "run-off") transcription assays have been used to obtain quantitative information about the relative rates of transcription of different genes in nuclei isolated from a particular tissue or organ. Basically, these assays exploit the activity of RNA polymerases to synthesize radiolabeled transcripts that then can be hybridized to filter-bound, cold, excess single-stranded DNA probes representing genes of interest. The protocol presented here streamlines, adapts, and optimizes nuclear run-on transcription assays for use in RNAi studies.

  8. Integration of light and photoperiodic signaling in transcriptional nuclear foci

    PubMed Central

    Kaiserli, Eirini; Paldi, Katalin; O'Donnell, Liz; Batalov, Olga; Pedmale, Ullas V.; Nusinow, Dmitri A.; Kay, Steve A.; Chory, Joanne

    2015-01-01

    Summary Light regulates major plant developmental transitions by orchestrating a series of nuclear events. This study uncovers the molecular function of the natural variant, TZP (Tandem Zinc-finger-Plus3), as a novel signal integrator of light and photoperiodic pathways in transcriptional nuclear foci. We report that TZP acts as a positive regulator of photoperiodic flowering via physical interactions with the red-light receptor phytochrome B (phyB). We demonstrate that TZP localizes in dynamic nuclear domains regulated by light quality and photoperiod. This study shows that phyB is indispensible not only for localizing TZP to transcriptionally active nuclear photobodies, but also for recruiting TZP on the promoter of the floral inducer FLOWERING LOCUS T (FT). Our findings signify a unique transcriptional regulatory role to the highly enigmatic plant nuclear photobodies, where TZP directly activates FT gene expression and promotes flowering. PMID:26555051

  9. Transcription termination by nuclear RNA polymerases

    PubMed Central

    Richard, Patricia; Manley, James L.

    2009-01-01

    Gene transcription in the cell nucleus is a complex and highly regulated process. Transcription in eukaryotes requires three distinct RNA polymerases, each of which employs its own mechanisms for initiation, elongation, and termination. Termination mechanisms vary considerably, ranging from relatively simple to exceptionally complex. In this review, we describe the present state of knowledge on how each of the three RNA polymerases terminates and how mechanisms are conserved, or vary, from yeast to human. PMID:19487567

  10. The murine Sry gene encodes a nuclear transcriptional activator

    SciTech Connect

    Dubin, R.A.; Ostrer, H.

    1994-09-01

    The Sry gene functions as a genetic switch in gonadal ridge initiating testis determination. The murine Sry and human SRY open reading frames (ORF) share a conserved 79 amino acid motif, the HMG-box, that binds DNA. Outside this region the two genes share no additional homology. These studies were undertaken to determine whether the Sry/SRY genes encode nuclear transcriptional regulators. As judged by the accumulation of lacZ-SRY hybrid proteins in the nucleus, both the human and murine SRY ORFs contain a nuclear localization signal. The murine Sry HMG-box selectively binds the sequence NACAAT in vitro when presented with a random pool of oligonucleotides and binds AACAAT with the highest affinity. The murine Sry ORF, when expressed in HeLa cells, activates transcription of a reporter gene containing multiple copies of the AACAAT binding site. Activation was observed for a GAL4-responsive gene when the murine Sry ORF was linked to the DNA-binding domain of GAL4. Using this system, the activation function was mapped to a C-terminal glutamine/histidine-rich domain. In addition, LexA-Sry fusion genes activated a LexA-responsive gene in yeast. In contrast, a GAL4-human SRY fusion gene did not cause transcriptional activation. These studies suggest that both the human and mouse SRY ORFs encode nuclear, DNA-binding proteins, and that the mouse Sry ORF can function as a transcriptional activator with separable DNA-binding and activator domains.

  11. Negative transcriptional regulation of mitochondrial transcription factor A (TFAM) by nuclear TFAM

    SciTech Connect

    Lee, Eun Jin; Kang, Young Cheol; Park, Wook-Ha; Jeong, Jae Hoon; Pak, Youngmi Kim

    2014-07-18

    Highlights: • TFAM localizes in nuclei and mitochondria of neuronal cells. • Nuclear TFAM does not bind the Tfam promoter. • Nuclear TFAM reduced the Tfam promoter activity via suppressing NRF-1 activity. • A novel self-negative feedback regulation of Tfam gene expression is explored. • FAM may play different roles depending on its subcellular localizations. - Abstract: The nuclear DNA-encoded mitochondrial transcription factor A (TFAM) is synthesized in cytoplasm and transported into mitochondria. TFAM enhances both transcription and replication of mitochondrial DNA. It is unclear, however, whether TFAM plays a role in regulating nuclear gene expression. Here, we demonstrated that TFAM was localized to the nucleus and mitochondria by immunostaining, subcellular fractionation, and TFAM-green fluorescent protein hybrid protein studies. In HT22 hippocampal neuronal cells, human TFAM (hTFAM) overexpression suppressed human Tfam promoter-mediated luciferase activity in a dose-dependent manner. The mitochondria targeting sequence-deficient hTFAM also repressed Tfam promoter activity to the same degree as hTFAM. It indicated that nuclear hTFAM suppressed Tfam expression without modulating mitochondrial activity. The repression required for nuclear respiratory factor-1 (NRF-1), but hTFAM did not bind to the NRF-1 binding site of its promoter. TFAM was co-immunoprecipitated with NRF-1. Taken together, we suggest that nuclear TFAM down-regulate its own gene expression as a NRF-1 repressor, showing that TFAM may play different roles depending on its subcellular localizations.

  12. A histone chaperone, DEK, transcriptionally coactivates a nuclear receptor

    PubMed Central

    Sawatsubashi, Shun; Murata, Takuya; Lim, Jinseon; Fujiki, Ryoji; Ito, Saya; Suzuki, Eriko; Tanabe, Masahiko; Zhao, Yue; Kimura, Shuhei; Fujiyama, Sally; Ueda, Takashi; Umetsu, Daiki; Ito, Takashi; Takeyama, Ken-ichi; Kato, Shigeaki

    2010-01-01

    Chromatin reorganization is essential for transcriptional control by sequence-specific transcription factors. However, the molecular link between transcriptional control and chromatin reconfiguration remains unclear. By colocalization of the nuclear ecdysone receptor (EcR) on the ecdysone-induced puff in the salivary gland, Drosophila DEK (dDEK) was genetically identified as a coactivator of EcR in both insect cells and intact flies. Biochemical purification and characterization of the complexes containing fly and human DEKs revealed that DEKs serve as histone chaperones via phosphorylation by forming complexes with casein kinase 2. Consistent with the preferential association of the DEK complex with histones enriched in active epigenetic marks, dDEK facilitated H3.3 assembly during puff formation. In some human myeloid leukemia patients, DEK was fused to CAN by chromosomal translocation. This mutation significantly reduced formation of the DEK complex, which is required for histone chaperone activity. Thus, the present study suggests that at least one histone chaperone can be categorized as a type of transcriptional coactivator for nuclear receptors. PMID:20040570

  13. Nuclear actin-binding proteins as modulators of gene transcription.

    PubMed

    Gettemans, Jan; Van Impe, Katrien; Delanote, Veerle; Hubert, Thomas; Vandekerckhove, Joël; De Corte, Veerle

    2005-10-01

    Dynamic transformations in the organization of the cellular microfilament system are the driving force behind fundamental biological processes such as cellular motility, cytokinesis, wound healing and secretion. Eukaryotic cells express a plethora of actin-binding proteins (ABPs) allowing cells to control the organization of the actin cytoskeleton in a flexible manner. These structural proteins were, not surprisingly, originally described as (major) constituents of the cytoplasm. However, in recent years, there has been a steady flow of reports detailing not only translocation of ABPs into and out of the nucleus but also describing their role in the nuclear compartment. This review focuses on recent developments pertaining to nucleocytoplasmic transport of ABPs, including their mode of translocation and nuclear function. In particular, evidence that structurally and functionally unrelated cytoplasmic ABPs regulate transcription activation by various nuclear (steroid hormone) receptors is steadily accruing. Furthermore, the recent finding that actin is a necessary component of the RNA polymerase II-containing preinitiation complex opens up new opportunities for nuclear ABPs in gene transcription regulation.

  14. Reshaping the transcriptional frontier: epigenetics and somatic cell nuclear transfer.

    PubMed

    Long, Charles R; Westhusin, Mark E; Golding, Michael C

    2014-02-01

    Somatic-cell nuclear transfer (SCNT) experiments have paved the way to the field of cellular reprogramming. The demonstrated ability to clone over 20 different species to date has proven that the technology is robust but very inefficient, and is prone to developmental anomalies. Yet, the offspring from cloned animals exhibit none of the abnormalities of their parents, suggesting the low efficiency and high developmental mortality are epigenetic in origin. The epigenetic barriers to reprogramming somatic cells into a totipotent embryo capable of developing into a viable offspring are significant and varied. Despite their intimate relationship, chromatin structure and transcription are often not uniformly reprogramed after nuclear transfer, and many cloned embryos develop gene expression profiles that are hybrids between the donor cell and an embryonic blastomere. Recent advances in cellular reprogramming suggest that alteration of donor-cell chromatin structure towards that found in an normal embryo is actually the rate-limiting step in successful development of SCNT embryos. Here we review the literature relevant to the transformation of a somatic-cell nucleus into an embryo capable of full-term development. Interestingly, while resetting somatic transcription and associated epigenetic marks are absolutely required for development of SCNT embryos, life does not demand perfection.

  15. Multiple nuclear localization signals function in the nuclear import of the transcription factor Nrf2.

    PubMed

    Theodore, Melanie; Kawai, Yumiko; Yang, Jianqi; Kleshchenko, Yuliya; Reddy, Sekhar P; Villalta, Fernando; Arinze, Ifeanyi J

    2008-04-04

    Nuclear factor erythroid 2-related factor 2 (Nrf2) mediates the transcriptional response of cells to oxidative stress and is translocated into the nucleus following, or concomitant with, its activation by electrophiles or reactive oxygen species. The mechanism of its translocation into the nucleus is not entirely elucidated. Here we have identified two novel nuclear localization signal (NLS) motifs in murine Nrf2, one located near the N-terminal region (amino acid residues 42-53) and the other (residues 587-593) located near the C-terminal region. Imaging of green fluorescent protein (GFP)-tagged Nrf2 revealed that mutation(s) in any of these sequences resulted in decreased nuclear fluorescence intensity compared with the wild-type Nrf2 when Nrf2 activation was induced with the electrophile tert-butylhydroquinone. The mutations also impaired Nrf2-induced transactivation of antioxidant response element-driven reporter gene expression to the same extent as the Nrf2 construct bearing mutation in a previously identified bipartite NLS that maps at residues 494-511. When linked to GFP or to GFP-PEPCK-C each of the novel NLS motifs was sufficient to drive nuclear translocation of the fusion proteins. Co-immunoprecipitation assays demonstrated that importins alpha5 and beta1 associate with Nrf2, an interaction that was blocked by the nuclear import inhibitor SN50. SN50 also blocked tert-butylhydroquinone-induced nuclear fluorescence of GFP-Nrf2 in cells transfected with wild-type GFP-Nrf2. Overall these results reveal that multiple NLS motifs in Nrf2 function in its nuclear translocation in response to pro-oxidant stimuli and that the importin alpha-beta heterodimer nuclear import receptor system plays a critical role in the import process.

  16. The Arabidopsis thaliana Nuclear Factor Y Transcription Factors

    PubMed Central

    Zhao, Hang; Wu, Di; Kong, Fanying; Lin, Ke; Zhang, Haishen; Li, Gang

    2017-01-01

    Nuclear factor Y (NF-Y) is an evolutionarily conserved trimeric transcription factor complex present in nearly all eukaryotes. The heterotrimeric NF-Y complex consists of three subunits, NF-YA, NF-YB, and NF-YC, and binds to the CCAAT box in the promoter regions of its target genes to regulate their expression. Yeast and mammal genomes generally have single genes with multiple splicing isoforms that encode each NF-Y subunit. By contrast, plant genomes generally have multi-gene families encoding each subunit and these genes are differentially expressed in various tissues or stages. Therefore, different subunit combinations can lead to a wide variety of NF-Y complexes in various tissues, stages, and growth conditions, indicating the potentially diverse functions of this complex in plants. Indeed, many recent studies have proved that the NF-Y complex plays multiple essential roles in plant growth, development, and stress responses. In this review, we highlight recent progress on NF-Y in Arabidopsis thaliana, including NF-Y protein structure, heterotrimeric complex formation, and the molecular mechanism by which NF-Y regulates downstream target gene expression. We then focus on its biological functions and underlying molecular mechanisms. Finally, possible directions for future research on NF-Y are also presented. PMID:28119722

  17. Nuclear import of a lipid-modified transcription factor

    PubMed Central

    Eisenhaber, Birgit; Sammer, Michaela; Lua, Wai Heng; Benetka, Wolfgang; Liew, Lai Ling; Yu, Weimiao; Lee, Hwee Kuan; Koranda, Manfred; Adhikari, Sharmila

    2011-01-01

    Lipid-modified transcription factors (TFs) are biomolecular oddities, since their reduced mobility and membrane attachment appear to contradict nuclear import required for their gene-regulatory function. NFAT5 isoform a (selected from an in silico screen for predicted lipid-modified TFs) is shown to contribute about half of all endogenous expression of human NFAT5 isoforms in the isotonic state. Wild-type NFAT5a protein is, indeed, myristoylated and palmitoylated on its transport to the plasmalemma via the endoplasmic reticulum and the Golgi. In contrast, its lipid anchor-deficient mutants as well as isoforms NFAT5b/c are diffusely localized in the cytoplasm without preference to vesicular structures. Quantitative/live microscopy shows the plasma membrane-bound fraction of NFAT5a moving into the nucleus upon osmotic stress despite the lipid anchoring. The mobilization mechanism is not based on proteolytic processing of the lipid-anchored N terminus but appears to involve reversible palmitoylation. Thus, NFAT5a is an example of TFs immobilized with lipid anchors at cyotoplasmic membranes in the resting state and that, nevertheless, can translocate into the nucleus upon signal induction. PMID:22071693

  18. The Arabidopsis thaliana Nuclear Factor Y Transcription Factors.

    PubMed

    Zhao, Hang; Wu, Di; Kong, Fanying; Lin, Ke; Zhang, Haishen; Li, Gang

    2016-01-01

    Nuclear factor Y (NF-Y) is an evolutionarily conserved trimeric transcription factor complex present in nearly all eukaryotes. The heterotrimeric NF-Y complex consists of three subunits, NF-YA, NF-YB, and NF-YC, and binds to the CCAAT box in the promoter regions of its target genes to regulate their expression. Yeast and mammal genomes generally have single genes with multiple splicing isoforms that encode each NF-Y subunit. By contrast, plant genomes generally have multi-gene families encoding each subunit and these genes are differentially expressed in various tissues or stages. Therefore, different subunit combinations can lead to a wide variety of NF-Y complexes in various tissues, stages, and growth conditions, indicating the potentially diverse functions of this complex in plants. Indeed, many recent studies have proved that the NF-Y complex plays multiple essential roles in plant growth, development, and stress responses. In this review, we highlight recent progress on NF-Y in Arabidopsis thaliana, including NF-Y protein structure, heterotrimeric complex formation, and the molecular mechanism by which NF-Y regulates downstream target gene expression. We then focus on its biological functions and underlying molecular mechanisms. Finally, possible directions for future research on NF-Y are also presented.

  19. Nur77 forms novel nuclear structures upon DNA damage that cause transcriptional arrest

    SciTech Connect

    Leseleuc, Louis de; Denis, Francois . E-mail: francois.denis@iaf.inrs.ca

    2006-05-15

    The orphan nuclear receptor Nur77 has been implicated in both growth and apoptosis, and its function and activity can be modulated by cellular redistribution. Green fluorescent protein-tagged Nur77 was used to evaluate the role of Nur77 intracellular redistribution in response to genotoxic stress. Selected DNA damaging agents and transcription inhibition lead to rapid redistribution of Nur77 into nuclear structures distinct from conventional nuclear bodies. These nuclear bodies formed transiently were tightly bound to the nuclear matrix and conditions that lead to their appearance were associated with Nur77 transcriptional inhibition. The formation of Nur77 nuclear bodies might be involved in programmed cell death modulation upon exposure to DNA damaging agents that inhibit transcription by sequestrating this proapoptotic factor in dense nuclear structures.

  20. The transcription factor nuclear factor-kappa B and cancer.

    PubMed

    Escárcega, R O; Fuentes-Alexandro, S; García-Carrasco, M; Gatica, A; Zamora, A

    2007-03-01

    Since the discovery of nuclear factor-kappa B (NF-kappaB) in 1986, many studies have been conducted showing the link between the NF-kappaB signalling pathway and control of the inflammatory response. Today it is well known that control of the inflammatory response and apoptosis is closely related to the activation of NF-kappaB. Three NF-kappaB activation pathways exist. The first (the classical pathway) is normally triggered in response to microbial and viral infections or exposure to pro-inflammatory cytokines that activate the tripartite IKK complex, leading to phosphorylation-induced IkappaB degradation and depends mainly on IKKbeta activity. The second (the alternative pathway), leads to selective activation of p52:RelB dimers by inducing the processing of the NF-kappaB2/p100 precursor protein, which mostly occurs as a heterodimer with RelB in the cytoplasm. This pathway is triggered by certain members of the tumour necrosis factor cytokine family, through selective activation of IKKalpha homodimers by the upstream kinase NIK. The third pathway is named CK2 and is IKK independent. NF-kappaB acts through the transcription of anti-apoptotic proteins, leading to increased proliferation of cells and tumour growth. It is also known that some drugs act directly in the inhibition of NF-kappaB, thus producing regulation of apoptosis; some examples are aspirin and corticosteroids. Here we review the role of NF-kappaB in the control of apoptosis, its link to oncogenesis, the evidence of several studies that show that NF-kappaB activation is closely related to different cancers, and finally the potential target of NF-kappaB as cancer therapy.

  1. Murine leukemia virus uses TREX components for efficient nuclear export of unspliced viral transcripts.

    PubMed

    Sakuma, Toshie; Tonne, Jason M; Ikeda, Yasuhiro

    2014-03-10

    Previously we reported that nuclear export of both unspliced and spliced murine leukemia virus (MLV) transcripts depends on the nuclear export factor (NXF1) pathway. Although the mRNA export complex TREX, which contains Aly/REF, UAP56, and the THO complex, is involved in the NXF1-mediated nuclear export of cellular mRNAs, its contribution to the export of MLV mRNA transcripts remains poorly understood. Here, we studied the involvement of TREX components in the export of MLV transcripts. Depletion of UAP56, but not Aly/REF, reduced the level of both unspliced and spliced viral transcripts in the cytoplasm. Interestingly, depletion of THO components, including THOC5 and THOC7, affected only unspliced viral transcripts in the cytoplasm. Moreover, the RNA immunoprecipitation assay showed that only the unspliced viral transcript interacted with THOC5. These results imply that MLV requires UAP56, THOC5 and THOC7, in addition to NXF1, for nuclear export of viral transcripts. Given that naturally intronless mRNAs, but not bulk mRNAs, require THOC5 for nuclear export, it is plausible that THOC5 plays a key role in the export of unspliced MLV transcripts.

  2. Nuclear gadgets in mitochondrial DNA replication and transcription.

    PubMed

    Clayton, D A

    1991-03-01

    In mammalian mitochondrial DNA, activation of the light-strand promoter mediates both priming of leading-strand replication and initiation of light-strand transcription. Accurate and efficient transcription requires at least two proteins: mitochondrial RNA polymerase and a separable transcription factor that can function across species boundaries. Subsequently, primer RNAs are cleaved by a site-specific ribonucleoprotein endoribonuclease that recognizes short, highly conserved sequence elements in the RNA substrate.

  3. Nuclear Matrix protein SMAR1 represses HIV-1 LTR mediated transcription through chromatin remodeling

    SciTech Connect

    Sreenath, Kadreppa; Pavithra, Lakshminarasimhan; Singh, Sandeep; Sinha, Surajit; Dash, Prasanta K.; Siddappa, Nagadenahalli B.; Ranga, Udaykumar; Mitra, Debashis; Chattopadhyay, Samit

    2010-04-25

    Nuclear Matrix and MARs have been implicated in the transcriptional regulation of host as well as viral genes but their precise role in HIV-1 transcription remains unclear. Here, we show that > 98% of HIV sequences contain consensus MAR element in their promoter. We show that SMAR1 binds to the LTR MAR and reinforces transcriptional silencing by tethering the LTR MAR to nuclear matrix. SMAR1 associated HDAC1-mSin3 corepressor complex is dislodged from the LTR upon cellular activation by PMA/TNFalpha leading to an increase in the acetylation and a reduction in the trimethylation of histones, associated with the recruitment of RNA Polymerase II on the LTR. Overexpression of SMAR1 lead to reduction in LTR mediated transcription, both in a Tat dependent and independent manner, resulting in a decreased virion production. These results demonstrate the role of SMAR1 in regulating viral transcription by alternative compartmentalization of LTR between the nuclear matrix and chromatin.

  4. A binding site for the transcription factor Grainyhead/Nuclear transcription factor-1 contributes to regulation of the Drosophila proliferating cell nuclear antigen gene promoter.

    PubMed

    Hayashi, Y; Yamagishi, M; Nishimoto, Y; Taguchi, O; Matsukage, A; Yamaguchi, M

    1999-12-03

    The Drosophila proliferating cell nuclear antigen promoter contains multiple transcriptional regulatory elements, including upstream regulatory element (URE), DNA replication-related element, E2F recognition sites, and three common regulatory factor for DNA replication and DNA replication-related element-binding factor genes recognition sites. In nuclear extracts of Drosophila embryos, we detected a protein factor, the URE-binding factor (UREF), that recognizes the nucleotide sequence 5'-AAACCAGTTGGCA located within URE. Analyses in Drosophila Kc cells and transgenic flies revealed that the UREF-binding site plays an important role in promoter activity both in cultured cells and in living flies. A yeast one-hybrid screen using URE as a bait allowed isolation of a cDNA encoding a transcription factor, Grainyhead/nuclear transcription factor-1 (GRH/NTF-1). The nucleotide sequence required for binding to GRH was indistinguishable from that for UREF detected in embryo nuclear extracts. Furthermore, a specific antibody to GRH reacted with UREF in embryo nuclear extracts. From these results we conclude that GRH is identical to UREF. Although GRH has been thought to be involved in regulation of differentiation-related genes, this study demonstrates, for the first time, involvement of a GRH-binding site in regulation of the DNA replication-related proliferating cell nuclear antigen gene.

  5. Nuclear stability and transcriptional directionality separate functionally distinct RNA species.

    PubMed

    Andersson, Robin; Refsing Andersen, Peter; Valen, Eivind; Core, Leighton J; Bornholdt, Jette; Boyd, Mette; Heick Jensen, Torben; Sandelin, Albin

    2014-11-12

    Mammalian genomes are pervasively transcribed, yielding a complex transcriptome with high variability in composition and cellular abundance. Although recent efforts have identified thousands of new long non-coding (lnc) RNAs and demonstrated a complex transcriptional repertoire produced by protein-coding (pc) genes, limited progress has been made in distinguishing functional RNA from spurious transcription events. This is partly due to present RNA classification, which is typically based on technical rather than biochemical criteria. Here we devise a strategy to systematically categorize human RNAs by their sensitivity to the ribonucleolytic RNA exosome complex and by the nature of their transcription initiation. These measures are surprisingly effective at correctly classifying annotated transcripts, including lncRNAs of known function. The approach also identifies uncharacterized stable lncRNAs, hidden among a vast majority of unstable transcripts. The predictive power of the approach promises to streamline the functional analysis of known and novel RNAs.

  6. Nuclear pore proteins regulate chromatin structure and transcriptional memory by a conserved mechanism.

    PubMed

    Light, William H; Brickner, Jason H

    2013-01-01

    Previous experience alters the rate of transcriptional induction of many genes in yeast and this phenomenon persists through several cell division cycles. This phenomenon is called epigenetic transcriptional memory. For the yeast gene INO1, transcriptional memory requires a physical interaction with the nuclear pore complex (NPC) and changes in the chromatin structure of the promoter. These changes lead to binding of a preinitiation form of RNA Polymerase II (RNAPII) to the INO1 promoter, bypassing the need to recruit RNAPII to the promoter during reactivation. In our recent study, we found that in human cells, hundreds of interferon-γ responsive genes exhibit a mechanistically similar form of transcriptional memory. Transcriptional memory requires a homologous nuclear pore protein in yeast and humans, which interacts with the promoters of genes that exhibit transcriptional memory and promotes both alteration of chromatin structure and binding of RNAPII. Whereas the interaction of yeast genes with nuclear pore proteins occurs at the NPC, the interaction of human genes with nuclear pore proteins occurs in the nucleoplasm. Thus, the interaction of nuclear pore proteins with genes plays an important and conserved role in affecting long-term epigenetic changes in transcriptional regulation.

  7. Nuclear actin activates human transcription factor genes including the OCT4 gene.

    PubMed

    Yamazaki, Shota; Yamamoto, Koji; Tokunaga, Makio; Sakata-Sogawa, Kumiko; Harata, Masahiko

    2015-01-01

    RNA microarray analyses revealed that nuclear actin activated many human transcription factor genes including OCT4, which is required for gene reprogramming. Oct4 is known to be activated by nuclear actin in Xenopus oocytes. Our findings imply that this process of OCT4 activation is conserved in vertebrates and among cell types and could be used for gene reprogramming of human cells.

  8. Bmal1 is a direct transcriptional target of the orphan nuclear receptor, NR2F1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Orphan nuclear receptor NR2F1 (also known as COUP-TFI, Chicken Ovalbumin Upstream Promoter Transcription Factor I) is a highly conserved member of the nuclear receptor superfamily. NR2F1 plays a critical role during embryonic development, particularly in the central and peripheral nervous systems a...

  9. Telomere-surrounding regions are transcription-permissive 3D nuclear compartments in human cells

    SciTech Connect

    Quina, Ana Sofia; Parreira, Leonor . E-mail: lparreir@igc.gulbenkian.pt

    2005-07-01

    Positioning of genes relative to nuclear heterochromatic compartments is thought to help regulate their transcriptional activity. Given that human subtelomeric regions are rich in highly expressed genes, we asked whether human telomeres are related to transcription-permissive nuclear compartments. To address this question, we investigated in the nuclei of normal human lymphocytes the spatial relations of two constitutively expressed genes (ACTB and RARA) and three nuclear transcripts (ACTB, IL2RA and TCRB) to telomeres and centromeres, as a function of gene activity and transcription levels. We observed that genes and gene transcripts locate close to telomere clusters and away from chromocenters upon activation of transcription. These findings, together with the observation that SC35 domains, which are enriched in pre-mRNA processing factors, are in close proximity to telomeres, indicate that telomere-neighboring regions are permissive to gene expression in human cells. Therefore, the associations of telomeres observed in the interphase nucleus might contribute, as opposed to chromocenters, for the establishment of transcription-permissive 3D nuclear compartments.

  10. Nuclear receptors and transcription factors in the development of fatty liver disease.

    PubMed

    Vluggens, Aurore; Reddy, Janardan K

    2012-12-01

    Liver regulates certain key aspects of lipid metabolism including de novo lipogenesis, fatty acid oxidation, and lipoprotein uptake and secretion. Disturbances in these hepatic functions can contribute to the development of fatty liver disease. An understanding of the regulatory mechanisms influencing hepatic lipid homeostasis and systemic energy balance is therefore of paramount importance in gaining insights that might be useful in the management of fatty liver disease. In this regard, emerging evidence indicates that certain members of the nuclear receptor superfamily and some key transcription coactivators function as intracellular sensors to orchestrate hepatic lipid metabolism. Dysregulation of nuclear receptor-mediated transcriptional signaling and perturbations in the levels of their cognate endogenous ligands play a prominent role in the development of fatty liver disease. The potential of nuclear receptors, transcription coactivators as well as enzymes that participate in the synthesis and degradation of endogenous nuclear receptor ligands, as effective therapeutic targets for fatty liver disease needs evaluation.

  11. Transcriptional coordination of hepatic autophagy by nutrient-sensing nuclear receptor PPARα and FXR

    PubMed Central

    2016-01-01

    Nuclear receptors are in general ligand-dependent transcription factors that control a variety of mammalian physiologies including development, differentiation, proliferation, and homeostasis. Recent studies have found that two nutrient-sensing nuclear receptors, peroxisome proliferator-activated receptor α and farnesoid x receptor, responding to fasting or feeding state, respectively are able to regulate autophagy, an evolutionarily conserved catabolic process involved in lysosomal degradation. In this review, we discuss the role of these nutrient-sensing nuclear receptors in an aspect of transcriptional regulation of autophagy, and how these nuclear receptor-driven transcriptional programs integrate lipophagy, a lipid autophagy with fatty acid oxidation to coordinate hepatic lipid metabolism in the fasted state of the liver. PMID:28164071

  12. Transcriptional coordination of hepatic autophagy by nutrient-sensing nuclear receptor PPARα and FXR.

    PubMed

    Lee, Jae Man

    2016-12-01

    Nuclear receptors are in general ligand-dependent transcription factors that control a variety of mammalian physiologies including development, differentiation, proliferation, and homeostasis. Recent studies have found that two nutrient-sensing nuclear receptors, peroxisome proliferator-activated receptor α and farnesoid x receptor, responding to fasting or feeding state, respectively are able to regulate autophagy, an evolutionarily conserved catabolic process involved in lysosomal degradation. In this review, we discuss the role of these nutrient-sensing nuclear receptors in an aspect of transcriptional regulation of autophagy, and how these nuclear receptor-driven transcriptional programs integrate lipophagy, a lipid autophagy with fatty acid oxidation to coordinate hepatic lipid metabolism in the fasted state of the liver.

  13. GATA transcription factors associate with a novel class of nuclear bodies in erythroblasts and megakaryocytes.

    PubMed Central

    Elefanty, A G; Antoniou, M; Custodio, N; Carmo-Fonseca, M; Grosveld, F G

    1996-01-01

    The nuclear distribution of GATA transcription factors in murine haemopoietic cells was examined by indirect immunofluorescence. Specific bright foci of GATA-1 fluorescence were observed in erythroleukaemia cells and primary murine erythroblasts and megakaryocytes, in addition to diffuse nucleoplasmic localization. These foci, which were preferentially found adjacent to nucleoli or at the nuclear periphery, did not represent sites of active transcription or binding of GATA-1 to consensus sites in the beta-globin loci. Immunoelectron microscopy demonstrated the presence of intensely labelled structures likely to represent the GATA-1 foci seen by immunofluorescence. The GATA-1 nuclear bodies differed from previously described nuclear structures and there was no co-localization with nuclear antigens involved in RNA processing or other ubiquitous (Spl, c-Jun and TBP) or haemopoietic (NF-E2) transcription factors. Interestingly, GATA-2 and GATA-3 proteins also localized to the same nuclear bodies in cell lines co-expressing GATA-1 and -2 or GATA-1 and -3 gene products. This pattern of distribution is, thus far, unique to the GATA transcription factors and suggests a protein-protein interaction with other components of the nuclear bodies via the GATA zinc finger domain. Images PMID:8617207

  14. Transcriptional regulation of human small nuclear RNA genes

    PubMed Central

    Jawdekar, Gauri W.; Henry, R. William

    2009-01-01

    The products of human snRNA genes have been frequently described as performing housekeeping functions and their synthesis refractory to regulation. However, recent studies have emphasized that snRNA and other related non-coding RNA molecules control multiple facets of the central dogma, and their regulated expression is critical to cellular homeostasis during normal growth and in response to stress. Human snRNA genes contain compact and yet powerful promoters that are recognized by increasingly well-characterized transcription factors, thus providing a premier model system to study gene regulation. This review summarizes many recent advances deciphering the mechanism by which the transcription of human snRNA and related genes are regulated. PMID:18442490

  15. Transcriptional regulation of human Paraoxonase 1 by nuclear receptors.

    PubMed

    Ponce-Ruiz, N; Murillo-González, F E; Rojas-García, A E; Mackness, Mike; Bernal-Hernández, Y Y; Barrón-Vivanco, B S; González-Arias, C A; Medina-Díaz, I M

    2017-02-20

    Paraoxonase 1 (PON1) is a calcium-dependent lactonase synthesized primarily in the liver and secreted into the plasma, where it is associates with high density lipoproteins (HDL). PON1 acts as antioxidant preventing low-density lipoprotein (LDL) oxidation, a process considered critical in the initiation and progression of atherosclerosis. Additionally, PON1 hydrolyzes and detoxifies some toxic metabolites of organophosphorus compounds (OPs). Thus, PON1 activity and expression levels are important for determining susceptibility to OPs intoxication and risk of developing diseases related to inflammation and oxidative stress. Increasing evidence has demonstrated the modulation of PON1 expression by many factors is due to interaction with nuclear receptors (NRs). Here, we briefly review the studies in this area and discuss the role of nuclear receptors in the regulation of PON1 expression, as well as how understanding these mechanisms may allow us to manipulate PON1 levels to improve drug efficacy and treat disease.

  16. Transcriptional competence of the integrated HIV-1 provirus at the nuclear periphery

    PubMed Central

    Dieudonné, Mariacarolina; Maiuri, Paolo; Biancotto, Chiara; Knezevich, Anna; Kula, Anna; Lusic, Marina; Marcello, Alessandro

    2009-01-01

    Spatial distribution of genes within the nucleus contributes to transcriptional control, allowing optimal gene expression as well as constitutive or regulated gene repression. Human immunodeficiency virus type 1 (HIV-1) integrates into host chromatin to transcribe and replicate its genome. Lymphocytes harbouring a quiescent but inducible provirus are a challenge to viral eradication in infected patients undergoing antiviral therapy. Therefore, our understanding of the contribution of sub-nuclear positioning to viral transcription may also have far-reaching implications in the pathology of the infection. To gain an insight into the conformation of chromatin at the site of HIV-1 integration, we investigated lymphocytes carrying a single latent provirus. In the silenced state, the provirus was consistently found at the nuclear periphery, associated in trans with a pericentromeric region of chromosome 12 in a significant number of quiescent cells. After induction of the transcription, this association was lost, although the location of the transcribing provirus remained peripheral. These results, extended to several other cell clones, unveil a novel mechanism of transcriptional silencing involved in HIV-1 post-transcriptional latency and reinforce the notion that gene transcription may also occur at the nuclear periphery. PMID:19478796

  17. Transcription and nuclear transport of CAG/CTG trinucleotide repeats in yeast.

    PubMed

    Fabre, Emmanuelle; Dujon, Bernard; Richard, Guy-Franck

    2002-08-15

    Trinucleotide repeats are involved in several neurological disorders in humans. DNA sequences containing CAG/CTG repeats are prone to slippage during replication and double-strand break repair. The effects of trinucleotide repeats on transcription and on nuclear export were analyzed in vivo in yeast. Transcription of a CAG/CTG trinucleotide repeat in the 3'-untranslated region of a URA3 reporter gene leads to transcription of messenger RNAs several kilobases longer than the expected size. These long mRNAs form more readily when CAG rather than CTG repeats are transcribed. CAG- or CUG-containing transcripts show a non-homogeneous cellular localization. We propose that long mRNAs result from transcription slippage, and discuss the possible implications for human diseases.

  18. Inhibition of CRM1-mediated nuclear export of transcription factors by leukemogenic NUP98 fusion proteins.

    PubMed

    Takeda, Akiko; Sarma, Nayan J; Abdul-Nabi, Anmaar M; Yaseen, Nabeel R

    2010-05-21

    NUP98 is a nucleoporin that plays complex roles in the nucleocytoplasmic trafficking of macromolecules. Rearrangements of the NUP98 gene in human leukemia result in the expression of numerous fusion oncoproteins whose effect on nucleocytoplasmic trafficking is poorly understood. The present study was undertaken to determine the effects of leukemogenic NUP98 fusion proteins on CRM1-mediated nuclear export. NUP98-HOXA9, a prototypic NUP98 fusion, inhibited the nuclear export of two known CRM1 substrates: mutated cytoplasmic nucleophosmin and HIV-1 Rev. In vitro binding assays revealed that NUP98-HOXA9 binds CRM1 through the FG repeat motif in a Ran-GTP-dependent manner similar to but stronger than the interaction between CRM1 and its export substrates. Two NUP98 fusions, NUP98-HOXA9 and NUP98-DDX10, whose fusion partners are structurally and functionally unrelated, interacted with endogenous CRM1 in myeloid cells as shown by co-immunoprecipitation. These leukemogenic NUP98 fusion proteins interacted with CRM1, Ran, and the nucleoporin NUP214 in a manner fundamentally different from that of wild-type NUP98. NUP98-HOXA9 and NUP98-DDX10 formed characteristic aggregates within the nuclei of a myeloid cell line and primary human CD34+ cells and caused aberrant localization of CRM1 to these aggregates. These NUP98 fusions caused nuclear accumulation of two transcription factors, NFAT and NFkappaB, that are regulated by CRM1-mediated export. The nuclear entrapment of NFAT and NFkappaB correlated with enhanced transcription from promoters responsive to these transcription factors. Taken together, the results suggest a new mechanism by which NUP98 fusions dysregulate transcription and cause leukemia, namely, inhibition of CRM1-mediated nuclear export with aberrant nuclear retention of transcriptional regulators.

  19. Nuclear DISC1 regulates CRE-mediated gene transcription and sleep homeostasis in the fruit fly

    PubMed Central

    Sawamura, Naoya; Ando, Tetsuya; Maruyama, Yasushi; Fujimuro, Masahiro; Mochizuki, Hiroaki; Honjo, Ken; Shimoda, Masami; Toda, Hirofumi; Sawamura-Yamamoto, Takako; Makuch, Lauren A; Hayashi, Akiko; Ishizuka, Koko; Cascella, Nicola G.; Kamiya, Atsushi; Ishida, Norio; Tomoda, Toshifumi; Hai, Tsonwin; Furukubo-Tokunaga, Katsuo; Sawa, Akira

    2009-01-01

    Disrupted-In-Schizophrenia-1 (DISC1) is one of major susceptibility factors for a wide range of mental illnesses, including schizophrenia, bipolar disorder, major depression, and autism spectrum conditions. DISC1 is located in several subcellular domains, such as the centrosome and the nucleus, and interacts with various proteins, including NudE-like (NUDEL/NDEL1) and activating transcription factor 4 (ATF4)/CREB2. Nevertheless, a role for DISC1 in vivo remains to be elucidated. Therefore, we have generated a Drosophila model for examining normal functions of DISC1 in living organisms. DISC1 transgenic flies with preferential accumulation of exogenous human DISC1 in the nucleus display disturbance in sleep homeostasis, which has been reportedly associated with CREB signaling/CRE-mediated gene transcription. Thus, in mammalian cells, we characterized nuclear DISC1, and identified a subset of nuclear DISC1 that co-localizes with the promyelocytic leukemia (PML) bodies, a nuclear compartment for gene transcription. Furthermore, we identified three functional cis-elements that regulate the nuclear localization of DISC1. We also report that DISC1 interacts with ATF4/CREB2 and a co-repressor N-CoR, modulating CRE-mediated gene transcription. PMID:18762802

  20. Nuclear DISC1 regulates CRE-mediated gene transcription and sleep homeostasis in the fruit fly.

    PubMed

    Sawamura, N; Ando, T; Maruyama, Y; Fujimuro, M; Mochizuki, H; Honjo, K; Shimoda, M; Toda, H; Sawamura-Yamamoto, T; Makuch, L A; Hayashi, A; Ishizuka, K; Cascella, N G; Kamiya, A; Ishida, N; Tomoda, T; Hai, T; Furukubo-Tokunaga, K; Sawa, A

    2008-12-01

    Disrupted-in-schizophrenia-1 (DISC1) is one of major susceptibility factors for a wide range of mental illnesses, including schizophrenia, bipolar disorder, major depression and autism spectrum conditions. DISC1 is located in several subcellular domains, such as the centrosome and the nucleus, and interacts with various proteins, including NudE-like (NUDEL/NDEL1) and activating transcription factor 4 (ATF4)/CREB2. Nevertheless, a role for DISC1 in vivo remains to be elucidated. Therefore, we have generated a Drosophila model for examining normal functions of DISC1 in living organisms. DISC1 transgenic flies with preferential accumulation of exogenous human DISC1 in the nucleus display disturbance in sleep homeostasis, which has been reportedly associated with CREB signaling/CRE-mediated gene transcription. Thus, in mammalian cells, we characterized nuclear DISC1, and identified a subset of nuclear DISC1 that colocalizes with the promyelocytic leukemia (PML) bodies, a nuclear compartment for gene transcription. Furthermore, we identified three functional cis-elements that regulate the nuclear localization of DISC1. We also report that DISC1 interacts with ATF4/CREB2 and a corepressor N-CoR, modulating CRE-mediated gene transcription.

  1. Simultaneous live imaging of the transcription and nuclear position of specific genes

    PubMed Central

    Ochiai, Hiroshi; Sugawara, Takeshi; Yamamoto, Takashi

    2015-01-01

    The relationship between genome organization and gene expression has recently been established. However, the relationships between spatial organization, dynamics, and transcriptional regulation of the genome remain unknown. In this study, we developed a live-imaging method for simultaneous measurements of the transcriptional activity and nuclear position of endogenous genes, which we termed the ‘Real-time Observation of Localization and EXpression (ROLEX)’ system. We demonstrated that ROLEX is highly specific and does not affect the expression level of the target gene. ROLEX enabled detection of sub-genome-wide mobility changes that depended on the state of Nanog transactivation in embryonic stem cells. We believe that the ROLEX system will become a powerful tool for exploring the relationship between transcription and nuclear dynamics in living cells. PMID:26092696

  2. Role of zinc finger structure in nuclear localization of transcription factor Sp1

    SciTech Connect

    Ito, Tatsuo; Azumano, Makiko; Uwatoko, Chisana; Itoh, Kohji Kuwahara, Jun

    2009-02-27

    Transcription factor Sp1 is localized in the nucleus and regulates gene expression. Our previous study demonstrated that the carboxyl terminal region of Sp1 containing 3-zinc finger region as DNA binding domain can also serve as nuclear localization signal (NLS). However, the nuclear transport mechanism of Sp1 has not been well understood. In this study, we performed a gene expression study on mutant Sp1 genes causing a set of amino acid substitutions in zinc finger domains to elucidate nuclear import activity. Nuclear localization of the GFP-fused mutant Sp1 proteins bearing concomitant substitutions in the first and third zinc fingers was highly inhibited. These mutant Sp1 proteins had also lost the binding ability as to the GC box sequence. The results suggest that the overall tertiary structure formed by the three zinc fingers is essential for nuclear localization of Sp1 as well as dispersed basic amino acids within the zinc fingers region.

  3. Feed-forward transcriptional programming by nuclear receptors: regulatory principles and therapeutic implications.

    PubMed

    Sasse, Sarah K; Gerber, Anthony N

    2015-01-01

    Nuclear receptors (NRs) are widely targeted to treat a range of human diseases. Feed-forward loops are an ancient mechanism through which single cell organisms organize transcriptional programming and modulate gene expression dynamics, but they have not been systematically studied as a regulatory paradigm for NR-mediated transcriptional responses. Here, we provide an overview of the basic properties of feed-forward loops as predicted by mathematical models and validated experimentally in single cell organisms. We review existing evidence implicating feed-forward loops as important in controlling clinically relevant transcriptional responses to estrogens, progestins, and glucocorticoids, among other NR ligands. We propose that feed-forward transcriptional circuits are a major mechanism through which NRs integrate signals, exert temporal control over gene regulation, and compartmentalize client transcriptomes into discrete subunits. Implications for the design and function of novel selective NR ligands are discussed.

  4. Mitochondrial-Nuclear Interactions Mediate Sex-Specific Transcriptional Profiles in Drosophila

    PubMed Central

    Mossman, Jim A.; Tross, Jennifer G.; Li, Nan; Wu, Zhijin; Rand, David M.

    2016-01-01

    The assembly and function of mitochondria require coordinated expression from two distinct genomes, the mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Mutations in either genome can be a source of phenotypic variation, yet their coexpression has been largely overlooked as a source of variation, particularly in the emerging paradigm of mitochondrial replacement therapy. Here we tested how the transcriptome responds to mtDNA and nDNA variation, along with mitonuclear interactions (mtDNA × nDNA) in Drosophila melanogaster. We used two mtDNA haplotypes that differ in a substantial number of single nucleotide polymorphisms, with >100 amino acid differences. We placed each haplotype on each of two D. melanogaster nuclear backgrounds and tested for transcription differences in both sexes. We found that large numbers of transcripts were differentially expressed between nuclear backgrounds, and that mtDNA type altered the expression of nDNA genes, suggesting a retrograde, trans effect of mitochondrial genotype. Females were generally more sensitive to genetic perturbation than males, and males demonstrated an asymmetrical effect of mtDNA in each nuclear background; mtDNA effects were nuclear-background specific. mtDNA-sensitive genes were not enriched in male- or female-limited expression space in either sex. Using a variety of differential expression analyses, we show the responses to mitonuclear covariation to be substantially different between the sexes, yet the mtDNA genes were consistently differentially expressed across nuclear backgrounds and sexes. Our results provide evidence that the main mtDNA effects can be consistent across nuclear backgrounds, but the interactions between mtDNA and nDNA can lead to sex-specific global transcript responses. PMID:27558138

  5. Quantification of nascent transcription by bromouridine immunocapture nuclear run-on RT-qPCR

    PubMed Central

    Roberts, Thomas C.; Hart, Jonathan R.; Kaikkonen, Minna U.; Weinberg, Marc S.; Vogt, Peter K.; Morris, Kevin V.

    2016-01-01

    Nuclear run-on (NRO) is a method that measures transcriptional activity via the quantification of biochemically labelled nascent RNA molecules derived from nuclear isolates. Widespread usage of this technique has been limited due to its technical difficulty relative to steady-state total mRNA analyses. Here we describe in detail a highly optimised and validated protocol for the quantification of transcriptional activity in human cell cultures using a modified bromouridine immunocapture NRO method and Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR). This method is rapid (the protocol can be completed in two days), cost-effective, exhibits negligible detection of background noise from unlabelled transcripts, requires no radioactive materials, and can be performed from as little as 500,000 nuclei. It also takes advantage of the high sensitivity, specificity and dynamic range of RT-qPCR. Protocol-specific issues such as appropriate qPCR assay design, data normalisation, changes in nuclear RNA content between experimental conditions, unequal label incorporation, and background signal determination are also discussed. PMID:26182239

  6. Post transcriptional regulation of chloroplast gene expression by nuclear encoded gene products

    SciTech Connect

    Kuchka, M.R.

    1992-01-01

    Many individual chloroplast genes require the products of a collection of nuclear genes for their successful expression. These nuclear gene products apparently work with great specificity, each committed to the expression of a single chloroplast gene. We have chosen as a model nuclear mutants of Chlamydomonas affected in different stages in the expression of the chloroplast encoded Photosystem II polypeptide, D2. We have made the progress in understanding how nuclear gene products affect the translation of the D2 encoding MRNA. Two nuclear genes are required for this process which have been mapped genetically. In contrast to other examples of nuclear control of translation in the chloroplast, these nuclear gene products appear to be required either for specific stages in translation elongation or for the post-translational stabilization of the nascent D2 protein. Pseudoreversion analysis has led us to a locus which may be directly involved in D2 expression. We have made considerable progress in pursuing the molecular basis of psbd MRNA stabilization. psbD 5' UTR specific transcripts have been synthesized in vitro and used in gel mobility shift assays. UV-crosslinking studies are underway to identify the transacting factors which bind to these sequences. The continued examination of these mutants will help us to understand how nuclear gene products work in this specific case of chloroplast gene expression, and will elucidate how two distinct genomes can interact generally.

  7. Assembly of the Nuclear Transcription and Processing Machinery: Cajal Bodies (Coiled Bodies) and Transcriptosomes

    PubMed Central

    Gall, Joseph G.; Bellini, Michel; Wu, Zheng’an; Murphy, Christine

    1999-01-01

    We have examined the distribution of RNA transcription and processing factors in the amphibian oocyte nucleus or germinal vesicle. RNA polymerase I (pol I), pol II, and pol III occur in the Cajal bodies (coiled bodies) along with various components required for transcription and processing of the three classes of nuclear transcripts: mRNA, rRNA, and pol III transcripts. Among these components are transcription factor IIF (TFIIF), TFIIS, splicing factors, the U7 small nuclear ribonucleoprotein particle, the stem–loop binding protein, SR proteins, cleavage and polyadenylation factors, small nucleolar RNAs, nucleolar proteins that are probably involved in pre-rRNA processing, and TFIIIA. Earlier studies and data presented here show that several of these components are first targeted to Cajal bodies when injected into the oocyte and only subsequently appear in the chromosomes or nucleoli, where transcription itself occurs. We suggest that pol I, pol II, and pol III transcription and processing components are preassembled in Cajal bodies before transport to the chromosomes and nucleoli. Most components of the pol II transcription and processing pathway that occur in Cajal bodies are also found in the many hundreds of B-snurposomes in the germinal vesicle. Electron microscopic images show that B-snurposomes consist primarily, if not exclusively, of 20- to 30-nm particles, which closely resemble the interchromatin granules described from sections of somatic nuclei. We suggest the name pol II transcriptosome for these particles to emphasize their content of factors involved in synthesis and processing of mRNA transcripts. We present a model in which pol I, pol II, and pol III transcriptosomes are assembled in the Cajal bodies before export to the nucleolus (pol I), to the B-snurposomes and eventually to the chromosomes (pol II), and directly to the chromosomes (pol III). The key feature of this model is the preassembly of the transcription and processing machinery into

  8. Urban renewal in the nucleus: is protein turnover by proteasomes absolutely required for nuclear receptor-regulated transcription?

    PubMed

    Nawaz, Zafar; O'Malley, Bert W

    2004-03-01

    The importance of the ubiquitin proteasome pathway in higher eukaryotes has been well established in cell cycle regulation, signal transduction, and cell differentiation, but has only recently been linked to nuclear hormone receptor-regulated gene transcription. Characterization of a number of ubiquitin proteasome pathway enzymes as coactivators and observations that several nuclear receptors are ubiquitinated and degraded in the course of their nuclear activities provide evidence that ubiquitin proteasome-mediated protein degradation plays an integral role in eukaryotic transcription. In addition to receptors, studies have revealed that coactivators are ubiquitinated and degraded via the proteasome. The notion that the ubiquitin proteasome pathway is involved in gene transcription is further strengthened by the fact that ubiquitin proteasome pathway enzymes are recruited to the promoters of target genes and that proteasome-dependent degradation of nuclear receptors is required for efficient transcriptional activity. These findings suggest that protein degradation is coupled with nuclear receptor coactivation activity. It is possible that the ubiquitin proteasome pathway modulates transcription by promoting remodeling and turnover of the nuclear receptor-transcription complex. In this review, we discus the possible role of the ubiquitin proteasome pathway in nuclear hormone receptor-regulated gene transcription.

  9. A novel bipartite nuclear localization signal with an atypically long linker in DOF transcription factors.

    PubMed

    Krebs, Jonas; Mueller-Roeber, Bernd; Ruzicic, Slobodan

    2010-05-01

    Large molecules require a nuclear localization signal (NLS) for translocation into the nucleus. Classical NLSs are rich in basic amino acids and they represent three groups, based on their structural features: SV40 T-antigen-type, yeast mating factor Matalpha-2-type, and bipartite NLSs. DNA-binding-with-one-finger (DOF) transcription factors play important roles in plants, and although their nuclear localization has been demonstrated in several cases, public protein localization prediction tools fail to detect NLS motifs in these proteins. Here, we demonstrate that an atypical bipartite NLS with a 17 amino acid long linker between its flanking basic regions directs Arabidopsis thaliana DOF proteins to the cell nucleus. The novel bipartite NLS is highly conserved in plant DOF transcription factors, including the single DOF protein in the green alga Chlamydomonas reinhardtii.

  10. Nuclear rRNA transcript processing versus internal transcribed spacer secondary structure.

    PubMed

    Coleman, Annette W

    2015-03-01

    rRNA is one of the few universal features of life, making it uniquely suited to assess phylogenetic relationships. The processing of the initial polycistronic rRNA transcript is also a conserved process, involving numerous cleavage events and the generation of secondary structures. The secondary structure of the internal transcribed spacer (ITS) regions of nuclear rRNA transcripts are well known for a wide variety of eukaryotes and have been used to aid in the alignment of these sequences for phylogenetic comparisons. By contrast, study of the processing of the initial rRNA transcripts has been largely limited to yeast, mice, rats, and humans. Here I examine the known cleavage sites in the two ITS regions and their positions relative to the secondary structure. A better understanding of the conservation of secondary structures and cleavage sites within the ITS regions will improve evolutionary inferences based on these sequences.

  11. Transcription-dependent nuclear localization of DAZAP1 requires an N-terminal signal

    SciTech Connect

    Lin, Yi-Tzu; Wen, Wan-Ching; Yen, Pauline H.

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer DAZAP1 shuttles between the nucleus and the cytoplasm. Black-Right-Pointing-Pointer DAZAP1 accumulates in the cytoplasm when the nuclear transcription is inhibited. Black-Right-Pointing-Pointer DAZAP1's transcription-dependent nuclear localization requires N-terminal N42. Black-Right-Pointing-Pointer SLIRP binds to N42 and may be involved in the process. -- Abstract: Deleted in Azoospermia Associated Protein 1 (DAZAP1) is a ubiquitous hnRNP protein required for normal development and spermatogenesis. It resides predominantly in the nucleus and moves between the nucleus and the cytoplasm via a ZNS shuttling signal at its C-terminus. DAZAP1 accumulates in the cytoplasm when RNA polymerase II activity is inhibited by actinomycin D. Here we report the mapping of a 42-amino acid segment (N42) at the N-terminus of DAZAP1 that is both necessary and sufficient for its transcription-dependent nuclear localization. In addition, using a yeast two-hybrid system, we have identified SLIRP as a N42-binding protein which may regulate DAZAP1 subcellular localization.

  12. Dynamic sorting of nuclear components into distinct nucleolar caps during transcriptional inhibition.

    PubMed

    Shav-Tal, Yaron; Blechman, Janna; Darzacq, Xavier; Montagna, Cristina; Dye, Billy T; Patton, James G; Singer, Robert H; Zipori, Dov

    2005-05-01

    Nucleolar segregation is observed under some physiological conditions of transcriptional arrest. This process can be mimicked by transcriptional arrest after actinomycin D treatment leading to the segregation of nucleolar components and the formation of unique structures termed nucleolar caps surrounding a central body. These nucleolar caps have been proposed to arise from the segregation of nucleolar components. We show that contrary to prevailing notion, a group of nucleoplasmic proteins, mostly RNA binding proteins, relocalized from the nucleoplasm to a specific nucleolar cap during transcriptional inhibition. For instance, an exclusively nucleoplasmic protein, the splicing factor PSF, localized to nucleolar caps under these conditions. This structure also contained pre-rRNA transcripts, but other caps contained either nucleolar proteins, PML, or Cajal body proteins and in addition nucleolar or Cajal body RNAs. In contrast to the capping of the nucleoplasmic components, nucleolar granular component proteins dispersed into the nucleoplasm, although at least two (p14/ARF and MRP RNA) were retained in the central body. The nucleolar caps are dynamic structures as determined using photobleaching and require energy for their formation. These findings demonstrate that the process of nucleolar segregation and capping involves energy-dependent repositioning of nuclear proteins and RNAs and emphasize the dynamic characteristics of nuclear domain formation in response to cellular stress.

  13. Phosphatidylinositol 3-kinase/Akt signaling enhances nuclear localization and transcriptional activity of BRCA1

    SciTech Connect

    Hinton, Cimona V.; Fitzgerald, Latricia D.; Thompson, Marilyn E. . E-mail: methompson@mmc.edu

    2007-05-15

    Signaling pathways involved in regulating nuclear-cytoplasmic distribution of BRCA1 have not been previously reported. Here, we provide evidence that heregulin {beta}1-induced activation of the Akt pathway increases the nuclear content of BRCA1. First, treatment of T47D breast cancer cells with heregulin {beta}1 results in a two-fold increase in nuclear BRCA1 as assessed by FACS analysis, immunoblotting and immunofluorescence. This heregulin-induced increase in nuclear BRCA1 is blocked by siRNA-mediated down-regulation of Akt. Second, mutation of threonine 509 in BRCA1, the site of Akt phosphorylation, to an alanine, attenuates the ability of heregulin to induce BRCA1 nuclear accumulation. These data suggest that Akt-catalyzed phosphorylation of BRCA1 is required for the heregulin-regulated nuclear concentration of BRCA1. Because most functions ascribed to BRCA1 occur within the nucleus, we postulated that phosphorylation-dependent nuclear accumulation of BRCA1 would result in enhanced nuclear activity, specifically transcriptional activity, of BRCA1. This postulate is affirmed by our observation that the ability of BRCA1 to transactivate GADD45 promoter constructs was enhanced in T47D cells treated with heregulin {beta}1. Furthermore, the heterologous expression of BRCA1 in HCC1937 human breast cancer cells, which have constitutively active Akt, also induces GADD45 promoter activity, whereas the expression of BRCA1 in which threonine 509 has been mutated to an alanine is able to only minimally induce promoter activity. These findings implicate Akt in upstream events leading to BRCA1 nuclear localization and function.

  14. O-GlcNAc-glycosylation of {beta}-catenin regulates its nuclear localization and transcriptional activity

    SciTech Connect

    Sayat, Ria; Leber, Brian; Grubac, Vanja; Wiltshire, Lesley; Persad, Sujata

    2008-09-10

    {beta}-catenin plays a role in intracellular adhesion and regulating gene expression. The latter role is associated with its oncogenic properties. Phosphorylation of {beta}-catenin controls its intracellular expression but mechanism/s that regulates the nuclear localization of {beta}-catenin is unknown. We demonstrate that O-GlcNAc glycosylation (O-GlcNAcylation) of {beta}-catenin negatively regulates its levels in the nucleus. We show that normal prostate cells (PNT1A) have significantly higher amounts of O-GlcNAcylated {beta}-catenin compared to prostate cancer (CaP) cells. The total nuclear levels of {beta}-catenin are higher in the CaP cells than PNT1A but only a minimal fraction of the nuclear {beta}-catenin in the CaP cells are O-GlcNAcylated. Increasing the levels of O-GlcNAcylated {beta}-catenin in the CaP cells with PUGNAc (O- (2-acetamido-2-deoxy-D-gluco-pyranosylidene) amino-N-phenylcarbamate) treatment is associated with a progressive decrease in the levels of {beta}-catenin in the nucleus. TOPFlash reporter assay and mRNA expressions of {beta}-catenin's target genes indicate that O-GlcNAcylation of {beta}-catenin results in a decrease in its transcriptional activity. We define a novel modification of {beta}-catenin that regulates its nuclear localization and transcriptional function.

  15. Direct visualization of the co-transcriptional assembly of a nuclear body by noncoding RNAs.

    PubMed

    Mao, Yuntao S; Sunwoo, Hongjae; Zhang, Bin; Spector, David L

    2011-01-01

    The cell nucleus is a highly compartmentalized organelle harbouring a variety of dynamic membraneless nuclear bodies. How these subnuclear domains are established and maintained is not well understood. Here, we investigate the molecular mechanism of how one nuclear body, the paraspeckle, is assembled and organized. Paraspeckles are discrete ribonucleoprotein bodies found in mammalian cells and implicated in nuclear retention of hyperedited mRNAs. We developed a live-cell imaging system that allows for the inducible transcription of Men ɛ/β (also known as Neat1; ref. 12) noncoding RNAs (ncRNAs) and the direct visualization of the recruitment of paraspeckle proteins. Using this system, we demonstrate that Men ɛ/β ncRNAs are essential to initiate the de novo assembly of paraspeckles. These newly formed structures effectively harbour nuclear-retained mRNAs confirming that they are bona fide functional paraspeckles. By three independent approaches, we show that it is the act of Men ɛ/β transcription, but not ncRNAs alone, that regulates paraspeckle maintenance. Finally, fluorescence recovery after photobleaching (FRAP) analyses supported a critical structural role for Men ɛ/β ncRNAs in paraspeckle organization. This study establishes a model in which Men ɛ/β ncRNAs serve as a platform to recruit proteins to assemble paraspeckles.

  16. Drosophila SAF-B Links the Nuclear Matrix, Chromosomes, and Transcriptional Activity

    PubMed Central

    Alfonso-Parra, Catalina; Maggert, Keith A.

    2010-01-01

    Induction of gene expression is correlated with alterations in nuclear organization, including proximity to other active genes, to the nuclear cortex, and to cytologically distinct domains of the nucleus. Chromosomes are tethered to the insoluble nuclear scaffold/matrix through interaction with Scaffold/Matrix Attachment Region (SAR/MAR) binding proteins. Identification and characterization of proteins involved in establishing or maintaining chromosome-scaffold interactions is necessary to understand how the nucleus is organized and how dynamic changes in attachment are correlated with alterations in gene expression. We identified and characterized one such scaffold attachment factor, a Drosophila homolog of mammalian SAF-B. The large nuclei and chromosomes of Drosophila have allowed us to show that SAF-B inhabits distinct subnuclear compartments, forms weblike continua in nuclei of salivary glands, and interacts with discrete chromosomal loci in interphase nuclei. These interactions appear mediated either by DNA-protein interactions, or through RNA-protein interactions that can be altered during changes in gene expression programs. Extraction of soluble nuclear proteins and DNA leaves SAF-B intact, showing that this scaffold/matrix-attachment protein is a durable component of the nuclear matrix. Together, we have shown that SAF-B links the nuclear scaffold, chromosomes, and transcriptional activity. PMID:20422039

  17. Transcriptional regulation is affected by subnuclear targeting of reporter plasmids to PML nuclear bodies.

    PubMed

    Block, Gregory J; Eskiw, Christopher H; Dellaire, Graham; Bazett-Jones, David P

    2006-12-01

    Whereas the PML protein has been reported to have both transcriptional coactivator and corepressor potential, the contribution of the PML nuclear body (PML NB) itself to transcriptional regulation is not well understood. Here we demonstrate that plasmid DNA artificially tethered to PML or the PML NB-targeting domain of Sp100 is preferentially localized to PML NBs. Using the tethering technique, we targeted a simian virus 40 promoter-driven luciferase reporter plasmid to PML NBs, resulting in the repression of the transgene transcriptional activity. Conversely, the tethering of a cytomegalovirus promoter-containing reporter plasmid resulted in activation. Targeting a minimal eukaryotic promoter did not affect its activity. The expression of targeted promoters could be modulated by altering the cellular concentration of PML NB components, including Sp100 and isoforms of the PML protein. Finally, we demonstrate that ICP0, the promiscuous herpes simplex virus transactivator, increases the level of transcriptional activation of plasmid DNA tethered to the PML NB. We conclude that when PML NB components are artificially tethered to reporter plasmids, the PML NB contributes to the regulation of the tethered DNA in a promoter-dependent manner. Our findings demonstrate that transient transcription assays are sensitive to the subnuclear localization of the transgene plasmid.

  18. Nuclear mRNA quality control in yeast is mediated by Nrd1 co-transcriptional recruitment, as revealed by the targeting of Rho-induced aberrant transcripts

    PubMed Central

    Honorine, Romy; Mosrin-Huaman, Christine; Hervouet-Coste, Nadège; Libri, Domenico; Rahmouni, A. Rachid

    2011-01-01

    The production of mature export-competent transcripts is under the surveillance of quality control steps where aberrant mRNP molecules resulting from inappropriate or inefficient processing and packaging reactions are subject to exosome-mediated degradation. Previously, we have shown that the heterologous expression of bacterial Rho factor in yeast interferes in normal mRNP biogenesis leading to the production of full-length yet aberrant transcripts that are degraded by the nuclear exosome with ensuing growth defect. Here, we took advantage of this new tool to investigate the molecular mechanisms by which an integrated system recognizes aberrancies at each step of mRNP biogenesis and targets the defective molecules for destruction. We show that the targeting and degradation of Rho-induced aberrant transcripts is associated with a large increase of Nrd1 recruitment to the transcription complex via its CID and RRM domains and a concomitant enrichment of exosome component Rrp6 association. The targeting and degradation of the aberrant transcripts is suppressed by the overproduction of Pcf11 or its isolated CID domain, through a competition with Nrd1 for recruitment by the transcription complex. Altogether, our results support a model in which a stimulation of Nrd1 co-transcriptional recruitment coordinates the recognition and removal of aberrant transcripts by promoting the attachment of the nuclear mRNA degradation machinery. PMID:21113025

  19. An active nuclear retention signal in the glucocorticoid receptor functions as a strong inducer of transcriptional activation.

    PubMed

    Carrigan, Amanda; Walther, Rhian F; Salem, Houssein Abdou; Wu, Dongmei; Atlas, Ella; Lefebvre, Yvonne A; Haché, Robert J G

    2007-04-13

    The glucocorticoid receptor (GR) cycles between a naive chaperone-complexed form in the cytoplasm and a transcriptionally active steroid-bound nuclear form. Nuclear import of GR occurs rapidly and is mediated through the importin alpha/beta karyopherin import pathway. By contrast, nuclear export of GR occurs only slowly under most conditions, despite a dependence on active signaling. In this study we have defined a nuclear retention signal (NRS) in the hinge region of GR that actively opposes the nuclear export of GR as well as the nuclear export mediated through an ectopic CRM1-dependent nuclear export signal (NES). The GR NRS overlaps closely with the basic NL1 nuclear localization signal (NLS) but can be distinguished from NL1 by targeted mutagenesis. Substitution of the classical NLS from SV40 T antigen for the GR NL1 results in a receptor in which nuclear export is accelerated. Remarkably, although the SV40-modified GR remains predominantly nuclear in the presence of steroid and is recruited to transcriptional regulatory regions indistinguishably from wild-type GR, the substitution dramatically weakens the ability of GR to activate transcription of a mouse mammary tumor virus reporter gene. These results suggest that active nuclear retention of GR plays an integral role in glucocorticoid signaling.

  20. Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor Hepatocyte nuclear factor 4 alpha.

    PubMed

    Davison, James M; Lickwar, Colin R; Song, Lingyun; Breton, Ghislain; Crawford, Gregory E; Rawls, John F

    2017-04-06

    Microbiota influence diverse aspects of intestinal physiology and disease in part by controlling tissue-specific transcription of host genes. However, host genomic mechanisms mediating microbial control of intestinal gene expression are poorly understood. Hepatocyte nuclear factor 4 (HNF4) is the most ancient family of nuclear receptor transcription factors with important roles in human metabolic and inflammatory bowel diseases, but a role in host response to microbes is unknown. Using an unbiased screening strategy, we found that zebrafish Hnf4a specifically binds and activates a microbiota-suppressed intestinal epithelial transcriptional enhancer. Genetic analysis revealed that zebrafish hnf4a activates nearly half of the genes that are suppressed by microbiota, suggesting microbiota negatively regulate Hnf4a. In support, analysis of genomic architecture in mouse intestinal epithelial cells disclosed that microbiota colonization leads to activation or inactivation of hundreds of enhancers along with drastic genome-wide reduction of HNF4A and HNF4G occupancy. Interspecies meta-analysis suggested interactions between HNF4A and microbiota promote gene expression patterns associated with human inflammatory bowel diseases. These results indicate a critical and conserved role for HNF4A in maintaining intestinal homeostasis in response to microbiota.

  1. Nuclear actin depolymerization in transcriptionally active avian and amphibian oocytes leads to collapse of intranuclear structures

    PubMed Central

    Maslova, Antonina; Krasikova, Alla

    2012-01-01

    Actin, which is normally depleted in the nuclei of somatic cells, accumulates in high amounts in giant nuclei of amphibian oocytes. The supramolecular organization and functions of this nuclear pool of actin in growing vertebrate oocyte are controversial. Here, we investigated the role of nuclear actin in the maintenance of the spatial architecture of intranuclear structures in avian and amphibian growing oocytes. A meshwork of filamentous actin was not detected in freshly isolated or fixed oocyte nuclei of Xenopus, chicken or quail. We found that the actin meshwork inside the oocyte nucleus could be induced by phalloidin treatment. Actin polymerization is demonstrated to be required to stabilize the specific spatial organization of nuclear structures in avian and amphibian growing oocytes. In experiments with the actin depolymerizing drugs cytochalasin D and latrunculin A, we showed that disassembly of nuclear actin polymers led to chromosome condensation and their transportation to a limited space within the oocyte nucleus. Experimentally induced “collapsing” of chromosomes and nuclear bodies, together with global inhibition of transcription, strongly resembled the process of karyosphere formation during oocyte growth. PMID:22572951

  2. Post transcriptional regulation of chloroplast gene expression by nuclear encoded gene products

    SciTech Connect

    Kuchka, M.R.

    1992-01-01

    The following is a review of research accomplished in the first two years of funding for the above mentioned project. The work performed is a molecular characterization of nuclear mutants of Chlamydomonas reinhardtii which are deficient in different stages in the post-transcriptional expression of a single chloroplast encoded polypeptide, the D2 protein of Photosystem II. Our long-term goals are to understand the molecular mechanisms by which nuclear gene products affect the expression of chloroplast genes. Specifically, we which to understand how specific nuclear gene products affect the turnover rate of the D2 encoding mRNA (psbD), how other nuclear encoded factors work to promote the translation of psbD mRNA and/or stabilize the D2 protein, and what the role of the D2 protein itself is in Photosystem II assembly and in the control of expression of other chloroplast genes. This progress report will be organized into four major sections concerning (I) The characterization of nuclear mutants affected in D2 translation/turnover, (II) The study of trans-acting factors which associate with the 5{prime} end of the psbD mRNA, (III) In vitro mutagenesis of the psbD gene, and (IV) Additional studies.

  3. Zinc Coordination Is Required for and Regulates Transcription Activation by Epstein-Barr Nuclear Antigen 1

    PubMed Central

    Aras, Siddhesh; Singh, Gyanendra; Johnston, Kenneth; Foster, Timothy; Aiyar, Ashok

    2009-01-01

    Epstein-Barr Nuclear Antigen 1 (EBNA1) is essential for Epstein-Barr virus to immortalize naïve B-cells. Upon binding a cluster of 20 cognate binding-sites termed the family of repeats, EBNA1 transactivates promoters for EBV genes that are required for immortalization. A small domain, termed UR1, that is 25 amino-acids in length, has been identified previously as essential for EBNA1 to activate transcription. In this study, we have elucidated how UR1 contributes to EBNA1's ability to transactivate. We show that zinc is necessary for EBNA1 to activate transcription, and that UR1 coordinates zinc through a pair of essential cysteines contained within it. UR1 dimerizes upon coordinating zinc, indicating that EBNA1 contains a second dimerization interface in its amino-terminus. There is a strong correlation between UR1-mediated dimerization and EBNA1's ability to transactivate cooperatively. Point mutants of EBNA1 that disrupt zinc coordination also prevent self-association, and do not activate transcription cooperatively. Further, we demonstrate that UR1 acts as a molecular sensor that regulates the ability of EBNA1 to activate transcription in response to changes in redox and oxygen partial pressure (pO2). Mild oxidative stress mimicking such environmental changes decreases EBNA1-dependent transcription in a lymphoblastoid cell-line. Coincident with a reduction in EBNA1-dependent transcription, reductions are observed in EBNA2 and LMP1 protein levels. Although these changes do not affect LCL survival, treated cells accumulate in G0/G1. These findings are discussed in the context of EBV latency in body compartments that differ strikingly in their pO2 and redox potential. PMID:19521517

  4. Nuclear transcription factor Nrf2 suppresses prostate cancer cells growth and migration through upregulating ferroportin.

    PubMed

    Xue, Dong; Zhou, Cuixing; Shi, Yunbo; Lu, Hao; Xu, Renfang; He, Xiaozhou

    2016-11-29

    VTo investigate the effect of nuclear transcription factor Nrf2 on the transcription of Ferroportin (FPN) in prostate cancer cells, and the regulation mechanisms of FPN on cell viability, migration and apoptosis of prostate cancer cells.Empty vectors, pEGFPC1-Nrf2, pEGFPC1-FPN, Si-FPN and Si-Nrf2 were transfected into prostate cancer cell line PC3. The expression of mRNA and protein were measured by real time-PCR (RT-PCR) and western blot. Cell viability, migration, cycle and apoptosis were tested by CCK-8 assay, wound healing and flow cytometry, respectively. The interaction between FPN and Nrf2 was confirmed by chromatin immunoprecipitation (CHIP) assay.The viability, migration and mitosis of PC3 cells could be repressed by over-expressed FPN, with decreased intracellular ferritin. The CHIP assay demonstrated that Nrf2 is one transcription factor of FPN and promotes its transcription. With the increase of Nrf2 in PC3 cells, the viability, migration ability and concentration of ferritin were suppressed, while the apoptosis rate was increased. The above effects were counteracted by down-regulating FPN.FPN could inhibit the prostate cancer cell viability, migration and mitosis, which is also related to a decrease of intracellular ferritin content. In conclusion, Nrf2 suppresses prostate cancer cells viability, migration, and mitosis through upregulating FPN.

  5. Nuclear transcription factor Nrf2 suppresses prostate cancer cells growth and migration through upregulating ferroportin

    PubMed Central

    Xue, Dong; Zhou, Cuixing; Shi, Yunbo; Lu, Hao; Xu, Renfang; He, Xiaozhou

    2016-01-01

    VTo investigate the effect of nuclear transcription factor Nrf2 on the transcription of Ferroportin (FPN) in prostate cancer cells, and the regulation mechanisms of FPN on cell viability, migration and apoptosis of prostate cancer cells. Empty vectors, pEGFPC1-Nrf2, pEGFPC1-FPN, Si-FPN and Si-Nrf2 were transfected into prostate cancer cell line PC3. The expression of mRNA and protein were measured by real time-PCR (RT-PCR) and western blot. Cell viability, migration, cycle and apoptosis were tested by CCK-8 assay, wound healing and flow cytometry, respectively. The interaction between FPN and Nrf2 was confirmed by chromatin immunoprecipitation (CHIP) assay. The viability, migration and mitosis of PC3 cells could be repressed by over-expressed FPN, with decreased intracellular ferritin. The CHIP assay demonstrated that Nrf2 is one transcription factor of FPN and promotes its transcription. With the increase of Nrf2 in PC3 cells, the viability, migration ability and concentration of ferritin were suppressed, while the apoptosis rate was increased. The above effects were counteracted by down-regulating FPN. FPN could inhibit the prostate cancer cell viability, migration and mitosis, which is also related to a decrease of intracellular ferritin content. In conclusion, Nrf2 suppresses prostate cancer cells viability, migration, and mitosis through upregulating FPN. PMID:27788496

  6. Identifying Novel Transcriptional and Epigenetic Features of Nuclear Lamina-associated Genes.

    PubMed

    Wu, Feinan; Yao, Jie

    2017-12-01

    Because a large portion of the mammalian genome is associated with the nuclear lamina (NL), it is interesting to study how native genes resided there are transcribed and regulated. In this study, we report unique transcriptional and epigenetic features of nearly 3,500 NL-associated genes (NL genes). Promoter regions of active NL genes are often excluded from NL-association, suggesting that NL-promoter interactions may repress transcription. Active NL genes with higher RNA polymerase II (Pol II) recruitment levels tend to display Pol II promoter-proximal pausing, while Pol II recruitment and Pol II pausing are not correlated among non-NL genes. At the genome-wide scale, NL-association and H3K27me3 distinguishes two large gene classes with low transcriptional activities. Notably, NL-association is anti-correlated with both transcription and active histone mark levels among genes not significantly enriched with H3K9me3 or H3K27me3, suggesting that NL-association may represent a novel gene repression pathway. Interestingly, an NL gene subgroup is not significantly enriched with H3K9me3 or H3K27me3 and is transcribed at higher levels than the rest of NL genes. Furthermore, we identified distal enhancers associated with active NL genes and reported their epigenetic features.

  7. Gene loops function to maintain transcriptional memory through interaction with the nuclear pore complex

    PubMed Central

    Tan-Wong, Sue Mei; Wijayatilake, Hashanthi D.; Proudfoot, Nick J.

    2009-01-01

    Inducible genes in yeast retain a “memory” of recent transcriptional activity during periods of short-term repression, allowing them to be reactivated faster when reinduced. This confers a rapid and versatile gene expression response to the environment. We demonstrate that this memory mechanism is associated with gene loop interactions between the promoter and 3′ end of the responsive genes HXK1 and GAL1∷FMP27. The maintenance of these memory gene loops (MGLs) during intervening periods of transcriptional repression is required for faster RNA polymerase II (Pol II) recruitment to the genes upon reinduction, thereby facilitating faster mRNA accumulation. Notably, a sua7-1 mutant or the endogenous INO1 gene that lacks this MGL does not display such faster reinduction. Furthermore, these MGLs interact with the nuclear pore complex through association with myosin-like protein 1 (Mlp1). An mlp1Δ strain does not maintain MGLs, and concomitantly loses transcriptional memory. We predict that gene loop conformations enhance gene expression by facilitating rapid transcriptional response to changing environmental conditions. PMID:19933151

  8. Identification, localization, transcription, and sequence analysis of the Choristoneura fumiferana nuclear polyhedrosis virus DNA polymerase gene.

    PubMed

    Liu, J J; Carstens, E B

    1995-06-01

    The location of the Choristoneura fumiferana baculovirus DNA polymerase gene was determined by hybridization analysis using a probe prepared from the previously identified polymerase gene from the Autographa californica multiple nuclear polyhedrosis virus. DNA sequence analysis revealed that the Choristoneura fumiferana baculovirus DNA polymerase gene consists of 2970 base pairs encoding 990 amino acids (114.2 kDa). Transcriptional analysis demonstrated that overlapping transcripts of 3.2 and 4.6 kb, first detected at 6 hr postinfection, potentially coded for the DNA polymerase gene. The major transcription starts sites, identified at 6 hr postinfection, mapped to baculovirus consensus early start sites CGTGCTCA and CAGT. The relatively low level and late initiation of the DNA polymerase gene coupled with our previous data on the temporal control of DNA replication and late gene synthesis (Liu and Carstens, 1993) suggests that the low virulence of the spruce budworm baculovirus may be related to the regulation of its gene expression at the transcriptional level.

  9. Cell-free production of transducible transcription factors for nuclear reprogramming

    PubMed Central

    Yang, William C.; Patel, Kedar G.; Lee, Jieun; Ghebremariam, Yohannes T.; Wong, H. Edward; Cooke, John P.; Swartz, James R.

    2011-01-01

    Ectopic expression of a defined set of transcription factors chosen from Oct3/4, Sox2, c-Myc, Klf4, Nanog, and Lin28 can directly reprogram somatic cells to pluripotency. These reprogrammed cells are referred to as induced pluripotent stem cells (iPSCs). To date, iPSCs have been successfully generated using lentiviruses, retroviruses, adenoviruses, plasmids, transposons, and recombinant proteins. Nucleic acid-based approaches raise concerns about genomic instability. In contrast, a protein-based approach for iPSC generation can avoid DNA integration concerns as well as provide greater control over the concentration, timing, and sequence of transcription factor stimulation. Researchers recently demonstrated that polyarginine peptide conjugation can deliver recombinant protein reprogramming factor (RF) cargoes into cells and reprogram somatic cells into iPSCs. However, the protein-based approach requires a significant amount of protein for the reprogramming process. Producing fusion reprogramming factors in the large amounts required for this approach using traditional heterologous in vivo production methods is difficult and cumbersome since toxicity, product aggregation, and proteolysis by endogenous proteases limit yields. In this work, we show that cell-free protein synthesis (CFPS) is a viable option for producing soluble and functional transducible transcription factors for nuclear reprogramming. We used an E. coli-based cell-free protein synthesis system to express the above set of six human RFs as fusion proteins, each with a nona-arginine (R9) protein transduction domain. Using the flexibility offered by the CFPS platform, we successfully addressed proteolysis and protein solubility problems to produce full-length and soluble R9-RF fusions. We subsequently showed that R9-Oct3/4, R9-Sox2, and R9-Nanog exhibit cognate DNA binding activities, R9-Nanog translocates across the plasma and nuclear membranes, and R9-Sox2 exerts transcriptional activity on a known

  10. Inositol polyphosphate multikinase (IPMK) in transcriptional regulation and nuclear inositide metabolism

    PubMed Central

    Malabanan, M. Merced; Blind, Raymond D.

    2017-01-01

    Inositol polyphosphate multikinase (IPMK, ipk2, Arg82, ArgRIII) is an inositide kinase with unusually flexible substrate specificity and the capacity to partake in many functional protein–protein interactions (PPIs). By merging these two activities, IPMK is able to execute gene regulatory functions that are very unique and only now beginning to be recognized. In this short review, we present a brief history of IPMK, describe the structural biology of the enzyme and highlight a few recent discoveries that have shed more light on the role IPMK plays in inositide metabolism, nuclear signalling and transcriptional regulation. PMID:26862216

  11. Transcriptional activation of NAD{sup +}-dependent protein deacetylase SIRT1 by nuclear receptor TLX

    SciTech Connect

    Iwahara, Naotoshi; Hisahara, Shin; Hayashi, Takashi; Horio, Yoshiyuki

    2009-09-04

    An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD{sup +}-dependent protein deacetylase, is highly expressed in the NPCs and participates in neurogenesis. Here, we found that TLX colocalized with SIRT1 and knockdown of TLX by small interfering RNAs decreased SIRT1 levels in NPCs. TLX increased the SIRT1 expression by binding to the newly identified TLX-activating element in the SIRT1 gene promoter in HEK293 cells. Thus, TLX is an inducer of SIRT1 and may contribute to neurogenesis both as a transactivator and as a repressor.

  12. Non-nuclear Pool of Splicing Factor SFPQ Regulates Axonal Transcripts Required for Normal Motor Development.

    PubMed

    Thomas-Jinu, Swapna; Gordon, Patricia M; Fielding, Triona; Taylor, Richard; Smith, Bradley N; Snowden, Victoria; Blanc, Eric; Vance, Caroline; Topp, Simon; Wong, Chun-Hao; Bielen, Holger; Williams, Katherine L; McCann, Emily P; Nicholson, Garth A; Pan-Vazquez, Alejandro; Fox, Archa H; Bond, Charles S; Talbot, William S; Blair, Ian P; Shaw, Christopher E; Houart, Corinne

    2017-04-04

    Recent progress revealed the complexity of RNA processing and its association to human disorders. Here, we unveil a new facet of this complexity. Complete loss of function of the ubiquitous splicing factor SFPQ affects zebrafish motoneuron differentiation cell autonomously. In addition to its nuclear localization, the protein unexpectedly localizes to motor axons. The cytosolic version of SFPQ abolishes motor axonal defects, rescuing key transcripts, and restores motility in the paralyzed sfpq null mutants, indicating a non-nuclear processing role in motor axons. Novel variants affecting the conserved coiled-coil domain, so far exclusively found in fALS exomes, specifically affect the ability of SFPQ to localize in axons. They broadly rescue morphology and motility in the zebrafish mutant, but alter motor axon morphology, demonstrating functional requirement for axonal SFPQ. Altogether, we uncover the axonal function of the splicing factor SFPQ in motor development and highlight the importance of the coiled-coil domain in this process.

  13. Agonist ligands mediate the transcriptional response of nuclear receptor heterodimers through distinct stoichiometric assemblies with coactivators.

    PubMed

    Pavlin, Mark Remec; Brunzelle, Joseph S; Fernandez, Elias J

    2014-09-05

    The constitutive androstane (CAR) and retinoid X receptors (RXR) are ligand-mediated transcription factors of the nuclear receptor protein superfamily. Functional CAR:RXR heterodimers recruit coactivator proteins, such as the steroid receptor coactivator-1 (SRC1). Here, we show that agonist ligands can potentiate transactivation through both coactivator binding sites on CAR:RXR, which distinctly bind two SRC1 molecules. We also observe that SRC1 transitions from a structurally plastic to a compact form upon binding CAR:RXR. Using small angle x-ray scattering (SAXS) we show that the CAR(tcp):RXR(9c)·SRC1 complex can encompass two SRC1 molecules compared with the CAR(tcp):RXR·SRC1, which binds only a single SRC1. Moreover, sedimentation coefficients and molecular weights determined by analytical ultracentrifugation confirm the SAXS model. Cell-based transcription assays show that disrupting the SRC1 binding site on RXR alters the transactivation by CAR:RXR. These data suggest a broader role for RXR within heterodimers, whereas offering multiple strategies for the assembly of the transcription complex.

  14. DNA Damage-induced Heterogeneous Nuclear Ribonucleoprotein K SUMOylation Regulates p53 Transcriptional Activation*

    PubMed Central

    Pelisch, Federico; Pozzi, Berta; Risso, Guillermo; Muñoz, Manuel Javier; Srebrow, Anabella

    2012-01-01

    Heterogeneous nuclear ribonucleoprotein (hnRNP) K is a nucleocytoplasmic shuttling protein that is a key player in the p53-triggered DNA damage response, acting as a cofactor for p53 in response to DNA damage. hnRNP K is a substrate of the ubiquitin E3 ligase MDM2 and, upon DNA damage, is de-ubiquitylated. In sharp contrast with the role and consequences of the other post-translational modifications, nothing is known about the role of SUMO conjugation to hnRNP K in p53 transcriptional co-activation. In the present work, we show that hnRNP K is modified by SUMO in lysine 422 within its KH3 domain, and sumoylation is regulated by the E3 ligase Pc2/CBX4. Most interestingly, DNA damage stimulates hnRNP K sumoylation through Pc2 E3 activity, and this modification is required for p53 transcriptional activation. Abrogation of hnRNP K sumoylation leads to an aberrant regulation of the p53 target gene p21. Our findings link the DNA damage-induced Pc2 activation to the p53 transcriptional co-activation through hnRNP K sumoylation. PMID:22825850

  15. Modulation of Epstein–Barr Virus Nuclear Antigen 2-dependent transcription by protein arginine methyltransferase 5

    SciTech Connect

    Liu, Cheng-Der; Cheng, Chi-Ping; Fang, Jia-Shih; Chen, Ling-Chih; Zhao, Bo; Kieff, Elliott; Peng, Chih-Wen

    2013-01-18

    Highlights: ► Catalytic active PRMT5 substantially binds to the EBNA2 RG domain. ► PRMT5 augments the EBNA2-dependent transcription. ► PRMT5 triggers the symmetric dimethylation of the EBNA2 RG domain. ► PRMT5 enhances the promoter occupancy of EBNA2 on its target promoters. -- Abstract: Epstein–Barr Virus Nuclear Antigen (EBNA) 2 features an Arginine–Glycine repeat (RG) domain at amino acid positions 335–360, which is a known target for protein arginine methyltransferaser 5 (PRMT5). In this study, we performed protein affinity pull-down assays to demonstrate that endogenous PRMT5 derived from lymphoblastoid cells specifically associated with the protein bait GST-E2 RG. Transfection of a plasmid expressing PRMT5 induced a 2.5- to 3-fold increase in EBNA2-dependent transcription of both the LMP1 promoter in AKATA cells, which contain the EBV genome endogenously, and a Cp-Luc reporter plasmid in BJAB cells, which are EBV negative. Furthermore, we showed that there was a 2-fold enrichment of EBNA2 occupancy in target promoters in the presence of exogenous PRMT5. Taken together, we show that PRMT5 triggers the symmetric dimethylation of EBNA2 RG domain to coordinate with EBNA2-mediated transcription. This modulation suggests that PRMT5 may play a role in latent EBV infection.

  16. Nuclear F-actin enhances the transcriptional activity of β-catenin by increasing its nuclear localization and binding to chromatin.

    PubMed

    Yamazaki, Shota; Yamamoto, Koji; de Lanerolle, Primal; Harata, Masahiko

    2016-04-01

    Actin plays multiple roles both in the cytoplasm and in the nucleus. Cytoplasmic actin, in addition to its structural role in the cytoskeleton, also contributes to the subcellular localization of transcription factors by interacting with them or their partners. The transcriptional cofactor β-catenin, which acts as an intracellular transducer of canonical Wnt signaling, indirectly associates with the cytoplasmic filamentous actin (F-actin). Recently, it has been observed that F-actin is transiently formed within the nucleus in response to serum stimulation and integrin signaling, and also during gene reprogramming. Despite these earlier observations, information about the function of nuclear F-actin is poorly defined. Here, by facilitating the accumulation of nuclear actin artificially, we demonstrate that polymerizing nuclear actin enhanced the nuclear accumulation and transcriptional function of β-catenin. Our results also show that the nuclear F-actin colocalizes with β-catenin and enhances the binding of β-catenin to the downstream target genes of the Wnt/β-catenin signaling pathway, including the genes for the cell cycle regulators c-myc and cyclin D, and the OCT4 gene. Nuclear F-actin itself also associated with these genes. Since Wnt/β-catenin signaling has important roles in cell differentiation and pluripotency, our observations suggest that nuclear F-actin formed during these biological processes is involved in regulating Wnt/β-catenin signaling.

  17. A Photo-Degradable Gene Delivery System for Enhanced Nuclear Gene Transcription

    PubMed Central

    Hoyoung, Lee; Yeji, Kim; Patrick G., Schweickert; Stephen F., Konieczny; You-Yeon, Won

    2013-01-01

    There currently exists a significant gap in our understanding of how the detailed chemical characteristics of polycation gene carriers influence their delivery performances in overcoming an important cellular-level transport barrier, i.e., intranuclear gene transcription. In this study, a UV-degradable gene carrier material (ENE4-1) was synthesized by crosslinking low molecular weight branched polyethylenimine (bPEI-2k) molecules using UV-cleavable o-nitrobenzyl urethane (NBU) as the linker molecule. NBU degrades upon exposure to mild UV irradiation. Therefore, this UV-degradable carrier allows us to control the chemical characteristics of the polymer/DNA complex (polyplex) particles at desired locations within the intracellular environment. By using this photolytic DNA carrier, we found that the exact timing of the UV degradation significantly influences the gene transfection efficiencies of ENE4-1/DNA(pGL2) polyplexes in HeLa cells. Interestingly, even if the polyplexes were UV-degraded at different intracellular locations/times, their nuclear entry efficiency was not influenced by the location/timing of UV degradation. The UV treatment did not influence the size or binding strength of the polyplexes. However, we confirmed that the degradation of the carrier molecules impacts the chemical characteristics of the polyplexes (it produces carbamic acid and nitrosobenzyl aldehyde groups on ENE4-1). We believe that these anionic acid groups enhance the interaction of the polyplexes with nuclear transcription proteins and thus the final gene expression levels; this effect was found to occur, even though UV irradiation itself has a general effect of reducing transfection efficiencies. Excess (uncomplexed) ENE4-1 polymers appear to not play any role in the UV-enhanced gene transcription phenomenon. PMID:24172855

  18. Inhibition of hepatitis B virus (HBV) by LNA-mediated nuclear interference with HBV DNA transcription

    SciTech Connect

    Sun, Zhen; Xiang, Wenqing; Guo, Yajuan; Chen, Zhi; Liu, Wei; Lu, Daru

    2011-06-10

    Highlights: {yields} LNA-modified oligonucleotides can pass through the plasma membrane of cultured cells even without using transfection machinery. {yields} LNA-modified oligonucleotides passed efficiently across the cell membrane, and lipid-coating facilitated translocation from the cytoplasm to the nucleus. {yields} LNA-oligonucleotide designed to target nuclear HBV DNA efficiently suppresses HBV replication and transcription in cultured hepatic cells. -- Abstract: Silencing target genes with small regulatory RNAs is widely used to investigate gene function and therapeutic drug development. Recently, triplex-based approaches have provided another attractive means to achieve targeted gene regulation and gene manipulation at the molecular and cellular levels. Nuclear entry of oligonucleotides and enhancement of their affinity to the DNA targets are key points of such approaches. In this study, we developed lipid-based transport of a locked-nucleic-acid (LNA)-modified oligonucleotide for hepatitis B virus (HBV) DNA interference in human hepatocytes expressing HBV genomic DNA. In these cells, the LNA-modified oligonucleotides passed efficiently across the cell membrane, and lipid-coating facilitated translocation from the cytoplasm to the nucleus. The oligonucleotide specifically targeting HBV DNA clearly interfered with HBV DNA transcription as shown by a block in pregenomic RNA (pgRNA) production. The HBV DNA-targeted oligonucleotide suppressed HBV DNA replication and HBV protein production more efficiently than small interfering RNAs directed to the pgRNA. These results demonstrate that fusion with lipid can carry LNA-modified oligonucleotides to the nucleus where they regulate gene expression. Interfering with HBV DNA transcription by LNA-modified oligonucleotides has strong potential as a new strategy for HBV inhibition.

  19. Discovery of Transcriptional Targets Regulated by Nuclear Receptors Using a Probabilistic Graphical Model.

    PubMed

    Lee, Mikyung; Huang, Ruili; Tong, Weida

    2016-03-01

    Nuclear receptors (NRs) are ligand-activated transcriptional regulators that play vital roles in key biological processes such as growth, differentiation, metabolism, reproduction, and morphogenesis. Disruption of NRs can result in adverse health effects such as NR-mediated endocrine disruption. A comprehensive understanding of core transcriptional targets regulated by NRs helps to elucidate their key biological processes in both toxicological and therapeutic aspects. In this study, we applied a probabilistic graphical model to identify the transcriptional targets of NRs and the biological processes they govern. The Tox21 program profiled a collection of approximate 10 000 environmental chemicals and drugs against a panel of human NRs in a quantitative high-throughput screening format for their NR disruption potential. The Japanese Toxicogenomics Project, one of the most comprehensive efforts in the field of toxicogenomics, generated large-scale gene expression profiles on the effect of 131 compounds (in its first phase of study) at various doses, and different durations, and their combinations. We applied author-topic model to these 2 toxicological datasets, which consists of 11 NRs run in either agonist and/or antagonist mode (18 assays total) and 203 in vitro human gene expression profiles connected by 52 shared drugs. As a result, a set of clusters (topics), which consists of a set of NRs and their associated target genes were determined. Various transcriptional targets of the NRs were identified by assays run in either agonist or antagonist mode. Our results were validated by functional analysis and compared with TRANSFAC data. In summary, our approach resulted in effective identification of associated/affected NRs and their target genes, providing biologically meaningful hypothesis embedded in their relationships.

  20. Nuclear transcription factors: a new approach to enhancing cellular responses to ALA-mediated photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Maytin, Edward V.; Anand, Sanjay; Sato, Nobuyuki; Moore, Brian; Mack, Judith; Gasbarre, Christopher; Keevey, Samantha; Ortel, Bernhard; Sinha, Alok; Khachemoune, Amor

    2006-02-01

    Photodynamic therapy (PDT) using aminolevulinic acid (ALA) relies upon the uptake of ALA into cancer cells, where it is converted into a porphyrin intermediate, protoporphyrin IX (PpIX) that is highly photosensitizing. For large or resistant tumors, however, ALA/PDT is often not completely effective due to inadequate PpIX levels. Therefore, new approaches to enhance the intracellular production of PpIX are sought. Here, we describe a general approach to improve intracellular PpIX accumulation via manipulations that increase the expression of an enzyme, coproporphyrinogen oxidase (CPO), that is rate-determining for PpIX production. We show that nuclear hormones that promote terminal differentiation, e.g. vitamin D or androgens, can also increase the accumulation of PpIX and the amount of killing of the target cells upon exposure to light. These hormones bind to intracellular hormone receptors that translocate to the nucleus, where they act as transcription factors to increase the expression of target genes. We have found that several other transcription factors associated with terminal differentiation, including members of the CCAAT enhancer binding (C/EBP) family, and a homeobox protein named Hoxb13, are also capable of enhancing PpIX accumulation. These latter transcription factors appear to interact directly with the CPO gene promoter, resulting in enhanced CPO transcriptional activity. Our data in several different cell systems, including epithelial cells of the skin and prostate cancer cells, indicate that enhancement of CPO expression and PpIX accumulation represents a viable new approach toward improving the efficacy of ALA/PDT.

  1. A novel function of the mitochondrial transcription factor Mtf1 in fission yeast; Mtf1 regulates the nuclear transcription of srk1.

    PubMed

    Sun, Wenxia; Wang, Zhe; Jiang, Hengyi; Zhang, Jing; Bähler, Jürg; Chen, Dongrong; Murchie, Alastair I H

    2011-04-01

    In eukaryotic cells, Mtf1 and its homologues function as mitochondrial transcription factors for the mitochondrial RNA polymerase in the mitochondrion. Here we show that in fission yeast Mtf1 exerts a non-mitochondrial function as a nuclear factor that regulates transcription of srk1, which is a kinase involved in the stress response and cell cycle progression. We first found Mtf1 expression in the nucleus. A ChIP-chip approach identified srk1 as a putative Mtf1 target gene. Over expression of Mtf1 induced transcription of the srk1 gene and Mtf1 deletion led to a reduction in transcription of the srk1 gene in vivo. Mtf1 overexpression causes cell elongation in a srk1 dependent manner. Mtf1 overexpression can cause cytoplasmic accumulation of Cdc25. We also provide biochemical evidence that Mtf1 binds to the upstream sequence of srk1. This is the first evidence that a mitochondrial transcription factor Mtf1 can regulate a nuclear gene. Mtf1 may also have a role in cell cycle progression.

  2. RGS12TS-S Localizes at Nuclear Matrix-Associated Subnuclear Structures and Represses Transcription: Structural Requirements for Subnuclear Targeting and Transcriptional Repression

    PubMed Central

    Chatterjee, Tapan K.; Fisher, Rory A.

    2002-01-01

    RGS12TS-S, an 1,157-amino-acid RGS protein (regulator of G protein signaling), is a nuclear protein that exhibits a unique pattern of subnuclear organization into nuclear foci or dots when expressed endogenously or ectopically. We now report that RGS12TS-S is a nuclear matrix protein and identify structural determinants that target this protein to the nuclear matrix and to discrete subnuclear sites. We also determine the relationship between RGS12TS-S-decorated nuclear dots and known subnuclear domains involved in control of gene expression and provide the first evidence that RGS12TS-S is functionally involved in the regulation of transcription and cell cycle events. A novel nuclear matrix-targeting sequence was identified that is distinct from a second novel motif needed for targeting RGS12TS-S to nuclear dots. RGS12TS-S nuclear dots were distinct from Cajal bodies, SC-35 domains, promyelocytic leukemia protein nuclear bodies, Polycomb group domains, and DNA replication sites. However, RGS12TS-S inhibited S-phase DNA synthesis in various tumor cell lines independently of Rb and p53 proteins, and its prolonged expression promoted formation of multinucleated cells. Expression of RGS12TS-S dramatically reduced bromo-UTP incorporation into sites of transcription. RGS12TS-S, when tethered to a Gal4 DNA binding domain, dramatically inhibited basal transcription from a Gal4-E1b TATA promoter in a histone deacetylase-independent manner. Structural analysis revealed a role for the unique N-terminal domain of RGS12TS-S in its transcriptional repressor and cell cycle-regulating activities and showed that the RGS domain was dispensable for these functions. These results provide novel insights into the structure and function of RGS12TS-S in the nucleus and demonstrate that RGS12TS-S possesses biological activities distinct from those of other members of the RGS protein family. PMID:12024043

  3. Transcription of Nrdp1 by the androgen receptor is regulated by nuclear Filamin A in prostate cancer

    PubMed Central

    Savoy, Rosalinda M.; Chen, Liqun; Siddiqui, Salma; Melgoza, Frank U.; Durbin-Johnson, Blythe; Drake, Christiana; Jathal, Maitreyee K.; Bose, Swagata; Steele, Thomas M.; Mooso, Benjamin A.; D’Abronzo, Leandro S.; Fry, William H.; Carraway, Kermit L.; Mudryj, Maria; Ghosh, Paramita M.

    2015-01-01

    Prostate cancer (PCa) progression is regulated by the androgen receptor (AR); however, patients undergoing androgen deprivation therapy (ADT) for disseminated PCa eventually develop castration resistant PCa (CRPC). Studies showed that AR, a transcription factor, occupies distinct genomic loci in CRPC compared to hormone-naïve PCa; however, the cause for this distinction was unknown. The E3 ubiquitin ligase Nrdp1 is a model AR target modulated by androgens in hormone-naïve PCa but not in CRPC. Using Nrdp1, we investigated how AR switches transcription programs during CRPC progression. The proximal Nrdp1 promoter contains an androgen response element (ARE); we demonstrated AR binding to this ARE in androgen-sensitive PCa. Analysis of hormone-naive human prostatectomy specimens revealed correlation between Nrdp1 and AR expression, supporting AR regulation of Nrdp1 levels in androgen-sensitive tissue. However, despite sustained AR levels, AR binding to the Nrdp1 promoter and Nrdp1 expression were suppressed in CRPC. Elucidation of the suppression mechanism demonstrated correlation of Nrdp1 levels with nuclear localization of the scaffolding protein Filamin A (FlnA) which, as we previously showed, is itself repressed following ADT in many CRPC tumors. Restoration of nuclear FlnA in CRPC stimulated AR binding to Nrdp1 ARE, increased its transcription, and augmented Nrdp1 protein expression and responsiveness to ADT, indicating that nuclear FlnA controls AR-mediated androgen-sensitive Nrdp1 transcription. Expressions of other AR-regulated genes lost in CRPC were also re-established by nuclear FlnA. Thus our data demonstrate that nuclear FlnA promotes androgen-dependent AR-regulated transcription in PCa, while loss of nuclear FlnA in CRPC alters the AR-regulated transcription program. PMID:25759396

  4. Transcription of Nrdp1 by the androgen receptor is regulated by nuclear filamin A in prostate cancer.

    PubMed

    Savoy, Rosalinda M; Chen, Liqun; Siddiqui, Salma; Melgoza, Frank U; Durbin-Johnson, Blythe; Drake, Christiana; Jathal, Maitreyee K; Bose, Swagata; Steele, Thomas M; Mooso, Benjamin A; D'Abronzo, Leandro S; Fry, William H; Carraway, Kermit L; Mudryj, Maria; Ghosh, Paramita M

    2015-06-01

    Prostate cancer (PCa) progression is regulated by the androgen receptor (AR); however, patients undergoing androgen-deprivation therapy (ADT) for disseminated PCa eventually develop castration-resistant PCa (CRPC). Results of previous studies indicated that AR, a transcription factor, occupies distinct genomic loci in CRPC compared with hormone-naïve PCa; however, the cause of this distinction was unknown. The E3 ubiquitin ligase Nrdp1 is a model AR target modulated by androgens in hormone-naïve PCa but not in CRPC. Using Nrdp1, we investigated how AR switches transcription programs during CRPC progression. The proximal Nrdp1 promoter contains an androgen response element (ARE); we demonstrated AR binding to this ARE in androgen-sensitive PCa. Analysis of hormone-naive human prostatectomy specimens revealed correlation between Nrdp1 and AR expression, supporting AR regulation of NRDP1 levels in androgen-sensitive tissue. However, despite sustained AR levels, AR binding to the Nrdp1 promoter and Nrdp1 expression were suppressed in CRPC. Elucidation of the suppression mechanism demonstrated correlation of NRDP1 levels with nuclear localization of the scaffolding protein filamin A (FLNA) which, as we previously showed, is itself repressed following ADT in many CRPC tumors. Restoration of nuclear FLNA in CRPC stimulated AR binding to Nrdp1 ARE, increased its transcription, and augmented NRDP1 protein expression and responsiveness to ADT, indicating that nuclear FLNA controls AR-mediated androgen-sensitive Nrdp1 transcription. Expression of other AR-regulated genes lost in CRPC was also re-established by nuclear FLNA. Thus, our results indicate that nuclear FLNA promotes androgen-dependent AR-regulated transcription in PCa, while loss of nuclear FLNA in CRPC alters the AR-regulated transcription program.

  5. Nuclear translocation uncovers the amyloid peptide Aβ42 as a regulator of gene transcription.

    PubMed

    Barucker, Christian; Harmeier, Anja; Weiske, Joerg; Fauler, Beatrix; Albring, Kai Frederik; Prokop, Stefan; Hildebrand, Peter; Lurz, Rudi; Heppner, Frank L; Huber, Otmar; Multhaup, Gerhard

    2014-07-18

    Although soluble species of the amyloid-β peptide Aβ42 correlate with disease symptoms in Alzheimer disease, little is known about the biological activities of amyloid-β (Aβ). Here, we show that Aβ peptides varying in lengths from 38 to 43 amino acids are internalized by cultured neuroblastoma cells and can be found in the nucleus. By three independent methods, we demonstrate direct detection of nuclear Aβ42 as follows: (i) biochemical analysis of nuclear fractions; (ii) detection of biotin-labeled Aβ in living cells by confocal laser scanning microscopy; and (iii) transmission electron microscopy of Aβ in cultured cells, as well as brain tissue of wild-type and transgenic APPPS1 mice (overexpression of amyloid precursor protein and presenilin 1 with Swedish and L166P mutations, respectively). Also, this study details a novel role for Aβ42 in nuclear signaling, distinct from the amyloid precursor protein intracellular domain. Chromatin immunoprecipitation showed that Aβ42 specifically interacts as a repressor of gene transcription with LRP1 and KAI1 promoters. By quantitative RT-PCR, we confirmed that mRNA levels of the examined candidate genes were exclusively decreased by the potentially neurotoxic Aβ42 wild-type peptide. Shorter peptides (Aβ38 or Aβ40) and other longer peptides (nontoxic Aβ42 G33A substitution or Aβ43) did not affect mRNA levels. Overall, our data indicate that the nuclear translocation of Aβ42 impacts gene regulation, and deleterious effects of Aβ42 in Alzheimer disease pathogenesis may be influenced by altering the expression profiles of disease-modifying genes.

  6. Interaction between the yeast mitochondrial and nuclear genomes influences the abundance of novel transcripts derived from the spacer region of the nuclear ribosomal DNA repeat.

    PubMed Central

    Parikh, V S; Conrad-Webb, H; Docherty, R; Butow, R A

    1989-01-01

    We have identified stable transcripts from the so-called nontranscribed spacer region (NTS) of the nuclear ribosomal DNA repeat in certain respiration-deficient strains of Saccharomyces cerevisiae. These RNAs, which are transcribed from the same strand as is the 37S rRNA precursor, are 500 to 800 nucleotides long and extend from the 5' end of the 5S rRNA gene to three major termination sites about 1,780, 1,830, and 1,870 nucleotides from the 3' end of the 26S rRNA gene. A survey of various wild-type and respiration-deficient strains showed that NTS transcript abundance depended on the mitochondrial genotype and a single codominant nuclear locus. In strains with that nuclear determinant, NTS transcripts were barely detected in [rho+] cells, were slightly more abundant in various mit- derivatives, and were most abundant in petites. However, in one petite that was hypersuppressive and contained a putative origin of replication (ori5) within its 757-base-pair mitochondrial genome, NTS transcripts were no more abundant than in [rho+] cells. The property of low NTS transcript abundance in the hypersuppressive petite was unstable, and spontaneous segregants that contained NTS transcripts as abundant as in the other petites examined could be obtained. Thus, respiration deficiency per se is not the major factor contributing to the accumulation of these unusual RNAs. Unlike RNA polymerase I transcripts, the abundant NTS RNAs were glucose repressible, fractionated as poly(A)+ RNAs, and were sensitive to inhibition by 10 micrograms of alpha-amanitin per ml, a concentration that had no effect on rRNA synthesis. Abundant NTS RNAs are therefore most likely derived by polymerase II transcription. Images PMID:2473390

  7. Superoxide dismutase 1 acts as a nuclear transcription factor to regulate oxidative stress resistance

    PubMed Central

    Tsang, Chi Kwan; Liu, Yuan; Thomas, Janice; Zhang, Yanjie; Zheng, X. F. Steven

    2015-01-01

    Summary Superoxide dismutase 1 (Sod1) has been known for nearly half a century for catalysis of superoxide to hydrogen peroxide. Here we report a new Sod1 function in oxidative signaling: in response to elevated endogenous and exogenous reactive oxygen species (ROS), Sod1 rapidly relocates into the nucleus, which is important for maintaining genomic stability. Interestingly, H2O2 is sufficient to promote Sod1 nuclear localization, indicating that it is responding to general ROS rather than Sod1 substrate superoxide. ROS signaling is mediated by Mec1/ATM and its effector Dun1/Cds1 kinase, through Dun1 interaction with Sod1 and regulation of Sod1 by phosphorylation at S60, 99. In the nucleus, Sod1 binds to the promoters and regulates the expression of oxidative resistance and repair genes. Altogether, our study unravels an unorthodox function of Sod1 as a transcription factor and elucidates the regulatory mechanism for its localization. PMID:24647101

  8. The Oct-1 POU-specific domain can stimulate small nuclear RNA gene transcription by stabilizing the basal transcription complex SNAPc.

    PubMed Central

    Mittal, V; Cleary, M A; Herr, W; Hernandez, N

    1996-01-01

    The RNA polymerase II and III human small nuclear RNA promoters have a common basal element, the proximal sequence element, which binds the TATA box-binding protein-containing complex SNAPc. They also contain an enhancer characterized by a highly conserved octamer sequence, which constitutes a binding site for the broadly expressed POU domain transcription factor Oct-1. The POU domain is a bipartite DNA-binding domain consisting of a POU-homeo (POUH) domain and a POU-specific (POUs) domain joined by a flexible linker. Here, we show that the Oct-1 POU domain but not the related Pit-1 POU domain can facilitate the binding of SNAPc to the proximal sequence element, and activate transcription. The effect is probably mediated by protein-protein contacts, and 1 of 30 amino acid differences between the Oct-1 and Pit-1 POUs domains is the key determinant for the differential interaction with SNAPc and the ability to activate transcription. These results show that a function that is the hallmark of activation domains, namely, recruitment of a basal transcription complex resulting in activation of transcription, can be performed by a DNA-binding domain. In this case, subtle changes between activator DNA-binding domains, as subtle as a single amino acid difference, can profoundly affect interaction with the basal transcription machinery. PMID:8628262

  9. Nuclear Factor of Activated T Cells Transcription Factor Nfatp Controls Superantigen-Induced Lethal Shock

    PubMed Central

    Tsytsykova, Alla V.; Goldfeld, Anne E.

    2000-01-01

    Tumor necrosis factor α (TNF-α) is the key mediator of superantigen-induced T cell lethal shock. Here, we show that nuclear factor of activated T cells transcription factor, NFATp, controls susceptibility to superantigen-induced lethal shock in mice through its activation of TNF-α gene transcription. In NFATp-deficient mice, T cell stimulation leads to delayed induction and attenuation of TNF-α mRNA levels, decreased TNF-α serum levels, and resistance to superantigen-induced lethal shock. By contrast, after lipopolysaccharide (LPS) challenge, serum levels of TNF-α and susceptibility to shock are unaffected. These results demonstrate that NFATp is an essential activator of immediate early TNF-α gene expression in T cells and they present in vivo evidence of the inducer- and cell type–specific regulation of TNF-α gene expression. Furthermore, they suggest NFATp as a potential selective target in the treatment of superantigen-induced lethal shock. PMID:10952728

  10. Nuclear respiratory factor 1 and endurance exercise promote human telomere transcription

    PubMed Central

    Diman, Aurélie; Boros, Joanna; Poulain, Florian; Rodriguez, Julie; Purnelle, Marin; Episkopou, Harikleia; Bertrand, Luc; Francaux, Marc; Deldicque, Louise; Decottignies, Anabelle

    2016-01-01

    DNA breaks activate the DNA damage response and, if left unrepaired, trigger cellular senescence. Telomeres are specialized nucleoprotein structures that protect chromosome ends from persistent DNA damage response activation. Whether protection can be enhanced to counteract the age-dependent decline in telomere integrity is a challenging question. Telomeric repeat–containing RNA (TERRA), which is transcribed from telomeres, emerged as important player in telomere integrity. However, how human telomere transcription is regulated is still largely unknown. We identify nuclear respiratory factor 1 and peroxisome proliferator–activated receptor γ coactivator 1α as regulators of human telomere transcription. In agreement with an upstream regulation of these factors by adenosine 5′-monophosphate (AMP)–activated protein kinase (AMPK), pharmacological activation of AMPK in cancer cell lines or in normal nonproliferating myotubes up-regulated TERRA, thereby linking metabolism to telomere fitness. Cycling endurance exercise, which is associated with AMPK activation, increased TERRA levels in skeletal muscle biopsies obtained from 10 healthy young volunteers. The data support the idea that exercise may protect against aging. PMID:27819056

  11. Nuclear factor I and epithelial cell-specific transcription of human papillomavirus type 16.

    PubMed Central

    Apt, D; Chong, T; Liu, Y; Bernard, H U

    1993-01-01

    The transcription of human papillomavirus type 16 (HPV-16) is mediated by the viral enhancer. Epithelial cell-specific activation is achieved by the cooperative interaction of apparently ubiquitous transcriptional factors. One of them, nuclear factor I (NFI), binds seven sites within the HPV-16 enhancer. Point mutations on enhancer fragments, which retain epithelial cell specificity, verify the functional contribution of NFI. In band shift experiments, the epithelial cell-derived NFI proteins CTF-1, CTF-2, and CTF-3 form a characteristic pattern of heterodimeric complexes which are observed in all epithelial cells tested. Divergence from this pattern in fibroblasts, liver cells, and lymphoid cells correlates with the lack of HPV-16 enhancer activation. The HPV-16 enhancer can be activated by CTF-1 in SL-2 cells, which lack NFI-like proteins. However, exogenous CTF-1 fails to overcome the inactivity of the viral enhancer in fibroblasts. Western immunoblot and supershift analysis shows that exogenously introduced CTF-1 proteins form different heterodimer complexes with the given subset of endogenous NFI proteins in epithelial or fibroblast cells. Polymerase chain reaction analysis and cDNA library screens identified the endogenous fibroblast type NFI as NFI-X, an NFI family member originally cloned from hamster liver cells. The strict correlation between the activation or lack of activation of the HPV-16 enhancer and cell-specific subsets of NFI proteins argues for the pivotal role of NFI binding sites in the epithelial cell-specific function of the viral enhancer. Images PMID:8392590

  12. Expression Profiles of the Nuclear Receptors and Their Transcriptional Coregulators During Differentiation of Neural Stem Cells

    PubMed Central

    Androutsellis-Theotokis, A.; Chrousos, G. P.; McKay, R. D.; DeCherney, A. H.; Kino, T.

    2013-01-01

    Neural stem cells (NSCs) are pluripotent precursors with the ability to proliferate and differentiate into 3 neural cell lineages, neurons, astrocytes and oligodendrocytes. Elucidation of the mechanisms underlying these biologic processes is essential for understanding both physiologic and pathologic neural development and regeneration after injury. Nuclear hormone receptors (NRs) and their transcriptional coregulators also play crucial roles in neural development, functions and fate. To identify key NRs and their transcriptional regulators in NSC differentiation, we examined mRNA expression of 49 NRs and many of their coregulators during differentiation (0–5 days) of mouse embryonic NSCs induced by withdrawal of fibroblast growth factor-2 (FGF2). 37 out of 49 NRs were expressed in NSCs before induction of differentiation, while receptors known to play major roles in neural development, such as THRα, RXRs, RORs, TRs, and COUPTFs, were highly expressed. CAR, which plays important roles in xenobiotic metabolism, was also highly expressed. FGF2 withdrawal induced mRNA expression of RORγ, RXRγ, and MR by over 20-fold. Most of the transcriptional coregulators examined were expressed basally and throughout differentiation without major changes, while FGF2 withdrawal strongly induced mRNA expression of several histone deacetylases (HDACs), including HDAC11. Dexamethasone and aldosterone, respectively a synthetic glucocorticoid and natural mineralocorticoid, increased NSC numbers and induced differentiation into neurons and astrocytes. These results indicate that the NRs and their coregulators are present and/or change their expression during NSC differentiation, suggesting that they may influence development of the central nervous system in the absence or presence of their ligands. PMID:22990992

  13. Visualization of nuclear localization of transcription factors with cyan and green fluorescent proteins in the red alga Porphyra yezoensis.

    PubMed

    Uji, Toshiki; Takahashi, Megumu; Saga, Naotsune; Mikami, Koji

    2010-04-01

    Transcription factors play a central role in expression of genomic information in all organisms. The objective of our study is to analyze the function of transcription factors in red algae. One way to analyze transcription factors in eukaryotic cells is to study their nuclear localization, as reported for land plants and green algae using fluorescent proteins. There is, however, no report documenting subcellular localization of transcription factors from red algae. In the present study, using the marine red alga Porphyra yezoensis, we confirmed for the first time successful expression of humanized fluorescent proteins (ZsGFP and ZsYFP) from a reef coral Zoanthus sp. and land plant-adapted sGFP(S65T) in gametophytic cells comparable to expression of AmCFP. Following molecular cloning and characterization of transcription factors DP-E2F-like 1 (PyDEL1), transcription elongation factor 1 (PyElf1) and multiprotein bridging factor 1 (PyMBF1), we then demonstrated that ZsGFP and AmCFP can be used to visualize nuclear localization of PyElf1 and PyMBF1. This is the first report to perform visualization of subcellular localization of transcription factors as genome-encoded proteins in red algae.

  14. Integration of light and circadian signals that regulate chloroplast transcription by a nuclear-encoded sigma factor.

    PubMed

    Belbin, Fiona E; Noordally, Zeenat B; Wetherill, Sarah J; Atkins, Kelly A; Franklin, Keara A; Dodd, Antony N

    2017-01-01

    We investigated the signalling pathways that regulate chloroplast transcription in response to environmental signals. One mechanism controlling plastid transcription involves nuclear-encoded sigma subunits of plastid-encoded plastid RNA polymerase. Transcripts encoding the sigma factor SIG5 are regulated by light and the circadian clock. However, the extent to which a chloroplast target of SIG5 is regulated by light-induced changes in SIG5 expression is unknown. Moreover, the photoreceptor signalling pathways underlying the circadian regulation of chloroplast transcription by SIG5 are unidentified. We monitored the regulation of chloroplast transcription in photoreceptor and sigma factor mutants under controlled light regimes in Arabidopsis thaliana. We established that a chloroplast transcriptional response to light intensity was mediated by SIG5; a chloroplast transcriptional response to the relative proportions of red and far red light was regulated by SIG5 through phytochrome and photosynthetic signals; and the circadian regulation of chloroplast transcription by SIG5 was predominantly dependent on blue light and cryptochrome. Our experiments reveal the extensive integration of signals concerning the light environment by a single sigma factor to regulate chloroplast transcription. This may originate from an evolutionarily ancient mechanism that protects photosynthetic bacteria from high light stress, which subsequently became integrated with higher plant phototransduction networks.

  15. Effects of primary metabolites of organophosphate flame retardants on transcriptional activity via human nuclear receptors.

    PubMed

    Kojima, Hiroyuki; Takeuchi, Shinji; Van den Eede, Nele; Covaci, Adrian

    2016-03-14

    Organophosphate flame retardants (OPFRs) have been used in a wide variety of applications and detected in several environmental matrices, including indoor air and dust. Continuous human exposure to these chemicals is of growing concern. In this study, the agonistic and/or antagonistic activities of 12 primary OPFR-metabolites against ten human nuclear receptors were examined using cell-based transcriptional assays, and compared to those of their parent compounds. As a result, 3-hydroxylphenyl diphenyl phosphate and 4-hydroxylphenyl diphenyl phosphate showed more potent estrogen receptor α (ERα) and ERβ agonistic activity than did their parent, triphenyl phosphate (TPHP). In addition, these hydroxylated TPHP-metabolites also showed ERβ antagonistic activity at higher concentrations and exhibited pregnane X receptor (PXR) agonistic activity as well as androgen receptor (AR) and glucocorticoid receptor (GR) antagonistic activities at similar levels to those of TPHP. Bis(2-butoxyethyl) 3'-hydroxy-2-butoxyethyl phosphate and 2-hydroxyethyl bis(2-butoxyethyl) phosphate act as PXR agonists at similar levels to their parent, tris(2-butoxyethyl) phosphate. On the other hand, seven diester OPFR-metabolites and 1-hydroxy-2-propyl bis(1-chloro-2-propyl) phosphate did not show any receptor activity. Taken together, these results suggest that hydroxylated TPHP-metabolites show increased estrogenicity compared to the parent compound, whereas the diester OPFR-metabolites may have limited nuclear receptor activity compared to their parent triester OPFRs.

  16. Capsaicinoids improve egg production by regulating ovary nuclear transcription factors against heat stress in quail.

    PubMed

    Sahin, N; Orhan, C; Tuzcu, M; Juturu, V; Sahin, K

    2016-12-12

    To examine the molecular mechanism of capsaicinoid supplementation from capsicum extract, laying Japanese quail (n = 180, 5 weeks old) were reared either at 22°C for 24 h/d (thermoneutral, TN) or at 34°C for 8 h/d (heat stress, HS) and fed on one of three diets containing 0, 25 or 50 mg of capsaicinoids per kilogram for 12 weeks (2 × 3 factorial arrangement). The results revealed that exposure to HS decreased feed consumption by 10.7% and egg production by 13.6%, increased serum and ovary malondialdehyde (MDA) levels by 66.9% and 88.1%, respectively, and reduced ovary superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities by 28.3%, 48.7% and 43.8%, respectively. There were magnifications in the ovary nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) levels by 42.4% and suppressions in nuclear factor (erythroid-derived 2)-like 2 (Nrf2), protein kinase B (Akt) and haem-oxygenase 1 (HO-1) levels by 29.2%, 38.2% and 30.7%, respectively, in heat-stressed quail. With increasing supplemental capsaicinoids, there were linear increases in egg production, antioxidant enzyme activity, linear decreases in ovary MDA and NF-κB levels and linear increases in ovary Nrf2, Akt and HO-1 levels at a greater extent in quail reared under TN condition than those reared under HS condition. Two-way treatment interactions showed that the degree of restorations in all response variables was more notable under the HS environment than under the TN environment as supplemental capsaicinoid level was increased. In conclusion, capsaicinoid supplementation alleviates oxidative stress through regulating the ovary nuclear transcription factors in heat-stressed quail.

  17. Split personality of transcription factors inside and outside the nuclear border.

    PubMed

    Naranjo, José R; Mellström, Britt

    2007-01-30

    Growing evidence indicates that transcription factors may have functions entirely distinct from the regulation of gene transcription. Here we describe three transcription factors that, when outside the nucleus, regulate calcium homeostasis by three independent but convergent mechanisms.

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

    PubMed

    Wada, Takeyoshi; Asahi, Toru; Sawamura, Naoya

    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 a 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.

  19. Transcription-dependent nucleolar cap localization and possible nuclear function of DExH RNA helicase RHAU

    SciTech Connect

    Iwamoto, Fumiko; Stadler, Michael; Chalupnikova, Katerina; Oakeley, Edward; Nagamine, Yoshikuni

    2008-04-01

    RHAU (RNA helicase associated with AU-rich element) is a DExH protein originally identified as a factor accelerating AU-rich element-mediated mRNA degradation. The discovery that RHAU is predominantly localized in the nucleus, despite mRNA degradation occurring in the cytoplasm, prompted us to consider the nuclear functions of RHAU. In HeLa cells, RHAU was found to be localized throughout the nucleoplasm with some concentrated in nuclear speckles. Transcriptional arrest altered the localization to nucleolar caps, where RHAU is closely localized with RNA helicases p68 and p72, suggesting that RHAU is involved in transcription-related RNA metabolism in the nucleus. To see whether RHAU affects global gene expression transcriptionally or posttranscriptionally, we performed microarray analysis using total RNA from RHAU-depleted HeLa cell lines, measuring both steady-state mRNA levels and mRNA half-lives by actinomycin D chase. There was no change in the half-lives of most transcripts whose steady-state levels were affected by RHAU knockdown, suggesting that these transcripts are subjected to transcriptional regulation. We propose that RHAU has a dual function, being involved in both the synthesis and degradation of mRNA in different subcellular compartments.

  20. Early nuclear exclusion of the transcription factor max is associated with retinal ganglion cell death independent of caspase activity.

    PubMed

    Petrs-Silva, Hilda; de Freitas, Fabíola G; Linden, Rafael; Chiarini, Luciana B

    2004-02-01

    We examined the behavior of the transcription factor Max during retrograde neuronal degeneration of retinal ganglion cells. Using immunohistochemistry, we found a progressive redistribution of full-length Max from the nucleus to the cytoplasm and dendrites of the ganglion cells following axon damage. Then, the axotomized cells lose all their content of Max, while undergoing nuclear pyknosis and apoptotic cell death. After treatment of retinal explants with either anisomycin or thapsigargin, the rate of nuclear exclusion of Max accompanied the rate of cell death as modulated by either drug. Treatment with a pan-caspase inhibitor abolished both TUNEL staining and immunoreactivity for activated caspase-3, but did not affect the subcellular redistribution of Max immunoreactivity after axotomy. The data show that nuclear exclusion of the transcription factor Max is an early event, which precedes and is independent of the activation of caspases, during apoptotic cell death in the central nervous system.

  1. Activation of the human mitochondrial transcription factor A gene by nuclear respiratory factors: a potential regulatory link between nuclear and mitochondrial gene expression in organelle biogenesis.

    PubMed Central

    Virbasius, J V; Scarpulla, R C

    1994-01-01

    Mitochondrial transcription factor A (mtTFA), the product of a nuclear gene, stimulates transcription from the two divergent mitochondrial promoters and is likely the principal activator of mitochondrial gene expression in vertebrates. Here we establish that the proximal promoter of the human mtTFA gene is highly dependent upon recognition sites for the nuclear respiratory factors, NRF-1 and NRF-2, for activity. These factors have been previously implicated in the activation of numerous nuclear genes that contribute to mitochondrial respiratory function. The affinity-purified factors from HeLa cells specifically bind to the mtTFA NRF-1 and NRF-2 sites through guanine nucleotide contacts that are characteristic for each site. Mutations in these contacts eliminate NRF-1 and NRF-2 binding and also dramatically reduce promoter activity in transfected cells. Although both factors contribute, NRF-1 binding appears to be the major determinant of promoter function. This dependence on NRF-1 activation is confirmed by in vitro transcription using highly purified recombinant proteins that display the same binding specificities as the HeLa cell factors. The activation of the mtTFA promoter by both NRF-1 and NRF-2 therefore provides a link between the expression of nuclear and mitochondrial genes and suggests a mechanism for their coordinate regulation during organelle biogenesis. Images PMID:8108407

  2. Nuclear localization domains of GATA activator Gln3 are required for transcription of target genes through dephosphorylation in Saccharomyces cerevisiae.

    PubMed

    Numamoto, Minori; Tagami, Shota; Ueda, Yusuke; Imabeppu, Yusuke; Sasano, Yu; Sugiyama, Minetaka; Maekawa, Hiromi; Harashima, Satoshi

    2015-08-01

    The GATA transcription activator Gln3 in the budding yeast (Saccharomyces cerevisiae) activates transcription of nitrogen catabolite repression (NCR)-sensitive genes. In cells grown in the presence of preferred nitrogen sources, Gln3 is phosphorylated in a TOR-dependent manner and localizes in the cytoplasm. In cells grown in non-preferred nitrogen medium or treated with rapamycin, Gln3 is dephosphorylated and is transported from the cytoplasm to the nucleus, thereby activating the transcription of NCR-sensitive genes. Caffeine treatment also induces dephosphorylation of Gln3 and its translocation to the nucleus and transcription of NCR-sensitive genes. However, the details of the mechanism by which phosphorylation controls Gln3 localization and transcriptional activity are unknown. Here, we focused on two regions of Gln3 with nuclear localization signal properties (NLS-K, and NLS-C) and one with nuclear export signal (NES). We constructed various mutants for our analyses: gln3 containing point mutations in all potential phosphoacceptor sites (Thr-339, Ser-344, Ser-347, Ser-355, Ser-391) in the NLS and NES regions to produce non-phosphorylatable (alanine) or mimic-phosphorylatable (aspartic acid) residues; and deletion mutants. We found that phosphorylation of Gln3 was impaired in all of these mutations and that the aspartic acid substitution mutants showed drastic reduction of Gln3-mediated transcriptional activity despite the fact that the mutations had no effect on nuclear localization of Gln3. Our observations suggest that these regions are required for transcription of target genes presumably through dephosphorylation.

  3. Lycopene activates antioxidant enzymes and nuclear transcription factor systems in heat-stressed broilers.

    PubMed

    Sahin, K; Orhan, C; Tuzcu, M; Sahin, N; Hayirli, A; Bilgili, S; Kucuk, O

    2016-05-01

    This study was conducted to evaluate the effects of dietary lycopene supplementation on growth performance, antioxidant status, and muscle nuclear transcription factor [Kelch like-ECH-associated protein 1 (Keap1) and (erythroid-derived 2)-like 2 (Nrf2)] expressions in broiler chickens exposed to heat stress (HS). A total of 180 one-day-old male broiler chicks (Ross 308) were assigned randomly to one of 2×3 factorially arranged treatments: two housing temperatures (22°C for 24 h/d; thermoneutral, TN or 34°C for 8 h/d HS) and three dietary lycopene levels (0, 200, or 400 mg/kg). Each treatment consisted of three replicates of 10 birds. Birds were reared to 42 d of age. Heat stress caused reductions in feed intake and weight gain by 12.2 and 20.7% and increased feed efficiency by 10.8% (P<0.0001 for all). Increasing dietary lycopene level improved performance in both environments. Birds reared under the HS environment had lower serum and muscle lycopene concentration (0.34 vs. 0.50 μg/mL and 2.80 vs. 2.13 μg/g), activities of superoxide dismutase (151 vs. 126 U/mL and 131 vs. 155 U/mg protein), glutathione peroxidase (184 vs. 154 U/mL and 1.39 vs. 1.74 U/mg protein), and higher malondialdehyde (MDA) concentration (0.53 vs. 0.83 μg/mL and 0.78 vs. 0.45 μg/ mg protein) than birds reared under the TN environment. Changes in levels of lycopene and MDA and activities of enzymes in serum and muscle varied by the environmental temperature as dietary lycopene level increased. Moreover, increasing dietary lycopene level suppressed muscle Keap1 expression and enhanced muscle Nrf2 expression, which had increased by 150% and decreased by 40%, respectively in response to HS. In conclusion, lycopene supplementation alleviates adverse effects of HS on performance through modulating expressions of stress-related nuclear transcription factors.

  4. A conserved RNA structural element within the hepatitis B virus post-transcriptional regulatory element enhance nuclear export of intronless transcripts and repress the splicing mechanism.

    PubMed

    Visootsat, Akasit; Payungporn, Sunchai; T-Thienprasert, Nattanan P

    2015-12-01

    Hepatitis B virus (HBV) infection is a primary cause of hepatocellular carcinoma and liver cirrhosis worldwide. To develop novel antiviral drugs, a better understanding of HBV gene expression regulation is vital. One important aspect is to understand how HBV hijacks the cellular machinery to export unspliced RNA from the nucleus. The HBV post-transcriptional regulatory element (HBV PRE) has been proposed to be the HBV RNA nuclear export element. However, the function remains controversial, and the core element is unclear. This study, therefore, aimed to identify functional regulatory elements within the HBV PRE and investigate their functions. Using bioinformatics programs based on sequence conservation and conserved RNA secondary structures, three regulatory elements were predicted, namely PRE 1151-1410, PRE 1520-1620 and PRE 1650-1684. PRE 1151-1410 significantly increased intronless and unspliced luciferase activity in both HepG2 and COS-7 cells. Likewise, PRE 1151-1410 significantly elevated intronless and unspliced HBV surface transcripts in liver cancer cells. Moreover, motif analysis predicted that PRE 1151-1410 contains several regulatory motifs. This study reported the roles of PRE 1151-1410 in intronless transcript nuclear export and the splicing mechanism. Additionally, these results provide knowledge in the field of HBV RNA regulation. Moreover, PRE 1151-1410 may be used to enhance the expression of other mRNAs in intronless reporter plasmids.

  5. Post-transcriptional regulation of meiotic genes by a nuclear RNA silencing complex

    PubMed Central

    Egan, Emily D.; Braun, Craig R.; Gygi, Steven P.; Moazed, Danesh

    2014-01-01

    RNA is a central component of gene-silencing pathways that regulate diverse cellular processes. In the fission yeast Schizosaccharomyces pombe, an RNA-based mechanism represses meiotic gene expression during vegetative growth. This pathway depends on the zinc finger protein Red1, which is required to degrade meiotic mRNAs as well as to target histone H3 lysine 9 (H3K9) methylation, a repressive chromatin mark, to a subset of meiotic genes. However, the mechanism of Red1 function is unknown. Here we use affinity purification and mass spectrometry to identify a Red1-containing nuclear RNA silencing (NURS) complex. In addition to Red1, this complex includes the Mtl1, Red5, Ars2, Rmn1, and Iss10 proteins and associates with several other complexes that are involved in either signaling or mediating RNA silencing. By analyzing the effects of gene knockouts and inducible knockdown alleles, we show that NURS subunits regulate RNA degradation and H3K9 methylation at meiotic genes. We also identify roles for individual NURS subunits in interactions with Mmi1, an RNA-binding protein that marks meiotic RNAs for destruction, and the nuclear exosome RNA degradation complex. Finally, we show that the levels of H3K9 methylation at meiotic genes are not sufficient to restrict RNA polymerase II access or repress gene expression during vegetative growth. Our results demonstrate that Red1 partners with other proteins to silence meiotic gene expression at the post-transcriptional level. Conservation of a NURS-like complex in human cells suggests that this pathway plays an ancient and fundamental role in RNA silencing. PMID:24713849

  6. Involvement of nuclear factor I transcription/replication factor in the early stage of chondrocytic differentiation.

    PubMed

    Uchihashi, Takayuki; Kimata, Masaaki; Tachikawa, Kanako; Koshimizu, Takao; Okada, Tomoko; Ihara-Watanabe, Miyuki; Sakai, Norio; Kogo, Mikihiko; Ozono, Keiichi; Michigami, Toshimi

    2007-12-01

    Gene-trap mutagenesis is based on the notion that the random insertion of a trapping vector may disturb the function of inserted genes. To identify the genes involved in chondrocytic differentiation, we applied this method to a murine mesenchymal cell line, ATDC5, which differentiate into mature chondrocytes in the presence of insulin, and isolated a clone in which the gene encoding a transcription/replication factor, nuclear factor I-B (NFIB), was trapped. In this particular clone, named #7-57, the trap vector pPT1-geo was inserted into intron 6 of the NFIB gene in one of the alleles. As a result, both wild-type NFIB and a mutant protein lacking the carboxyl-terminal transactivation/repression domain were expressed in the clone. Immunoprecipitation/Western blotting confirmed the interaction between wild-type NFIB and the truncated protein derived from the trapped allele, suggesting that the mutant protein formed a heterodimer with wild-type NFI proteins. When cultured in the differentiation medium, #7-57 exhibited impaired nodule formation and less accumulation of cartilageous matrices compared with the parental ATDC5 cells. In addition, the expression of marker genes for proliferating chondrocytes, including type II collagen (Col2a1), matrillin-1, and PTHrP, was reduced in the clone. The expression of SOX9 was also slightly decreased in the clone #7-57 compared with the parental cells. The overexpression of wild-type NFIB in parental ATDC5 cells resulted in the increased expression of Col2a1, and a series of reporter assays using a Col2a1 promoter/enhancer-luciferase construct demonstrated the transcriptional regulation of the gene by NFIB and the dominant-negative effect of the truncated mutant derived from the trapped allele. Interestingly, mutation in the SOX9-binding site in the 48-bp cis-element located in intron 1 failed to abolish the transactivation of Col2a1 gene by NFIB, suggesting that NFI regulates the transactivation of Col2a1, at least in part

  7. Amphibian interorder nuclear transfer embryos reveal conserved embryonic gene transcription, but deficient DNA replication or chromosome segregation.

    PubMed

    Narbonne, Patrick; Gurdon, John B

    2012-01-01

    Early interspecies nuclear transfer (iNT) experiments suggested that a foreign nucleus may become permanently damaged after a few rounds of cell division in the cytoplasm of another species. That is, in some distant species combinations, nucleocytoplasmic hybrid (cybrid) blastula nuclei can no longer support development, even if they are back-transferred into their own kind of egg cytoplasm. We monitored foreign DNA amplification and RNA production by quantitative PCR (qPCR) and RT-qPCR in interorder amphibian hybrids and cybrids formed by the transfer of newt (Pleurodeles waltl) embryonic nuclei into intact and enucleated frog (Xenopus laevis) eggs. We found a dramatic reduction in the expansion of foreign DNA and cell numbers in developing cybrid embryos that correlated with reduced gene transcription. Interestingly, expansion in cell numbers was rescued by the recipient species (Xenopus) maternal genome in iNT hybrids, but it did not improve P. waltl DNA expansion or gene transcription. Also, foreign gene transcripts, normalized to DNA copy numbers, were mostly normal in both iNT hybrids and cybrids. Thus, incomplete foreign DNA replication and/or chromosome segregation during cell division may be the major form of nuclear damage occurring as a result of nuclear replication in a foreign cytoplasmic environment. It also shows that the mechanisms of embryonic gene transcription are highly conserved across amphibians and may not be a major cause of cybrid lethality.

  8. Angiotensin II activates the proinflammatory transcription factor nuclear factor-kappaB in human monocytes.

    PubMed

    Kranzhöfer, R; Browatzki, M; Schmidt, J; Kübler, W

    1999-04-21

    The renin-angiotensin system may contribute to the pathogenesis of atherosclerosis. A common feature of all stages of atherosclerosis is inflammation of the vessel wall. The transcription factor nuclear factor-kappaB (NF-kappaB) participates in most signaling pathways involved in inflammation. This study therefore examined the effect of angiotensin (ANG) II on NF-kappaB activation in monocytic cells, a major cellular component of human atheroma, by electrophoretic mobility shift assay. ANG II, like TNFalpha, caused rapid activation of NF-kappaB in human mononuclear cells isolated from peripheral blood by Ficoll density gradient. This ANG II effect was blocked by the angiotensin AT1 receptor antagonist losartan. Specificity of ANG II-induced NF-kappaB activation was ascertained by supershift and competition experiments. Moreover, ANG II stimulated NF-kappaB activation in human monocytes, but not in lymphocytes from the same preparation. Together, the data demonstrate the ability of the vasoactive peptide ANG II to activate inflammatory pathways in human monocytes.

  9. Expression of prox1, lymphatic endothelial nuclear transcription factor, in Kaposiform hemangioendothelioma and tufted angioma.

    PubMed

    Le Huu, Aude Rimella; Jokinen, Chris H; Rubin, Brian P; Ruben, Brian P; Mihm, Martin C; Weiss, Sharon W; North, Paula E; Dadras, Soheil S

    2010-11-01

    Kaposiform hemangioendothelioma (KHE) and tufted angioma (TA) are rare tumors mainly occurring in early childhood. Our recent results showed that ectopic overexpression of human Prox1 gene, a lymphatic endothelial nuclear transcription factor, promoted an aggressive behavior in 2 murine models of KHE. This dramatic Prox1-induced phenotype prompted us to investigate immunohistochemical staining pattern of Prox1, podoplanin (D2-40), LYVE-1, and Prox1/CD34 as well as double immunofluorescent staining pattern of LYVE-1/CD31 in KHE and TA, compared with other pediatric vascular tumors. For this purpose, we examined 75 vascular lesions: KHE (n=18), TA (n=13), infantile hemangioma (n=13), pyogenic granuloma (n=18), and granulation tissue (n=13). Overall, KHE and TA shared an identical endothelial immunophenotype: the neoplastic spindle cells were Prox1, podoplanin, LYVE-1, CD31, and CD34, whereas endothelial cells within glomeruloid foci were Prox1, podoplanin, LYVE-1, CD31, and CD34. The lesional cells of all infantile hemangiomas and pyogenic granulomas were negative for Prox1 in the presence of positive internal control. These findings provide immunophenotypic evidence to support a preexisting notion that KHE and TA are closely related, if not identical. Overall, our results show, for the first time, that Prox1 is an immunohistochemical biomarker helpful in confirming the diagnosis of KHE/TA and in distinguishing it from infantile hemangioma and pyogenic granuloma.

  10. Rrp6: Integrated roles in nuclear RNA metabolism and transcription termination

    PubMed Central

    Fox, Melanie J.

    2015-01-01

    The yeast RNA exosome is a eukaryotic ribonuclease complex essential for RNA processing, surveillance and turnover. It is comprised of a barrel-shaped core and cap as well as a 3’–5’ ribonuclease known as Dis3 which contains both endo- and exonuclease domains. A second exonuclease, Rrp6, is added in the nucleus. Dis3 and Rrp6 have both shared and distinct roles in RNA metabolism, and this review will focus primarily on Rrp6 and the roles of the RNA exosome in the nucleus. The functions of the nuclear exosome are modulated by cofactors and interacting partners specific to each type of substrate. Generally, the cofactor TRAMP (Trf4/5-Air2/1-Mtr4 polyadenylation) complex helps unwind unstable RNAs, RNAs requiring processing such as rRNAs, tRNAs, or snRNAs or improperly processed RNAs and direct it toward the exosome. In yeast, Rrp6 interacts with Nrd1, the cap binding complex (CBC), and RNA Polymerase II to aid in nascent RNA processing, termination, and polyA tail length regulation. Recent studies have shown that proper termination and processing of short, non-coding RNAs by Rrp6 is particularly important for transcription regulation across the genome and has important implications for regulation of diverse processes at the cellular level. Loss of proper Rrp6 and exosome activity may contribute to various pathologies such as autoimmune disease, neurological disorders, and cancer. PMID:26612606

  11. Arsenic Trioxide Activate Transcription of Heme Oxygenase-1 by Promoting Nuclear Translocation of NFE2L2.

    PubMed

    Yue, Zhen; Zhong, Lingzhi; Mou, Yan; Wang, Xiaotong; Zhang, Haiying; Wang, Yang; Xia, Jianxin; Li, Ronggui; Wang, Zonggui

    2015-01-01

    In a previous study, we found that induced expression of Heme Oxygenase-1 (HO-1) is responsible for the resistance of human osteosarcoma MG63 cells to the chemotherapeutic agent arsenic trioxide (ATO). The present study was aimed at investigating the molecular mechanisms underlying the induction of HO-1 that occurs after exposure of MG63 cells to ATO. First, using RT-QPCT and Western-blot, we found that ATO strongly induced the expression of heme oxygenase-1 (HO-1) in these human osteosarcoma cells. Then by analyzing HO-1 mRNA of MG63 cells exposed to ATO in the presence and absence of a transcription inhibitor Actinomycin-D (Act-D), we demonstrated that ATO activates HO-1 expression in MG63 cells by regulating the transcription of the gene. Finally, through the analysis of the NFE2L2 protein levels among the total cellular and nuclear proteins by Western-blot and Immunocytochemical staning, we determined that ATO enhanced the nuclear translocation of nuclear factor erythroid 2-like 2 (NFE2L2), also known as Nrf2. From these results we have concluded that transcription activation of HO-1 resulting from the nuclear translocation of NFE2L2 is the underlying molecular mechanism for its high induction, which, in turn, is responsible for the resistance of human osteosarcoma cells to ATO treatment.

  12. The second AT-hook of the architectural transcription factor HMGA2 is determinant for nuclear localization and function

    PubMed Central

    Cattaruzzi, Giacomo; Altamura, Sandro; Tessari, Michela A.; Rustighi, Alessandra; Giancotti, Vincenzo; Pucillo, Carlo; Manfioletti, Guidalberto

    2007-01-01

    High Mobility Group A (HMGA) is a family of architectural nuclear factors which play an important role in neoplastic transformation. HMGA proteins are multifunctional factors that associate both with DNA and nuclear proteins that have been involved in several nuclear processes including transcription. HMGA localization is exclusively nuclear but, to date, the mechanism of nuclear import for these proteins remains unknown. Here, we report the identification and characterization of a nuclear localization signal (NLS) for HMGA2, a member of the HMGA family. The NLS overlaps with the second of the three AT-hooks, the DNA-binding domains characteristic for this group of proteins. The functionality of this NLS was demonstrated by its ability to target a heterologous β-galactosidase/green fluorescent protein fusion protein to the nucleus. Mutations to alanine of basic residues within the second AT-hook resulted in inhibition of HMGA2 nuclear localization and impairment of its function in activating the cyclin A promoter. In addition, HMGA2 was shown to directly interact with the nuclear import receptor importin-α2 via the second AT-hook. HMGA proteins are overexpressed and rearranged in a variety of tumors; our findings can thus help elucidating their role in neoplastic transformation. PMID:17324944

  13. Transcriptional Regulation by Nuclear Corepressors and PGC-1α: Implications for Mitochondrial Quality Control and Insulin Sensitivity

    PubMed Central

    Qi, Zhengtang; Ding, Shuzhe

    2012-01-01

    The peroxisome proliferator-activated receptors (PPARs) and estrogen-related receptor (ERRα) are ligand-activated nuclear receptors that coordinately regulate gene expression. Recent evidence suggests that nuclear corepressors, NCoR, RIP140, and SMRT, repress nuclear receptors-mediated transcriptional activity on specific promoters, and thus regulate insulin sensitivity, adipogenesis, mitochondrial number, and activity in vivo. Moreover, the coactivator PGC-1α that increases mitochondrial biogenesis during exercise and calorie restriction directly regulates autophagy in skeletal muscle and mitophagy in the pathogenesis of Parkinson's disease. In this paper, we discuss the PGC-1α's novel role in mitochondrial quality control and the role of nuclear corepressors in regulating insulin sensitivity and interacting with PGC-1α. PMID:23304112

  14. Morris Water Maze Training in Mice Elevates Hippocampal Levels of Transcription Factors Nuclear Factor (Erythroid-derived 2)-like 2 and Nuclear Factor Kappa B p65

    PubMed Central

    Snow, Wanda M.; Pahlavan, Payam S.; Djordjevic, Jelena; McAllister, Danielle; Platt, Eric E.; Alashmali, Shoug; Bernstein, Michael J.; Suh, Miyoung; Albensi, Benedict C.

    2015-01-01

    Research has identified several transcription factors that regulate activity-dependent plasticity and memory, with cAMP-response element binding protein (CREB) being the most well-studied. In neurons, CREB activation is influenced by the transcription factor nuclear factor kappa B (NF-κB), considered central to immunity but more recently implicated in memory. The transcription factor early growth response-2 (Egr-2), an NF-κB gene target, is also associated with learning and memory. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), an antioxidant transcription factor linked to NF-κB in pathological conditions, has not been studied in normal memory. Given that numerous transcription factors implicated in activity-dependent plasticity demonstrate connections to NF-κB, this study simultaneously evaluated protein levels of NF-κB, CREB, Egr-2, Nrf2, and actin in hippocampi from young (1 month-old) weanling CD1 mice after training in the Morris water maze, a hippocampal-dependent spatial memory task. After a 6-day acquisition period, time to locate the hidden platform decreased in the Morris water maze. Mice spent more time in the target vs. non-target quadrants of the maze, suggestive of recall of the platform location. Western blot data revealed a decrease in NF-κB p50 protein after training relative to controls, whereas NF-κB p65, Nrf2 and actin increased. Nrf2 levels were correlated with platform crosses in nearly all tested animals. These data demonstrate that training in a spatial memory task results in alterations in and associations with particular transcription factors in the hippocampus, including upregulation of NF-κB p65 and Nrf2. Training-induced increases in actin protein levels caution against its use as a loading control in immunoblot studies examining activity-dependent plasticity, learning, and memory. PMID:26635523

  15. A highly organized structure mediating nuclear localization of a Myb2 transcription factor in the protozoan parasite Trichomonas vaginalis.

    PubMed

    Chu, Chien-Hsin; Chang, Lung-Chun; Hsu, Hong-Ming; Wei, Shu-Yi; Liu, Hsing-Wei; Lee, Yu; Kuo, Chung-Chi; Indra, Dharmu; Chen, Chinpan; Ong, Shiou-Jeng; Tai, Jung-Hsiang

    2011-12-01

    Nuclear proteins usually contain specific peptide sequences, referred to as nuclear localization signals (NLSs), for nuclear import. These signals remain unexplored in the protozoan pathogen, Trichomonas vaginalis. The nuclear import of a Myb2 transcription factor was studied here using immunodetection of a hemagglutinin-tagged Myb2 overexpressed in the parasite. The tagged Myb2 was localized to the nucleus as punctate signals. With mutations of its polybasic sequences, 48KKQK51 and 61KR62, Myb2 was localized to the nucleus, but the signal was diffusive. When fused to a C-terminal non-nuclear protein, the Myb2 sequence spanning amino acid (aa) residues 48 to 143, which is embedded within the R2R3 DNA-binding domain (aa 40 to 156), was essential and sufficient for efficient nuclear import of a bacterial tetracycline repressor (TetR), and yet the transport efficiency was reduced with an additional fusion of a firefly luciferase to TetR, while classical NLSs from the simian virus 40 T-antigen had no function in this assay system. Myb2 nuclear import and DNA-binding activity were substantially perturbed with mutation of a conserved isoleucine (I74) in helix 2 to proline that altered secondary structure and ternary folding of the R2R3 domain. Disruption of DNA-binding activity alone by point mutation of a lysine residue, K51, preceding the structural domain had little effect on Myb2 nuclear localization, suggesting that nuclear translocation of Myb2, which requires an ordered structural domain, is independent of its DNA binding activity. These findings provide useful information for testing whether myriad Mybs in the parasite use a common module to regulate nuclear import.

  16. Nuclear respiratory factors 1 and 2 utilize similar glutamine-containing clusters of hydrophobic residues to activate transcription.

    PubMed Central

    Gugneja, S; Virbasius, C M; Scarpulla, R C

    1996-01-01

    Nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) are ubiquitous transcription factors that have been implicated in the control of nuclear genes required for respiration, heme biosynthesis, and mitochondrial DNA transcription and replication. Recently, both factors have been found to be major transcriptional determinants for a subset of these genes that define a class of simple promoters involved in respiratory chain expression. Here, functional domains required for transactivation by NRF-1 have been defined. An atypical nuclear localization signal resides in a conserved amino-terminal region adjacent to the DNA binding domain and consists of functionally redundant clusters of basic residues. A second domain in the carboxy-terminal half of the molecule is necessary for transcriptional activation. The activation domains of both NRF-1 and NRF-2 were extensively characterized by both deletion and alanine substitution mutagenesis. The results show that these domains do not fall into known classes defined by a preponderance of amino acid residues, including glutamines, prolines, or isoleucines, as found in other eukaryotic activators. Rather, in both factors, a series of tandemly arranged clusters of hydrophobic amino acids were required for activation. Although all of the functional clusters contain glutamines, the glutamines differ from the hydrophobic residues in that they are inconsequential for activation. Unlike the NRF-2 domain, which contains its essential hydrophobic motifs within 40 residues, the NRF-1 domain spans about 40% of the molecule and appears to have a bipartite structure. The findings indicate that NRF-1 and NRF-2 utilize similar hydrophobic structural motifs for activating transcription. PMID:8816484

  17. Acute β-Adrenergic Activation Triggers Nuclear Import of Histone Deacetylase 5 and Delays Gq-induced Transcriptional Activation*

    PubMed Central

    Chang, Chia-Wei Jenny; Lee, Linda; Yu, David; Dao, Khanha; Bossuyt, Julie; Bers, Donald M.

    2013-01-01

    During hemodynamic stress, catecholamines and neurohumoral stimuli may induce co-activation of Gq-coupled receptors and β-adrenergic receptors (β-AR), leading to cardiac remodeling. Dynamic regulation of histone deacetylase 5 (HDAC5), a transcriptional repressor, is crucial during stress signaling due to its role in epigenetic control of fetal gene markers. Little is known about its regulation during acute and chronic β-AR stimulation and its cross-interaction with Gq signaling in adult cardiac myocytes. Here, we evaluate the potential cross-talk between Gq-driven and β-AR mediated signaling at the level of nucleocytoplasmic shuttling of HDAC5. We show the translocation of GFP-tagged wild type HDAC5 or mutants (S279A and S279D) in response to β-AR or Gq agonists. Isoproterenol (ISO) or PKA activation results in strong nuclear accumulation of HDAC5 in contrast to nuclear export driven by Ca2+-calmodulin protein kinase II and protein kinase D. Moreover, nuclear accumulation of HDAC5 under acute ISO/PKA signaling is dependent on phosphorylation of Ser-279 and can block subsequent Gq-mediated nuclear HDAC5 export. Intriguingly, the attenuation of Gq-induced export is abolished after chronic PKA activation, yet nuclear HDAC5 remains elevated. Last, the effect of chronic β-AR signaling on HDAC5 translocation was examined in adult myocytes from a rabbit model of heart failure, where ISO-induced nuclear import is ablated, but Gq-agonist mediated export is preserved. Acute β-AR/PKA activation protects against hypertrophic signaling by delaying Gq-mediated transcriptional activation. This serves as a key physiological control switch before allowing genetic reprogramming via HDAC5 nuclear export during more severe stress, such as heart failure. PMID:23161540

  18. Gamma Interferon-Dependent Transcriptional Memory via Relocalization of a Gene Locus to PML Nuclear Bodies▿ †

    PubMed Central

    Gialitakis, Manolis; Arampatzi, Panagiota; Makatounakis, Takis; Papamatheakis, Joseph

    2010-01-01

    Memory of past cellular responses is an essential adaptation to repeating environmental stimuli. We addressed the question of whether gamma interferon (IFN-γ)-inducible transcription generates memory that sensitizes cells to a second stimulus. We have found that the major histocompatibility complex class II gene DRA is relocated to promyelocytic leukemia (PML) nuclear bodies upon induction with IFN-γ, and this topology is maintained long after transcription shut off. Concurrent interaction of PML protein with mixed-lineage leukemia generates a prolonged permissive chromatin state on the DRA gene characterized by high promoter histone H3 K4 dimethylation that facilitates rapid expression upon restimulation. We propose that the primary signal-induced transcription generates spatial and epigenetic memory that is maintained through several cell generations and endows the cell with increased responsiveness to future activation signals. PMID:20123968

  19. Nuclear translocation of doublecortin-like protein kinase and phosphorylation of a transcription factor JDP2

    SciTech Connect

    Nagamine, Tadashi; Nomada, Shohgo; Onouchi, Takashi; Kameshita, Isamu; Sueyoshi, Noriyuki

    2014-03-28

    Highlights: • Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase. • In living cells, DCLK was cleaved into two functional fragments. • zDCLK(kinase) was translocated into the nucleus by osmotic stresses. • Jun dimerization protein 2 (JDP2) was identified as zDCLK(kinase)-binding protein. • JDP2 was efficiently phosphorylated by zDCLK(kinase) only when histone was present. - Abstract: Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase predominantly expressed in brain. In a previous paper, we reported that zebrafish DCLK2 (zDCLK) was cleaved into two functional fragments; the N-terminal zDCLK(DC + SP) with microtubule-binding activity and the C-terminal zDCLK(kinase) with a Ser/Thr protein kinase activity. In this study, we demonstrated that zDCLK(kinase) was widely distributed in the cytoplasm and translocated into the nucleus when the cells were treated under hyperosmotic conditions with NaCl or mannitol. By two-hybrid screening using the C-terminal domain of DCLK, Jun dimerization protein 2 (JDP2), a nuclear transcription factor, was identified as zDCLK(kinase)-binding protein. Furthermore, JDP2 served as an efficient substrate for zDCLK(kinase) only when histone was present. These results suggest that the kinase fragment of DCLK is translocated into the nucleus upon hyperosmotic stresses and that the kinase efficiently phosphorylates JDP2, a possible target in the nucleus, with the aid of histones.

  20. Nuclear translocation of {alpha}N-catenin by the novel zinc finger transcriptional repressor ZASC1

    SciTech Connect

    Bogaerts, Sven; Vanlandschoot, Ann; Hengel, Jolanda van; Roy, Frans van . E-mail: F.Vanroy@dmbr.UGent.be

    2005-11-15

    Alpha-catenins anchor the transmembrane cell-cell adhesion molecule E-cadherin indirectly to the actin cytoskeleton through interaction with {beta}-catenin or plakoglobin. Three different {alpha}-catenins are known at present: {alpha}E-, {alpha}T-, and {alpha}N-catenin. Despite their different expression patterns, no functional differences between the {alpha}-catenins are known. In a yeast two-hybrid screening with {alpha}N-catenin as bait, we identified the Cys{sub 2}-His{sub 2} zinc finger protein ZASC1. The mRNA and protein of ZASC1 were ubiquitously expressed in various cell lines and human tissues. Our results suggest an association of the ZASC1 protein with DNA, and luciferase reporter assays revealed that ZASC1 is a transcriptional repressor. Upon transient overexpression, the ZASC1 protein localized in the nucleus, to where it was able to recruit cytoplasmic {alpha}N-catenin. Neither the highly related {alpha}E-catenin nor {alpha}T-catenin interacted with ZASC1. By interchanging parts of {alpha}N-catenin and {alpha}E-catenin cDNAs, we were able to narrow down the interaction region of {alpha}N-catenin to two limited amino-terminal regions. On the other hand, the interaction of ZASC1 with {alpha}N-catenin can be mediated by the domain comprising zinc fingers six to eight of ZASC1. The interaction and nuclear cotranslocation of a neural {alpha}-catenin with a putative proto-oncogene product as reported here provides novel insights into the signaling functions of {alpha}-catenins.

  1. Differential effects of culture and nuclear transfer on relative transcript levels of genes with key roles during preimplantation.

    PubMed

    Moreira, P N; Fernández-Gonzalez, R; Ramirez, M A; Pérez-Crespo, M; Rizos, D; Pintado, B; Gutiérrez-Adán, A

    2006-02-01

    It is well known that the preimplantation culture environment to which embryos are exposed influences the expression of developmentally important genes. Recently, it has been reported that MEMalpha, a culture medium commonly used for somatic cells, allows high rates of preimplantation development and development to term of mouse somatic cell nuclear transfer (SCNT) embryos. The objective of this study was to compare the differential effects of this medium and of the nuclear transfer procedure on the relative mRNA abundance of several genes with key roles during preimplantation. The relative mRNA levels of nine genes (Glut 1, Glut 5, G6PDH, Bax, Survivin, Gpx 1, Oct4, mTert and IGF2bp1) were quantified at blastocyst stage on cumulus cell cloned embryos cultured in MEMalpha, as well as on in vivo cultured and MEMalpha cultured controls. Only three of the nine transcripts analysed (Glut 5, Gpx 1 and Igf2bp1) were significantly down-regulated at blastocyst stage in in vitro produced controls. However, most genes analysed in our MEMalpha cultured cloned embryos showed altered transcription levels. Interestingly, between cloned and in vitro produced controls only the transcription levels measured for Glut 1 were significantly different. This result suggests that Glut 1 may be a good marker for embryo quality after cumulus cell nuclear transfer.

  2. Two proteolytic fragments of menin coordinate the nuclear transcription and postsynaptic clustering of neurotransmitter receptors during synaptogenesis between Lymnaea neurons

    PubMed Central

    Getz, Angela M.; Visser, Frank; Bell, Erin M.; Xu, Fenglian; Flynn, Nichole M.; Zaidi, Wali; Syed, Naweed I.

    2016-01-01

    Synapse formation and plasticity depend on nuclear transcription and site-specific protein targeting, but the molecular mechanisms that coordinate these steps have not been well defined. The MEN1 tumor suppressor gene, which encodes the protein menin, is known to induce synapse formation and plasticity in the CNS. This synaptogenic function has been conserved across evolution, however the underlying molecular mechanisms remain unidentified. Here, using central neurons from the invertebrate Lymnaea stagnalis, we demonstrate that menin coordinates subunit-specific transcriptional regulation and synaptic clustering of nicotinic acetylcholine receptors (nAChR) during neurotrophic factor (NTF)-dependent excitatory synaptogenesis, via two proteolytic fragments generated by calpain cleavage. Whereas menin is largely regarded as a nuclear protein, our data demonstrate a novel cytoplasmic function at central synapses. Furthermore, this study identifies a novel synaptogenic mechanism in which a single gene product coordinates the nuclear transcription and postsynaptic targeting of neurotransmitter receptors through distinct molecular functions of differentially localized proteolytic fragments. PMID:27538741

  3. Functional Analysis of Adenovirus Protein IX Identifies Domains Involved in Capsid Stability, Transcriptional Activity, and Nuclear Reorganization

    PubMed Central

    Rosa-Calatrava, Manuel; Grave, Linda; Puvion-Dutilleul, Francine; Chatton, Bruno; Kedinger, Claude

    2001-01-01

    The product of adenovirus (Ad) type 5 gene IX (pIX) is known to actively participate in the stability of the viral icosahedron, acting as a capsid cement. We have previously demonstrated that pIX is also a transcriptional activator of several viral and cellular TATA-containing promoters, likely contributing to the transactivation of the Ad expression program. By extensive mutagenesis, we have now delineated the functional domains involved in each of the pIX properties: residues 22 to 26 of the highly conserved N-terminal domain are crucial for incorporation of the protein into the virion; specific residues of the C-terminal leucine repeat are responsible for pIX interactions with itself and possibly other proteins, a property that is critical for pIX transcriptional activity. We also show that pIX takes part in the virus-induced nuclear reorganization of late infected cells: the protein induces, most likely through self-assembly, the formation of specific nuclear structures which appear as dispersed nuclear globules by immunofluorescence staining and as clear amorphous spherical inclusions by electron microscopy. The integrity of the leucine repeat appears to be essential for the formation and nuclear retention of these inclusions. Together, our results demonstrate the multifunctional nature of pIX and provide new insights into Ad biology. PMID:11435594

  4. Nuclear pore complex evolution: a trypanosome Mlp analogue functions in chromosomal segregation but lacks transcriptional barrier activity.

    PubMed

    Holden, Jennifer M; Koreny, Ludek; Obado, Samson; Ratushny, Alexander V; Chen, Wei-Ming; Chiang, Jung-Hsien; Kelly, Steven; Chait, Brian T; Aitchison, John D; Rout, Michael P; Field, Mark C

    2014-05-01

    The nuclear pore complex (NPC) has dual roles in nucleocytoplasmic transport and chromatin organization. In many eukaryotes the coiled-coil Mlp/Tpr proteins of the NPC nuclear basket have specific functions in interactions with chromatin and defining specialized regions of active transcription, whereas Mlp2 associates with the mitotic spindle/NPC in a cell cycle-dependent manner. We previously identified two putative Mlp-related proteins in African trypanosomes, TbNup110 and TbNup92, the latter of which associates with the spindle. We now provide evidence for independent ancestry for TbNup92/TbNup110 and Mlp/Tpr proteins. However, TbNup92 is required for correct chromosome segregation, with knockout cells exhibiting microaneuploidy and lowered fidelity of telomere segregation. Further, TbNup92 is intimately associated with the mitotic spindle and spindle anchor site but apparently has minimal roles in control of gene transcription, indicating that TbNup92 lacks major barrier activity. TbNup92 therefore acts as a functional analogue of Mlp/Tpr proteins, and, together with the lamina analogue NUP-1, represents a cohort of novel proteins operating at the nuclear periphery of trypanosomes, uncovering complex evolutionary trajectories for the NPC and nuclear lamina.

  5. Nuclear localization of a putative Phytophthora sojae bZIP1 transcription factor is mediated by multiple targeting motifs.

    PubMed

    Fang, Yufeng; Tyler, Brett M

    2017-02-18

    Oomycetes are fungal-like eukaryotic microbes in the kingdom Stramenopila. We recently found that the oomycete plant pathogen Phytophthora sojae uses nuclear localization signals (NLSs) for translocation of proteins into the nucleus that differ from conventional well-characterized NLSs from mammals and yeast. Here we have characterized in depth the nuclear localization signals of a P. sojae basic leucine zipper transcription factor, PsbZIP1. Nuclear localization of PsbZIP1 was determined by a central conserved region overlapping the DNA binding domain. Mutational analysis of this region identified four distinct elements that contributed multiplicatively to nuclear localization, but the conserved DNA binding residues were not required. Three of the elements showed autonomous NLS activity and the fourth served as a nuclear localization enhancer. Sequences within two of the nuclear localization elements defined a new form of bipartite NLS consisting of a triplet of basic residues followed by a tail of scattered basic amino acids. This article is protected by copyright. All rights reserved.

  6. Overexpression of the Transcriptional Repressor Complex BCL-6/BCoR Leads to Nuclear Aggregates Distinct from Classical Aggresomes

    PubMed Central

    Buchberger, Elisabeth; El Harchi, Miriam; Payrhuber, Dietmar; Zommer, Anna; Schauer, Dominic; Simonitsch-Klupp, Ingrid; Bilban, Martin; Brostjan, Christine

    2013-01-01

    Nuclear inclusions of aggregated proteins have primarily been characterized for molecules with aberrant poly-glutamine repeats and for mutated or structurally altered proteins. They were termed “nuclear aggresomes” and misfolding was shown to promote association with molecular chaperones and proteasomes. Here, we report that two components of a transcriptional repressor complex (BCL-6 and BCoR) of wildtype amino acid sequence can independently or jointly induce the formation of nuclear aggregates when overexpressed. The observation that the majority of cells rapidly downregulate BCL-6/BCoR levels, supports the notion that expression of these proteins is under tight control. The inclusions occur when BCL-6/BCoR expression exceeds 150-fold of endogenous levels. They preferentially develop in the nucleus by a gradual increase in aggregate size to form large, spheroid structures which are not associated with heat shock proteins or marked by ubiquitin. In contrast, we find the close association of BCL-6/BCoR inclusions with PML bodies and a reduction in aggregation upon the concomitant overexpression of histone deacetylases or heat shock protein 70. In summary, our data offer a perspective on nuclear aggregates distinct from classical “nuclear aggresomes”: Large complexes of spheroid structure can evolve in the nucleus without being marked by the cellular machinery for protein refolding and degradation. However, nuclear proteostasis can be restored by balancing the levels of chaperones. PMID:24146931

  7. Release of positive transcription elongation factor b (P-TEFb) from 7SK small nuclear ribonucleoprotein (snRNP) activates hexamethylene bisacetamide-inducible protein (HEXIM1) transcription.

    PubMed

    Liu, Pingyang; Xiang, Yanhui; Fujinaga, Koh; Bartholomeeusen, Koen; Nilson, Kyle A; Price, David H; Peterlin, B Matija

    2014-04-04

    By phosphorylating negative elongation factors and the C-terminal domain of RNA polymerase II (RNAPII), positive transcription elongation factor b (P-TEFb), which is composed of CycT1 or CycT2 and CDK9, activates eukaryotic transcription elongation. In growing cells, it is found in active and inactive forms. In the former, free P-TEFb is a potent transcriptional coactivator. In the latter, it is inhibited by HEXIM1 or HEXIM2 in the 7SK small nuclear ribonucleoprotein (snRNP), which contains, additionally, 7SK snRNA, methyl phosphate-capping enzyme (MePCE), and La-related protein 7 (LARP7). This P-TEFb equilibrium determines the state of growth and proliferation of the cell. In this study, the release of P-TEFb from the 7SK snRNP led to increased synthesis of HEXIM1 but not HEXIM2 in HeLa cells, and this occurred only from an unannotated, proximal promoter. ChIP with sequencing revealed P-TEFb-sensitive poised RNA polymerase II at this proximal but not the previously annotated distal HEXIM1 promoter. Its immediate upstream sequences were fused to luciferase reporters and were found to be responsive to many P-TEFb-releasing compounds. The superelongation complex subunits AF4/FMR2 family member 4 (AFF4) and elongation factor RNA polymerase II 2 (ELL2) were recruited to this proximal promoter after P-TEFb release and were required for its transcriptional effects. Thus, P-TEFb regulates its own equilibrium in cells, most likely to maintain optimal cellular homeostasis.

  8. Nuclear localization of γ-tubulin affects E2F transcriptional activity and S-phase progression

    PubMed Central

    Höög, Greta; Zarrizi, Reihaneh; von Stedingk, Kristoffer; Jonsson, Kristina; Alvarado-Kristensson, Maria

    2011-01-01

    We show that the centrosome- and microtubule-regulating protein γ-tubulin interacts with E2 promoter binding factors (E2Fs) to modulate E2F transcriptional activity and thereby control cell cycle progression. γ-Tubulin contains a C-terminal signal that results in its translocation to the nucleus during late G1 to early S phase. γ-Tubulin mutants showed that the C terminus interacts with the transcription factor E2F1 and that the E2F1–γ-tubulin complex is formed during the G1/S transition, when E2F1 is transcriptionally active. Furthermore, E2F transcriptional activity is altered by reduced expression of γ-tubulin or by complex formation between γ-tubulin and E2F1, E2F2, or E2F3, but not E2F6. In addition, the γ-tubulin C terminus encodes a DNA-binding domain that interacts with E2F-regulated promoters, resulting in γ-tubulin-mediated transient activation of E2Fs. Thus, we report a novel mechanism regulating the activity of E2Fs, which can help explain how these proteins affect cell cycle progression in mammalian cells.—Höög, G., Zarrizi, R., von Stedingk, K., Jonsson, K., Alvarado-Kristensson, M. Nuclear localization of γ-tubulin affects E2F transcriptional activity and S-phase progression. PMID:21788450

  9. Analysis of the Heat Shock Response in Mouse Liver Reveals Transcriptional Dependence on the Nuclear Receptor Peroxisome Proliferator-Activated Receptor alpha (PPARα)

    EPA Science Inventory

    BACKGROUND: The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha) regulates responses to chemical or physical stress in part by altering expression of genes involved in proteome maintenance. Many of these genes are also transcriptionally regulated by h...

  10. The Elongation Complex Components BRD4 and MLLT3/AF9 Are Transcriptional Coactivators of Nuclear Retinoid Receptors

    PubMed Central

    Flajollet, Sébastien; Rachez, Christophe; Ploton, Maheul; Schulz, Céline; Gallais, Rozenn; Métivier, Raphaël; Pawlak, Michal; Leray, Aymeric; Issulahi, Al Amine; Héliot, Laurent; Staels, Bart; Salbert, Gilles; Lefebvre, Philippe

    2013-01-01

    Nuclear all-trans retinoic acid receptors (RARs) initiate early transcriptional events which engage pluripotent cells to differentiate into specific lineages. RAR-controlled transactivation depends mostly on agonist-induced structural transitions in RAR C-terminus (AF-2), thus bridging coactivators or corepressors to chromatin, hence controlling preinitiation complex assembly. However, the contribution of other domains of RAR to its overall transcriptional activity remains poorly defined. A proteomic characterization of nuclear proteins interacting with RAR regions distinct from the AF-2 revealed unsuspected functional properties of the RAR N-terminus. Indeed, mass spectrometry fingerprinting identified the Bromodomain-containing protein 4 (BRD4) and ALL1-fused gene from chromosome 9 (AF9/MLLT3), known to associate with and regulates the activity of Positive Transcription Elongation Factor b (P-TEFb), as novel RAR coactivators. In addition to promoter sequences, RAR binds to genomic, transcribed regions of retinoid-regulated genes, in association with RNA polymerase II and as a function of P-TEFb activity. Knockdown of either AF9 or BRD4 expression affected differentially the neural differentiation of stem cell-like P19 cells. Clusters of retinoid-regulated genes were selectively dependent on BRD4 and/or AF9 expression, which correlated with RAR association to transcribed regions. Thus RAR establishes physical and functional links with components of the elongation complex, enabling the rapid retinoid-induced induction of genes required for neuronal differentiation. Our data thereby extends the previously known RAR interactome from classical transcriptional modulators to components of the elongation machinery, and unravel a functional role of RAR in transcriptional elongation. PMID:23762261

  11. Nuclear sequestration of COL1A1 mRNA transcript associated with type I osteogenesis imperfecta (OI)

    SciTech Connect

    Primorac, D.; Stover, M.L.; McKinstry, M.B.

    1994-09-01

    Previously we identified an OI type I patient with a splice donor mutation that resulted in intron 26 retention instead of exon skipping and sequestration of normal levels of the mutant transcript in the nuclear compartment. Intron retention was consistent with the exon definition hypothesis for splice site selection since the size of the exon-intron-exon unit was less than 300 bp. Furthermore, the retained intron contained in-frame stop codons which is thought to cause the mutant RNA to remain within the nucleus rather than appearing in the cytoplasm. To test these hypotheses, genomic fragments containing the normal sequence or the donor mutation were cloned into a collagen minigene and expressed in stably tansfected NIH 3T3 cells. None of the modifications to the normal intron altered the level of RNA that accumulated in the cytoplasm, as expected. However none of the modifications to the mutant intron allowed accumulation of normal levels of mRNA in the cytoplasm. Moreover, in contrast to our findings in the patient`s cells only low levels of mutant transcript were found in the nucleus; a fraction of the transcript did appear in the cytoplasm which had spliced the mutant donor site correctly. Nuclear run-on experiments demonstrated equal levels of transcription from each transgene. Expression of another donor mutation known to cause in-frame exon skipping in OI type IV was accurately reproduced in the minigene in transfected 3T3 cells. Our experience suggests that either mechanism can lead to formation of a null allele possibly related to the type of splicing events surrounding the potential stop codons. Understanding the rules governing inactivation of a collagen RNA transcript may be important in designing a strategy to inactivate a dominate negative mutation associated with the more severe forms of OI.

  12. The RNA Helicases AtMTR4 and HEN2 Target Specific Subsets of Nuclear Transcripts for Degradation by the Nuclear Exosome in Arabidopsis thaliana

    PubMed Central

    Lange, Heike; Zuber, Hélène; Sement, François M.; Chicher, Johana; Kuhn, Lauriane; Hammann, Philippe; Brunaud, Véronique; Bérard, Caroline; Bouteiller, Nathalie; Balzergue, Sandrine; Aubourg, Sébastien; Martin-Magniette, Marie-Laure; Vaucheret, Hervé; Gagliardi, Dominique

    2014-01-01

    The RNA exosome is the major 3′-5′ RNA degradation machine of eukaryotic cells and participates in processing, surveillance and turnover of both nuclear and cytoplasmic RNA. In both yeast and human, all nuclear functions of the exosome require the RNA helicase MTR4. We show that the Arabidopsis core exosome can associate with two related RNA helicases, AtMTR4 and HEN2. Reciprocal co-immunoprecipitation shows that each of the RNA helicases co-purifies with the exosome core complex and with distinct sets of specific proteins. While AtMTR4 is a predominantly nucleolar protein, HEN2 is located in the nucleoplasm and appears to be excluded from nucleoli. We have previously shown that the major role of AtMTR4 is the degradation of rRNA precursors and rRNA maturation by-products. Here, we demonstrate that HEN2 is involved in the degradation of a large number of polyadenylated nuclear exosome substrates such as snoRNA and miRNA precursors, incompletely spliced mRNAs, and spurious transcripts produced from pseudogenes and intergenic regions. Only a weak accumulation of these exosome substrate targets is observed in mtr4 mutants, suggesting that MTR4 can contribute, but plays rather a minor role for the degradation of non-ribosomal RNAs and cryptic transcripts in Arabidopsis. Consistently, transgene post-transcriptional gene silencing (PTGS) is marginally affected in mtr4 mutants, but increased in hen2 mutants, suggesting that it is mostly the nucleoplasmic exosome that degrades aberrant transgene RNAs to limit their entry in the PTGS pathway. Interestingly, HEN2 is conserved throughout green algae, mosses and land plants but absent from metazoans and other eukaryotic lineages. Our data indicate that, in contrast to human and yeast, plants have two functionally specialized RNA helicases that assist the exosome in the degradation of specific nucleolar and nucleoplasmic RNA populations, respectively. PMID:25144737

  13. Heterogeneous nuclear ribonucleoprotein A1 in health and neurodegenerative disease: from structural insights to post-transcriptional regulatory roles.

    PubMed

    Bekenstein, Uriya; Soreq, Hermona

    2013-09-01

    Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of conserved nuclear proteins that associate with nascent RNA polymerase II transcripts to yield hnRNP particles, playing key roles in mRNA metabolism, DNA-related functions and microRNA biogenesis. HnRNPs accompany transcripts from stages of transcriptional regulation through splicing and post-transcriptional regulation, and are believed to affect the majority of expressed genes in mammals. Most hnRNP mRNA transcripts undergo alternative splicing and post-translational modifications, to yield a remarkable diversity of proteins with numerous functional elements that work in concert in their multiple functions. Therefore, mis-regulation of hnRNPs leads to different maladies. Here, we focus on the role of one of the best-known members of this protein family, hnRNP A1 in RNA metabolism, and address recent works that note its multileveled involvement in several neurodegenerative disorders. Initially discovered as a DNA binding protein, hnRNP A1 includes two RNA recognition motifs, and post-translational modifications of these and other regions in this multifunctional protein alter both its nuclear pore shuttling properties and its RNA interactions and affect transcription, mRNA splicing and microRNA biogenesis. HnRNP A1 plays several key roles in neuronal functioning and its depletion, either due to debilitated cholinergic neurotransmission or under autoimmune reactions causes drastic changes in RNA metabolism. Consequently, hnRNP A1 decline contributes to the severity of symptoms in several neurodegenerative diseases, including Alzheimer's disease (AD), spinal muscular atrophy (SMA), fronto-temporal lobar degeneration (FTLD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), hereditary spastic paraparesis (HSP) and HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP). At the translational level, these properties of hnRNP A1 led to massive research efforts aimed at developing RNA

  14. Neuronal expression of nuclear transcription factor MafG in the rat medulla oblongata after baroreceptor stimulation.

    PubMed

    Kumaki, Iku; Yang, Dawei; Koibuchi, Noriyuki; Takayama, Kiyoshige

    2006-03-06

    The medulla oblongata is the site of central baroreceptive neurons in mammals. These neurons express specific basic-leucine zipper transcription factors (bZIP) after baroreceptor stimulation. Previously we showed that activation of baroreceptors induced expression of nuclear transcription factors c-Fos and FosB in central baroreceptive neurons. Here we studied the effects of baroreceptor stimulation on induction of MafG, a member of small Maf protein family that functions as dimeric partners for various bZIP transcription factors by forming transcription-regulating complexes, in the rat medulla oblongata. To determine whether gene expression of MafG is induced by stimulation of arterial baroreceptors, we examined the expression of its mRNA by semi-quantitative reverse transcription-PCR method and its gene product by immunohistochemistry. We found that the number of MafG transcripts increased significantly in the medulla oblongata after baroreceptor stimulation. MafG-immunoreactive neurons were distributed in the nucleus tractus solitarii, the dorsal motor nucleus of the vagus nerve, the ambiguous nucleus and the ventrolateral medulla. The numbers of MafG-immunoreactive neurons in these nuclei were significantly greater in test rats than in saline-injected control rats. We also found approximately 20% of MafG-immunoreactive neurons coexpress FosB after baroreceptor stimulation. Our results suggest that MafG cooperates with FosB to play critical roles as an immediate early gene in the signal transduction of cardiovascular regulation mediated by baroreceptive signals in the medulla oblongata.

  15. SRY-box-containing Gene 2 Regulation of Nuclear Receptor Tailless (Tlx) Transcription in Adult Neural Stem Cells*

    PubMed Central

    Shimozaki, Koji; Zhang, Chun-Li; Suh, Hoonkyo; Denli, Ahmet M.; Evans, Ronald M.; Gage, Fred H.

    2012-01-01

    Adult neurogenesis is maintained by self-renewable neural stem cells (NSCs). Their activity is regulated by multiple signaling pathways and key transcription factors. However, it has been unclear whether these factors interplay with each other at the molecular level. Here we show that SRY-box-containing gene 2 (Sox2) and nuclear receptor tailless (TLX) form a molecular network in adult NSCs. We observed that both Sox2 and TLX proteins bind to the upstream region of Tlx gene. Sox2 positively regulates Tlx expression, whereas the binding of TLX to its own promoter suppresses its transcriptional activity in luciferase reporter assays. Such TLX-mediated suppression can be antagonized by overexpressing wild-type Sox2 but not a mutant lacking the transcriptional activation domain. Furthermore, through regions involved in DNA-binding activity, Sox2 and TLX physically interact to form a complex on DNAs that contain a consensus binding site for TLX. Finally, depletion of Sox2 revealed the potential negative feedback loop of TLX expression that is antagonized by Sox2 in adult NSCs. These data suggest that Sox2 plays an important role in Tlx transcription in cultured adult NSCs. PMID:22194602

  16. Identification, molecular cloning, and transcription analysis of the Choristoneura fumiferana nuclear polyhedrosis virus spindle-like protein gene.

    PubMed

    Liu, J J; Carstens, E B

    1996-09-15

    The Choristoneura fumiferana nuclear polyhedrosis virus spindle-like protein (slp) gene has been identified and localized immediately downstream and in the same orientation as the CfMNPV DNA polymerase gene. The slp gene is 1101 bp long, predicted to code for a 366 amino acid (42.1 kDa) polypeptide. Transcriptional analysis revealed that the CfMNPV slp gene is expressed at late times postinfection, beginning at 24 hr postinfection and is most abundantly expressed after 36 hr. Transcription initiates within a single baculovirus consensus late start site sequence (GTAAG) at position -18 relative to the translation start codon. Based on amino acid comparisons, the CfMNPV gene is closely related to other similar baculovirus genes and distantly but recognizably related to the fusolin proteins of two entomopoxviruses. The conservation of amino acid sequence, glycosylation signals and specific domains throughout the protein suggest that this gene product may play an important role in insect DNA virus replication.

  17. Mutations in nuclear genes alter post-transcriptional regulation of mitochondrial genes.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nuclear gene products are required for the expression of mitochondrial genes and elaboration of functional mitochondrial protein complexes. To better understand the roles of these nuclear genes, we exploited the mitochondrial encoded S-type of cytoplasmic male sterility (CMS-S) and developed a nove...

  18. The transcriptional activity of hepatocyte nuclear factor 4 alpha is inhibited via phosphorylation by ERK1/2.

    PubMed

    Vető, Borbála; Bojcsuk, Dóra; Bacquet, Caroline; Kiss, Judit; Sipeki, Szabolcs; Martin, Ludovic; Buday, László; Bálint, Bálint L; Arányi, Tamás

    2017-01-01

    Hepatocyte nuclear factor 4 alpha (HNF4α) nuclear receptor is a master regulator of hepatocyte development, nutrient transport and metabolism. HNF4α is regulated both at the transcriptional and post-transcriptional levels by different mechanisms. Several kinases (PKA, PKC, AMPK) were shown to phosphorylate and decrease the activity of HNF4α. Activation of the ERK1/2 signalling pathway, inducing proliferation and survival, inhibits the expression of HNF4α. However, based on our previous results we hypothesized that HNF4α is also regulated at the post-transcriptional level by ERK1/2. Here we show that ERK1/2 is capable of directly phosphorylating HNF4α in vitro at several phosphorylation sites including residues previously shown to be targeted by other kinases, as well. Furthermore, we also demonstrate that phosphorylation of HNF4α leads to a reduced trans-activational capacity of the nuclear receptor in luciferase reporter gene assay. We confirm the functional relevance of these findings by demonstrating with ChIP-qPCR experiments that 30-minute activation of ERK1/2 leads to reduced chromatin binding of HNF4α. Accordingly, we have observed decreasing but not disappearing binding of HNF4α to the target genes. In addition, 24-hour activation of the pathway further decreased HNF4α chromatin binding to specific loci in ChIP-qPCR experiments, which confirms the previous reports on the decreased expression of the HNF4a gene due to ERK1/2 activation. Our data suggest that the ERK1/2 pathway plays an important role in the regulation of HNF4α-dependent hepatic gene expression.

  19. The transcriptional activity of hepatocyte nuclear factor 4 alpha is inhibited via phosphorylation by ERK1/2

    PubMed Central

    Bacquet, Caroline; Kiss, Judit; Sipeki, Szabolcs; Martin, Ludovic; Buday, László; Bálint, Bálint L.; Arányi, Tamás

    2017-01-01

    Hepatocyte nuclear factor 4 alpha (HNF4α) nuclear receptor is a master regulator of hepatocyte development, nutrient transport and metabolism. HNF4α is regulated both at the transcriptional and post-transcriptional levels by different mechanisms. Several kinases (PKA, PKC, AMPK) were shown to phosphorylate and decrease the activity of HNF4α. Activation of the ERK1/2 signalling pathway, inducing proliferation and survival, inhibits the expression of HNF4α. However, based on our previous results we hypothesized that HNF4α is also regulated at the post-transcriptional level by ERK1/2. Here we show that ERK1/2 is capable of directly phosphorylating HNF4α in vitro at several phosphorylation sites including residues previously shown to be targeted by other kinases, as well. Furthermore, we also demonstrate that phosphorylation of HNF4α leads to a reduced trans-activational capacity of the nuclear receptor in luciferase reporter gene assay. We confirm the functional relevance of these findings by demonstrating with ChIP-qPCR experiments that 30-minute activation of ERK1/2 leads to reduced chromatin binding of HNF4α. Accordingly, we have observed decreasing but not disappearing binding of HNF4α to the target genes. In addition, 24-hour activation of the pathway further decreased HNF4α chromatin binding to specific loci in ChIP-qPCR experiments, which confirms the previous reports on the decreased expression of the HNF4a gene due to ERK1/2 activation. Our data suggest that the ERK1/2 pathway plays an important role in the regulation of HNF4α-dependent hepatic gene expression. PMID:28196117

  20. Multiple NUCLEAR FACTOR Y Transcription Factors Respond to Abiotic Stress in Brassica napus L

    PubMed Central

    Xu, Li; Lin, Zhongyuan; Tao, Qing; Liang, Mingxiang; Zhao, Gengmao; Yin, Xiangzhen; Fu, Ruixin

    2014-01-01

    Members of the plant NUCLEAR FACTOR Y (NF-Y) family are composed of the NF-YA, NF-YB, and NF-YC subunits. In Brassica napus (canola), each of these subunits forms a multimember subfamily. Plant NF-Ys were reported to be involved in several abiotic stresses. In this study, we demonstrated that multiple members of thirty three BnNF-Ys responded rapidly to salinity, drought, or ABA treatments. Transcripts of five BnNF-YAs, seven BnNF-YBs, and two BnNF-YCs were up-regulated by salinity stress, whereas the expression of thirteen BnNF-YAs, ten BnNF-YBs, and four BnNF-YCs were induced by drought stress. Under NaCl treatments, the expression of one BnNF-YA10 and four NF-YBs (BnNF-YB3, BnNF-YB7, BnNF-YB10, and BnNF-YB14) were greatly increased. Under PEG treatments, the expression levels of four NF-YAs (BnNF-YA9, BnNF-YA10, BnNF-YA11, and BnNF-YA12) and five NF-YBs (BnNF-YB1, BnNF-YB8, BnNF-YB10, BnNF-YB13, and BnNF-YB14) were greatly induced. The expression profiles of 20 of the 27 salinity- or drought-induced BnNF-Ys were also affected by ABA treatment. The expression levels of six NF-YAs (BnNF-YA1, BnNF-YA7, BnNF-YA8, BnNF-YA9, BnNF-YA10, and BnNF-YA12) and seven BnNF-YB members (BnNF-YB2, BnNF-YB3, BnNF-YB7, BnNF-YB10, BnNF-YB11, BnNF-YB13, and BnNF-YB14) and two NF-YC members (BnNF-YC2 and BnNF-YC3) were greatly up-regulated by ABA treatments. Only a few BnNF-Ys were inhibited by the above three treatments. Several NF-Y subfamily members exhibited collinear expression patterns. The promoters of all stress-responsive BnNF-Ys harbored at least two types of stress-related cis-elements, such as ABRE, DRE, MYB, or MYC. The cis-element organization of BnNF-Ys was similar to that of Arabidopsis thaliana, and the promoter regions exhibited higher levels of nucleotide sequence identity with Brassica rapa than with Brassica oleracea. This work represents an entry point for investigating the roles of canola NF-Y proteins during abiotic stress responses and provides insight into

  1. Multiple NUCLEAR FACTOR Y transcription factors respond to abiotic stress in Brassica napus L.

    PubMed

    Xu, Li; Lin, Zhongyuan; Tao, Qing; Liang, Mingxiang; Zhao, Gengmao; Yin, Xiangzhen; Fu, Ruixin

    2014-01-01

    Members of the plant NUCLEAR FACTOR Y (NF-Y) family are composed of the NF-YA, NF-YB, and NF-YC subunits. In Brassica napus (canola), each of these subunits forms a multimember subfamily. Plant NF-Ys were reported to be involved in several abiotic stresses. In this study, we demonstrated that multiple members of thirty three BnNF-Ys responded rapidly to salinity, drought, or ABA treatments. Transcripts of five BnNF-YAs, seven BnNF-YBs, and two BnNF-YCs were up-regulated by salinity stress, whereas the expression of thirteen BnNF-YAs, ten BnNF-YBs, and four BnNF-YCs were induced by drought stress. Under NaCl treatments, the expression of one BnNF-YA10 and four NF-YBs (BnNF-YB3, BnNF-YB7, BnNF-YB10, and BnNF-YB14) were greatly increased. Under PEG treatments, the expression levels of four NF-YAs (BnNF-YA9, BnNF-YA10, BnNF-YA11, and BnNF-YA12) and five NF-YBs (BnNF-YB1, BnNF-YB8, BnNF-YB10, BnNF-YB13, and BnNF-YB14) were greatly induced. The expression profiles of 20 of the 27 salinity- or drought-induced BnNF-Ys were also affected by ABA treatment. The expression levels of six NF-YAs (BnNF-YA1, BnNF-YA7, BnNF-YA8, BnNF-YA9, BnNF-YA10, and BnNF-YA12) and seven BnNF-YB members (BnNF-YB2, BnNF-YB3, BnNF-YB7, BnNF-YB10, BnNF-YB11, BnNF-YB13, and BnNF-YB14) and two NF-YC members (BnNF-YC2 and BnNF-YC3) were greatly up-regulated by ABA treatments. Only a few BnNF-Ys were inhibited by the above three treatments. Several NF-Y subfamily members exhibited collinear expression patterns. The promoters of all stress-responsive BnNF-Ys harbored at least two types of stress-related cis-elements, such as ABRE, DRE, MYB, or MYC. The cis-element organization of BnNF-Ys was similar to that of Arabidopsis thaliana, and the promoter regions exhibited higher levels of nucleotide sequence identity with Brassica rapa than with Brassica oleracea. This work represents an entry point for investigating the roles of canola NF-Y proteins during abiotic stress responses and provides insight into

  2. Structural dynamics of the cell nucleus: basis for morphology modulation of nuclear calcium signaling and gene transcription.

    PubMed

    Queisser, Gillian; Wiegert, Simon; Bading, Hilmar

    2011-01-01

    Neuronal morphology plays an essential role in signal processing in the brain. Individual neurons can undergo use-dependent changes in their shape and connectivity, which affects how intracellular processes are regulated and how signals are transferred from one cell to another in a neuronal network. Calcium is one of the most important intracellular second messengers regulating cellular morphologies and functions. In neurons, intracellular calcium levels are controlled by ion channels in the plasma membrane such as NMDA receptors (NMDARs), voltage-gated calcium channels (VGCCs) and certain α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) as well as by calcium exchange pathways between the cytosol and internal calcium stores including the endoplasmic reticulum and mitochondria. Synaptic activity and the subsequent opening of ligand and/or voltage-gated calcium channels can initiate cytosolic calcium transients which propagate towards the cell soma and enter the nucleus via its nuclear pore complexes (NPCs) embedded in the nuclear envelope. We recently described the discovery that in hippocampal neurons the morphology of the nucleus affects the calcium dynamics within the nucleus. Here we propose that nuclear infoldings determine whether a nucleus functions as an integrator or detector of oscillating calcium signals. We outline possible ties between nuclear mophology and transcriptional activity and discuss the importance of extending the approach to whole cell calcium signal modeling in order to understand synapse-to-nucleus communication in healthy and dysfunctional neurons.

  3. Viral and host cellular transcription in Autographa californica nuclear polyhedrosis virus-infected gypsy moth cell lines.

    PubMed Central

    Guzo, D; Rathburn, H; Guthrie, K; Dougherty, E

    1992-01-01

    Infection of two gypsy moth cell lines (IPLB-Ld652Y and IPLB-LdFB) by the Autographa californica multiple-enveloped nuclear polyhedrosis virus (AcMNPV) is characterized by extremely attenuated viral protein synthesis followed by a total arrest of all viral and cellular protein production. In this study, AcMNPV- and host cell-specific transcription were examined. Overall levels of viral RNAs in infected gypsy moth cells were, at most measured times, comparable to RNA levels from an infected cell line (TN-368) permissive for AcMNPV replication. Northern blot (RNA) analyses using viral and host gene-specific probes revealed predominantly normal-length virus- and cell-specific transcripts postinfection. Transport of viral RNAs from the nucleus to the cytoplasm and transcript stability in infected gypsy moth cells also appeared normal compared with similar parameters for AcMNPV-infected TN-368 cells. Host cellular and viral mRNAs extracted from gypsy moth and TN-368 cells at various times postinfection and translated in vitro yielded similar spectra of host and viral proteins. Treatment of infected gypsy moth cells with the DNA synthesis inhibitor aphidicolin eliminated the total protein synthesis shutoff in infected IPLB-LdFB cells but had no effect on protein synthesis inhibition in infected IPLB-Ld652Y cells. The apparent selective block in the translation of viral transcripts early in infection and the absence of normal translation or transcription of host cellular genes at later times is discussed. Images PMID:1560533

  4. Anti-Nuclear Antibody Production and Autoimmunity in Transgenic Mice that Over-Express the Transcription Factor Bright

    PubMed Central

    Shankar, Malini; Nixon, Jamee C.; Maier, Shannon; Workman, Jennifer; Farris, A. Darise; Webb, Carol F.

    2009-01-01

    The B cell-restricted transcription factor, Bright, up-regulates immunoglobulin heavy chain transcription three- to seven-fold in activated B cells in vitro. Bright function is dependent upon both active Bruton’s tyrosine kinase and its substrate, the transcription factor, TFII-I. In mouse and human B lymphocytes, Bright transcription is down regulated in mature B cells, and its expression is tightly regulated during B cell differentiation. To determine how Bright expression affects B cell development, transgenic mice were generated that express Bright constitutively in all B lineage cells. These mice exhibited increases in total B220+ B lymphocyte lineage cells in the bone marrow, but the relative percentages of the individual subpopulations were not altered. Splenic immature transitional B cells were significantly expanded both in total cell numbers and as increased percentages of cells relative to other B cell subpopulations. Serum immunoglobulin levels, particularly IgG isotypes, were increased slightly in the Bright transgenic mice compared to littermate controls. However, immunization studies suggest that responses to all foreign antigens were not increased globally. Moreover, four week-old Bright transgenic mice produced anti-nuclear antibodies. Older animals developed antibody deposits in the kidney glomeruli, but did not succumb to further autoimmune sequelae. These data indicate that enhanced Bright expression results in failure to maintain B cell tolerance and suggest a previously unappreciated role for Bright regulation in immature B cells. Bright is the first B cell-restricted transcription factor demonstrated to induce autoimmunity. Therefore, the Bright transgenics provide a novel model system for future analyses of B cell autoreactivity. PMID:17312145

  5. Dexamethasone inhibits human interleukin 2 but not interleukin 2 receptor gene expression in vitro at the level of nuclear transcription.

    PubMed Central

    Boumpas, D T; Anastassiou, E D; Older, S A; Tsokos, G C; Nelson, D L; Balow, J E

    1991-01-01

    Glucocorticosteroids have an inhibitory effect on the expression of interleukin 2 (IL-2) and interleukin 2 receptor (IL-2R) genes. To determine the mechanisms of this inhibition, human T lymphocytes were stimulated with mitogens in the presence of dexamethasone. Nuclear transcription run-off assays showed that high doses of dexamethasone inhibited the transcription of the IL-2 gene but not that of the IL-2R gene. Post-transcriptionally, high doses of dexamethasone (10(-4) M) were required to inhibit IL-2R mRNA levels by 50%, whereas lower doses (10(-6) M) inhibited by greater than 70% the accumulation of IL-2 mRNA. IL-2 mRNA half-life decreased in the presence of dexamethasone (10(-6) M) by approximately 50%. At the protein product level, dexamethasone inhibited both IL-2 production, as well as cell surface and soluble forms of IL-2R. IL-2R gene expression was inhibited for at least 72 h after exposure of cells to dexamethasone. In the presence of exogenous IL-2, dexamethasone failed to exert a significant effect on the production of IL-2R protein. These data indicate that dexamethasone has a greater effect on the expression of the IL-2 gene than on the IL-2R gene. Dexamethasone both inhibits transcription of the IL-2 gene and decreases the stability of IL-2 mRNA. The effect of dexamethasone on the IL-2R gene is post-transcriptional and may result indirectly from decreased IL-2 production. Images PMID:2022743

  6. Total cellular protein presence of the transcription factor IRF8 does not necessarily correlate with its nuclear presence.

    PubMed

    Minderman, Hans; Maguire, Orla; O'Loughlin, Kieran L; Muhitch, Jason; Wallace, Paul K; Abrams, Scott I

    2017-01-01

    The transcription factor interferon regulatory factor-8 (IRF8) plays an essential role in myeloid differentiation and lineage commitment, based largely on molecular and genetic studies. The detection of IRF8 in specific cell populations by flow cytometry (FCM) has the potential to provide new insights into normal and pathologic myelopoiesis, but critical validation of this protein-based approach, particularly in human samples, is lacking. In this study, the assessment of total cellular IRF8 presence was compared to its specific nuclear presence as assessed by imaging flow cytometry (IFC) analysis. Peptide neutralization of the IRF8-specific antibody that has been predominantly used to date in the literature served as a negative control for the immunofluorescent labeling. Expression of total IRF8 was analyzed by total cellular fluorescence analogous to the mean fluorescence intensity readout of conventional FCM. Additionally, specific nuclear fluorescence and the similarity score between the nuclear image (DAPI) and the corresponding IRF8 image for each cell were analyzed as parameters for nuclear localization of IRF8. IFC showed that peptide blocking eliminated binding of the IRF8 antibody in the nucleus. It also reduced cytoplasmic binding of the antibody but not to the extent observed in the nucleus. In agreement with the similarity score data, the total cellular IRF8 as well as nuclear IRF8 intensities decreased with peptide blocking. In healthy donor peripheral blood subpopulations and a positive control cell line (THP-1), the assessment of IRF8 by total cellular presence correlated well with its specific nuclear presence and correlated with the known distribution of IRF8 in these cells. In clinical samples of myeloid-derived suppressors cells derived from patients with renal carcinoma, however, total cellular IRF8 did not necessarily correlate with its nuclear presence. Discordance was primarily associated with peptide blocking having a proportionally greater

  7. The C-terminal domain-phosphorylated IIO form of RNA polymerase II is associated with the transcription repressor NC2 (Dr1/DRAP1) and is required for transcription activation in human nuclear extracts.

    PubMed

    Castaño, E; Gross, P; Wang, Z; Roeder, R G; Oelgeschläger, T

    2000-06-20

    Activation of class II gene transcription may involve alleviation of transcription repression as well as stimulation of the assembly and function of the general RNA polymerase (RNAP) II transcription machinery. Here, we investigated whether activator-reversible transcription repression by NC2 (Dr1/DRAP1) contributes to maximum induction levels in unfractionated HeLa nuclear extracts. Surprisingly, we found that depletion of NC2 does not significantly affect basal transcription, but dramatically reduces activated transcription. Immunoblot analyses revealed that the loss of activator function coincides with selective removal of the C-terminal domain (CTD)-hyperphosphorylated RNAP IIO along with NC2. Coimmunoprecipitation experiments with purified factors confirmed that NC2 interacts with RNAP IIO, but not with the unphosphorylated or hypophosphorylated RNAP IIA or CTD-less RNAP IIB forms. Finally, we demonstrate that, in contrast to previously published observations in cell-free systems reconstituted with purified factors, only the CTD-phosphorylated form of RNAP II can mediate activator function in the context of unfractionated HeLa nuclear extracts. These findings reveal an unexpected link between NC2 and transcription activation and suggest that regulation of RNAP II transcription through reversible CTD phosphorylation might be more complex than previously proposed.

  8. Cytoplasmic and nuclear quality control and turnover of single-stranded RNA modulate post-transcriptional gene silencing in plants.

    PubMed

    Moreno, Ana Beatriz; Martínez de Alba, Angel Emilio; Bardou, Florian; Crespi, Martin D; Vaucheret, Hervé; Maizel, Alexis; Mallory, Allison C

    2013-04-01

    Eukaryotic RNA quality control (RQC) uses both endonucleolytic and exonucleolytic degradation to eliminate dysfunctional RNAs. In addition, endogenous and exogenous RNAs are degraded through post-transcriptional gene silencing (PTGS), which is triggered by the production of double-stranded (ds)RNAs and proceeds through short-interfering (si)RNA-directed ARGONAUTE-mediated endonucleolytic cleavage. Compromising cytoplasmic or nuclear 5'-3' exoribonuclease function enhances sense-transgene (S)-PTGS in Arabidopsis, suggesting that these pathways compete for similar RNA substrates. Here, we show that impairing nonsense-mediated decay, deadenylation or exosome activity enhanced S-PTGS, which requires host RNA-dependent RNA polymerase 6 (RDR6/SGS2/SDE1) and SUPPRESSOR OF GENE SILENCING 3 (SGS3) for the transformation of single-stranded RNA into dsRNA to trigger PTGS. However, these RQC mutations had no effect on inverted-repeat-PTGS, which directly produces hairpin dsRNA through transcription. Moreover, we show that these RQC factors are nuclear and cytoplasmic and are found in two RNA degradation foci in the cytoplasm: siRNA-bodies and processing-bodies. We propose a model of single-stranded RNA tug-of-war between RQC and S-PTGS that ensures the correct partitioning of RNA substrates among these RNA degradation pathways.

  9. Gene Resistance to Transcriptional Reprogramming following Nuclear Transfer Is Directly Mediated by Multiple Chromatin-Repressive Pathways.

    PubMed

    Jullien, Jerome; Vodnala, Munender; Pasque, Vincent; Oikawa, Mami; Miyamoto, Kei; Allen, George; David, Sarah Anne; Brochard, Vincent; Wang, Stan; Bradshaw, Charles; Koseki, Haruhiko; Sartorelli, Vittorio; Beaujean, Nathalie; Gurdon, John

    2017-03-02

    Understanding the mechanism of resistance of genes to reactivation will help improve the success of nuclear reprogramming. Using mouse embryonic fibroblast nuclei with normal or reduced DNA methylation in combination with chromatin modifiers able to erase H3K9me3, H3K27me3, and H2AK119ub1 from transplanted nuclei, we reveal the basis for resistance of genes to transcriptional reprogramming by oocyte factors. A majority of genes is affected by more than one type of treatment, suggesting that resistance can require repression through multiple epigenetic mechanisms. We classify resistant genes according to their sensitivity to 11 chromatin modifier combinations, revealing the existence of synergistic as well as adverse effects of chromatin modifiers on removal of resistance. We further demonstrate that the chromatin modifier USP21 reduces resistance through its H2AK119 deubiquitylation activity. Finally, we provide evidence that H2A ubiquitylation also contributes to resistance to transcriptional reprogramming in mouse nuclear transfer embryos.

  10. Epstein-Barr virus nuclear antigen 3A protein regulates CDKN2B transcription via interaction with MIZ-1.

    PubMed

    Bazot, Quentin; Deschamps, Thibaut; Tafforeau, Lionel; Siouda, Maha; Leblanc, Pascal; Harth-Hertle, Marie L; Rabourdin-Combe, Chantal; Lotteau, Vincent; Kempkes, Bettina; Tommasino, Massimo; Gruffat, Henri; Manet, Evelyne

    2014-09-01

    The Epstein-Barr virus (EBV) nuclear antigen 3 family of protein is critical for the EBV-induced primary B-cell growth transformation process. Using a yeast two-hybrid screen we identified 22 novel cellular partners of the EBNA3s. Most importantly, among the newly identified partners, five are known to play direct and important roles in transcriptional regulation. Of these, the Myc-interacting zinc finger protein-1 (MIZ-1) is a transcription factor initially characterized as a binding partner of MYC. MIZ-1 activates the transcription of a number of target genes including the cell cycle inhibitor CDKN2B. Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus. Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A. Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1.

  11. Sensitive detection of transcription factors in cell nuclear extracts by using a molecular beacons based amplification strategy.

    PubMed

    Zhang, Kai; Wang, Ke; Zhu, Xue; Xie, Minhao

    2016-03-15

    Monitoring transcription factor (TF) levels provides an important assessment of the state of cell populations. Unfortunately, traditional methods for monitoring TF concentration are generally cumbersome and time-consuming. We developed an ultrasensitive one-pot TF detection method that uses target-molecular beacons-dependent amplification (TMDA) fluorescence strategy to circumvent the aforementioned limitations in TF detection. In this assay, we employed a DNA1/DNA2 duplex as the reporting probe and a stem-loop DNA molecular beacon (MB) as the signaling probe. The integration of protein-DNA1/DNA2 duplex and exonuclease III (Exo III) digestion can convert the detection of transcription factors to the detection of reporter oligonucleotides. The subsequent hybridization of the reporter oligonucleotides with the molecular beacons opens the stem-loop structure. The formation of the DNA complex triggers amplification reaction and the recovery of the fluorescence. This assay exhibits high sensitivity with a detection limit of 2.2 pM and a detection range of 3 orders of magnitude, which is superior to most currently used methods for transcription factor detection. More importantly, this method is suitable for the direct detection of TFs in crude nuclear extracts of cancer cells.

  12. Epstein–Barr virus nuclear antigen 3A protein regulates CDKN2B transcription via interaction with MIZ-1

    PubMed Central

    Bazot, Quentin; Deschamps, Thibaut; Tafforeau, Lionel; Siouda, Maha; Leblanc, Pascal; Harth-Hertle, Marie L.; Rabourdin-Combe, Chantal; Lotteau, Vincent; Kempkes, Bettina; Tommasino, Massimo; Gruffat, Henri; Manet, Evelyne

    2014-01-01

    The Epstein–Barr virus (EBV) nuclear antigen 3 family of protein is critical for the EBV-induced primary B-cell growth transformation process. Using a yeast two-hybrid screen we identified 22 novel cellular partners of the EBNA3s. Most importantly, among the newly identified partners, five are known to play direct and important roles in transcriptional regulation. Of these, the Myc-interacting zinc finger protein-1 (MIZ-1) is a transcription factor initially characterized as a binding partner of MYC. MIZ-1 activates the transcription of a number of target genes including the cell cycle inhibitor CDKN2B. Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus. Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A. Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1. PMID:25092922

  13. Nuclear Perilipin 5 integrates lipid droplet lipolysis with PGC-1α/SIRT1-dependent transcriptional regulation of mitochondrial function

    PubMed Central

    Gallardo-Montejano, Violeta I.; Saxena, Geetu; Kusminski, Christine M.; Yang, Chaofeng; McAfee, John L.; Hahner, Lisa; Hoch, Kathleen; Dubinsky, William; Narkar, Vihang A.; Bickel, Perry E.

    2016-01-01

    Dysfunctional cellular lipid metabolism contributes to common chronic human diseases, including type 2 diabetes, obesity, fatty liver disease and diabetic cardiomyopathy. How cells balance lipid storage and mitochondrial oxidative capacity is poorly understood. Here we identify the lipid droplet protein Perilipin 5 as a catecholamine-triggered interaction partner of PGC-1α. We report that during catecholamine-stimulated lipolysis, Perilipin 5 is phosphorylated by protein kinase A and forms transcriptional complexes with PGC-1α and SIRT1 in the nucleus. Perilipin 5 promotes PGC-1α co-activator function by disinhibiting SIRT1 deacetylase activity. We show by gain-and-loss of function studies in cells that nuclear Perilipin 5 promotes transcription of genes that mediate mitochondrial biogenesis and oxidative function. We propose that Perilipin 5 is an important molecular link that couples the coordinated catecholamine activation of the PKA pathway and of lipid droplet lipolysis with transcriptional regulation to promote efficient fatty acid catabolism and prevent mitochondrial dysfunction. PMID:27554864

  14. Expression of Nuclear Transcription Factor Kappa B in Locally Advanced Human Cervical Cancer Treated With Definitive Chemoradiation

    SciTech Connect

    Garg, Amit K.; Jhingran, Anuja; Klopp, Ann H.; Aggarwal, Bharat B.; Kunnumakkara, Ajai B.; Broadus, Russell R.; Eifel, Patricia J.; Buchholz, Thomas A.

    2010-12-01

    Purpose: Nuclear factor kappa B (NF-{kappa}B), a transcriptional factor that has been shown to be constitutively active in cervical cancer, is part of an important pathway leading to treatment resistance in many tumor types. The purpose of our study was to determine whether expression of NF-{kappa}B in pretreatment specimens and specimens taken shortly after treatment initiation correlated with outcome in cervical cancer patients treated with definitive chemoradiation. Methods and Materials: Eighteen patients with locally advanced cervical cancer were enrolled in a study in which cervical biopsy specimens were obtained before radiation therapy and 48 h after treatment initiation. Matched biopsy specimens from 16 of these patients were available and evaluated for the nuclear expression of NF-{kappa}B protein by immunohistochemical staining. Results: After a median follow-up of 43 months, there were 9 total treatment failures. Nuclear staining for NF-{kappa}B was positive in 3 of 16 pretreatment biopsy specimens (19%) and 5 of 16 postradiation biopsy specimens (31%). Pretreatment expression of NF-{kappa}B nuclear staining correlated with increased rates of local-regional failure (100% vs. 23%, p = 0.01), distant failure (100% vs. 38%, p = 0.055), disease-specific mortality (100% vs. 31%, p = 0.03), and overall mortality (100% vs. 38%, p = 0.055). Conclusions: Our data suggest that pretreatment nuclear expression of NF-{kappa}B may be associated with a poor outcome for cervical cancer patients treated with chemoradiation. Although these data require validation in a larger group of patients, the results support the continued study of the relationship between NF-{kappa}B and outcome in patients treated for carcinoma of the cervix.

  15. Transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR.

    PubMed

    Calkin, Anna C; Tontonoz, Peter

    2012-03-14

    Nuclear receptors are integrators of hormonal and nutritional signals, mediating changes to metabolic pathways within the body. Given that modulation of lipid and glucose metabolism has been linked to diseases including type 2 diabetes, obesity and atherosclerosis, a greater understanding of pathways that regulate metabolism in physiology and disease is crucial. The liver X receptors (LXRs) and the farnesoid X receptors (FXRs) are activated by oxysterols and bile acids, respectively. Mounting evidence indicates that these nuclear receptors have essential roles, not only in the regulation of cholesterol and bile acid metabolism but also in the integration of sterol, fatty acid and glucose metabolism.

  16. Transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR

    PubMed Central

    2013-01-01

    Nuclear receptors are integrators of hormonal and nutritional signals, mediating changes to metabolic pathways within the body. Given that modulation of lipid and glucose metabolism has been linked to diseases including type 2 diabetes, obesity and atherosclerosis, a greater understanding of pathways that regulate metabolism in physiology and disease is crucial. The liver X receptors (LXRs) and the farnesoid X receptors (FXRs) are activated by oxysterols and bile acids, respectively. Mounting evidence indicates that these nuclear receptors have essential roles, not only in the regulation of cholesterol and bile acid metabolism but also in the integration of sterol, fatty acid and glucose metabolism. PMID:22414897

  17. Transcription Factor EB Is Selectively Reduced in the Nuclear Fractions of Alzheimer's and Amyotrophic Lateral Sclerosis Brains

    PubMed Central

    Wang, Hongjie

    2016-01-01

    Multiple studies suggest that autophagy is strongly dysregulated in Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), as evidenced by accumulation of numerous autophagosomes, lysosomes with discontinuous membranes, and aggregated proteins in the patients' brains. Transcription factor EB (TFEB) was recently discovered to be a master regulator of lysosome biogenesis and autophagy. To examine whether aberrant autophagy in AD and ALS is due to alterations in TFEB expression, we systematically quantified the levels of TFEB in these brains by immunoblotting. Interestingly, cytoplasmic fractions of AD brains showed increased levels of normalized (to tubulin) TFEB only at Braak stage IV (61%, p < 0.01). Most importantly, normalized (to lamin) TFEB levels in the nuclear fractions were consistently reduced starting from Braak stage IV (52%, p < 0.01), stage V (67%, p < 0.01), and stage VI (85%, p < 0.01) when compared to normal control (NC) brains. In the ALS brains also, nuclear TFEB levels were reduced by 62% (p < 0.001). These data suggest that nuclear TFEB is selectively lost in ALS as well as AD brains, in which TFEB reduction was Braak-stage-dependent. Taken together, the observed reductions in TFEB protein levels may be responsible for the widely reported autophagy defects in these disorders. PMID:27433468

  18. Nuclear role of WASp in gene transcription is uncoupled from its ARP2/3-dependent cytoplasmic role in actin polymerization.

    PubMed

    Sadhukhan, Sanjoy; Sarkar, Koustav; Taylor, Matthew; Candotti, Fabio; Vyas, Yatin M

    2014-07-01

    Defects in Wiskott-Aldrich Syndrome protein (WASp) underlie development of WAS, an X-linked immunodeficiency and autoimmunity disorder of childhood. Nucleation-promoting factors (NPFs) of the WASp family generate F-actin in the cytosol via the VCA (verprolin-homology, cofilin-homology, and acidic) domain and support RNA polymerase II-dependent transcription in the nucleus. Whether nuclear-WASp requires the integration of its actin-related protein (ARP)2/3-dependent cytoplasmic function to reprogram gene transcription, however, remains unresolved. Using the model of human TH cell differentiation, we find that WASp has a functional nuclear localizing and nuclear exit sequences, and accordingly, its effects on transcription are controlled mainly at the level of its nuclear entry and exit via the nuclear pore. Human WASp does not use its VCA-dependent, ARP2/3-driven, cytoplasmic effector mechanisms to support histone H3K4 methyltransferase activity in the nucleus of TH1-skewed cells. Accordingly, an isolated deficiency of nuclear-WASp is sufficient to impair the transcriptional reprogramming of TBX21 and IFNG promoters in TH1-skewed cells, whereas an isolated deficiency of cytosolic-WASp does not impair this process. In contrast, nuclear presence of WASp in TH2-skewed cells is small, and its loss does not impair transcriptional reprogramming of GATA3 and IL4 promoters. Our study unveils an ARP2/3:VCA-independent function of nuclear-WASp in TH1 gene activation that is uncoupled from its cytoplasmic role in actin polymerization.

  19. A conserved family of nuclear phosphoproteins localized to sites of polymerase II transcription

    PubMed Central

    1991-01-01

    An antibody was identified previously that recognizes sites of polymerase II transcription on lampbrush chromosomes, puffs on polytene chromosomes, and many small granules in the nucleoplasm of all cells tested. This antibody binds a conserved family of phosphorylated polypeptides in vertebrate and invertebrate cells. We developed a method for purifying these proteins that involves differential solubility in MgCl2. We isolated a Drosophila cDNA encoding one of the proteins using information obtained from microsequencing. In vivo expression studies show that this protein is concentrated on sites of polymerase II transcription and that it is highly phosphorylated. The protein shares a high degree of homology with proteins involved in alternative splicing of pre-mRNA suggesting the possibility that this protein plays a role in pre-mRNA splicing. PMID:1717489

  20. Structural and calorimetric studies demonstrate that the hepatocyte nuclear factor 1β (HNF1β) transcription factor is imported into the nucleus via a monopartite NLS sequence.

    PubMed

    Wiedmann, Mareike M; Aibara, Shintaro; Spring, David R; Stewart, Murray; Brenton, James D

    2016-09-01

    The transcription factor hepatocyte nuclear factor 1β (HNF1β) is ubiquitously overexpressed in ovarian clear cell carcinoma (CCC) and is a potential therapeutic target. To explore potential approaches that block HNF1β transcription we have identified and characterised extensively the nuclear localisation signal (NLS) for HNF1β and its interactions with the nuclear protein import receptor, Importin-α. Pull-down assays demonstrated that the DNA binding domain of HNF1β interacted with a spectrum of Importin-α isoforms and deletion constructs tagged with eGFP confirmed that the HNF1β (229)KKMRRNR(235) sequence was essential for nuclear localisation. We further characterised the interaction between the NLS and Importin-α using complementary biophysical techniques and have determined the 2.4Å resolution crystal structure of the HNF1β NLS peptide bound to Importin-α. The functional, biochemical, and structural characterisation of the nuclear localisation signal present on HNF1β and its interaction with the nuclear import protein Importin-α provide the basis for the development of compounds targeting transcription factor HNF1β via its nuclear import pathway.

  1. Nuclear life of the voltage-gated Cacnb4 subunit and its role in gene transcription regulation.

    PubMed

    Ronjat, Michel; Kiyonaka, Shigeki; Barbado, Maud; De Waard, Michel; Mori, Yasuo

    2013-01-01

    The pore-forming subunit of voltage-gated calcium channels is associated to auxiliary subunits among which the cytoplasmic β subunit. The different isoforms of this subunit control both the plasma membrane targeting and the biophysical properties of the channel moiety. In a recent study, we demonstrated that the Cacnb4 (β 4) isoform is at the center of a new signaling pathway that connects neuronal excitability and gene transcription. This mechanism relies on nuclear targeting of β 4 triggered by neuronal electrical stimulation. This re-localization of β 4 is promoted by its interaction with Ppp2r5d a regulatory subunit of PP2A in complex with PP2A itself. The formation, as well as the nuclear translocation, of the β 4/ Ppp2r5d/ PP2A complex is totally impaired by the premature R482X stops mutation of β 4 that has been previously associated with juvenile epilepsy. Taking as a case study the tyrosine hydroxylase gene that is strongly upregulated in brain of lethargic mice, deficient for β 4 expression, we deciphered the molecular steps presiding to this signaling pathway. Here we show that expression of wild-type β 4 in HEK293 cells results in the regulation of several genes, while expression of the mutated β 4 (β 1-481) produces a different set of gene regulation. Several genes regulated by β 4 in HEK293 cells were also regulated upon neuronal differentiation of NG108-15 cells that induces nuclear translocation of β 4 suggesting a link between β 4 nuclear targeting and gene regulation.

  2. PIASy Represses CCAAT/Enhancer-binding Protein δ (C/EBPδ) Transcriptional Activity by Sequestering C/EBPδ to the Nuclear Periphery*

    PubMed Central

    Zhou, Shanggen; Si, Junling; Liu, Tong; DeWille, James W.

    2008-01-01

    CCAAT/enhancer binding proteinδ (C/EBPδ) plays a key role in mammary epithelial cell G0 growth arrest, and “loss of function” alterations in C/EBPδ have been reported in breast cancer and acute myeloid leukemia. C/EBPδ is regulated at the transcriptional, post-transcriptional, and post-translational levels, suggesting tight control of C/EBPδ content and function. Protein inhibitors of activated STATs (PIASs) regulate a growing number of transcription factors, including C/EBPs. HC11 nontransformed mammary epithelial cells express PIAS3, PIASxβ, and PIASy, and all three PIAS family members repress C/EBPδ transcriptional activity. PIASy is the most potent, however, repressing C/EBPδ transcriptional activity by >80%. PIASy repression of C/EBPδ transcriptional activity is dependent upon interaction between the highly conserved PIASy N-terminal nuclear matrix binding domain (SAPD) and the C/EBPδ transactivation domain (TAD). PIASy repression of C/EBPδ transcriptional activity is independent of histone deacetylase activity, PIASy E3 SUMO ligase activity, and C/EBPδ sumoylation status. PIASy expression is associated with C/EBPδ translocation from nuclear foci, where C/EBPδ co-localizes with p300, to the nuclear periphery. PIASy-mediated translocation of C/EBPδ is dependent upon the PIASy SAPD and C/EBPδ TAD. PIASy reduces the expression of C/EBPδ adhesion-related target genes and enhances repopulation of open areas within a cell monolayer in the in vitro “scratch” assay. These results demonstrate that PIASy represses C/EBPδ by a mechanism that requires interaction between the PIASy SAPD and C/EBPδ TAD and does not require PIASy SUMO ligase activity or C/EBPδ sumoylation. PIASy alters C/EBPδ nuclear localization, reduces C/EBPδ transcriptional activity, and enhances cell proliferation/migration. PMID:18477566

  3. Nuclear-encoded factors involved in post-transcriptional processing and modification of mitochondrial tRNAs in human disease

    PubMed Central

    Powell, Christopher A.; Nicholls, Thomas J.; Minczuk, Michal

    2015-01-01

    The human mitochondrial genome (mtDNA) encodes 22 tRNAs (mt-tRNAs) that are necessary for the intraorganellar translation of the 13 mtDNA-encoded subunits of the mitochondrial respiratory chain complexes. Maturation of mt-tRNAs involves 5′ and 3′ nucleolytic excision from precursor RNAs, as well as extensive post-transcriptional modifications. Recent data suggest that over 7% of all mt-tRNA residues in mammals undergo post-transcriptional modification, with over 30 different modified mt-tRNA positions so far described. These processing and modification steps are necessary for proper mt-tRNA function, and are performed by dedicated, nuclear-encoded enzymes. Recent growing evidence suggests that mutations in these nuclear genes (nDNA), leading to incorrect maturation of mt-tRNAs, are a cause of human mitochondrial disease. Furthermore, mtDNA mutations in mt-tRNA genes, which may also affect mt-tRNA function, processing, and modification, are also frequently associated with human disease. In theory, all pathogenic mt-tRNA variants should be expected to affect only a single process, which is mitochondrial translation, albeit to various extents. However, the clinical manifestations of mitochondrial disorders linked to mutations in mt-tRNAs are extremely heterogeneous, ranging from defects of a single tissue to complex multisystem disorders. This review focuses on the current knowledge of nDNA coding for proteins involved in mt-tRNA maturation that have been linked to human mitochondrial pathologies. We further discuss the possibility that tissue specific regulation of mt-tRNA modifying enzymes could play an important role in the clinical heterogeneity observed for mitochondrial diseases caused by mutations in mt-tRNA genes. PMID:25806043

  4. Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope.

    PubMed

    Tsai, Shang-Yi A; Chuang, Jian-Ying; Tsai, Meng-Shan; Wang, Xiao-Fei; Xi, Zheng-Xiong; Hung, Jan-Jong; Chang, Wen-Chang; Bonci, Antonello; Su, Tsung-Ping

    2015-11-24

    The sigma-1 receptor (Sig-1R) chaperone at the endoplasmic reticulum (ER) plays important roles in cellular regulation. Here we found a new function of Sig-1R, in that it translocates from the ER to the nuclear envelope (NE) to recruit chromatin-remodeling molecules and regulate the gene transcription thereof. Sig-1Rs mainly reside at the ER-mitochondrion interface. However, on stimulation by agonists such as cocaine, Sig-1Rs translocate from ER to the NE, where Sig-1Rs bind NE protein emerin and recruit chromatin-remodeling molecules, including lamin A/C, barrier-to-autointegration factor (BAF), and histone deacetylase (HDAC), to form a complex with the gene repressor specific protein 3 (Sp3). Knockdown of Sig-1Rs attenuates the complex formation. Cocaine was found to suppress the gene expression of monoamine oxidase B (MAOB) in the brain of wild-type but not Sig-1R knockout mouse. A single dose of cocaine (20 mg/kg) in rats suppresses the level of MAOB at nuclear accumbens without affecting the level of dopamine transporter. Daily injections of cocaine in rats caused behavioral sensitization. Withdrawal from cocaine in cocaine-sensitized rats induced an apparent time-dependent rebound of the MAOB protein level to about 200% over control on day 14 after withdrawal. Treatment of cocaine-withdrawn rats with the MAOB inhibitor deprenyl completely alleviated the behavioral sensitization to cocaine. Our results demonstrate a role of Sig-1R in transcriptional regulation and suggest cocaine may work through this newly discovered genomic action to achieve its addictive action. Results also suggest the MAOB inhibitor deprenyl as a therapeutic agent to block certain actions of cocaine during withdrawal.

  5. Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope

    PubMed Central

    Tsai, Shang-Yi A.; Chuang, Jian-Ying; Tsai, Meng-Shan; Wang, Xiao-fei; Hung, Jan-Jong; Chang, Wen-Chang; Bonci, Antonello; Su, Tsung-Ping

    2015-01-01

    The sigma-1 receptor (Sig-1R) chaperone at the endoplasmic reticulum (ER) plays important roles in cellular regulation. Here we found a new function of Sig-1R, in that it translocates from the ER to the nuclear envelope (NE) to recruit chromatin-remodeling molecules and regulate the gene transcription thereof. Sig-1Rs mainly reside at the ER–mitochondrion interface. However, on stimulation by agonists such as cocaine, Sig-1Rs translocate from ER to the NE, where Sig-1Rs bind NE protein emerin and recruit chromatin-remodeling molecules, including lamin A/C, barrier-to-autointegration factor (BAF), and histone deacetylase (HDAC), to form a complex with the gene repressor specific protein 3 (Sp3). Knockdown of Sig-1Rs attenuates the complex formation. Cocaine was found to suppress the gene expression of monoamine oxidase B (MAOB) in the brain of wild-type but not Sig-1R knockout mouse. A single dose of cocaine (20 mg/kg) in rats suppresses the level of MAOB at nuclear accumbens without affecting the level of dopamine transporter. Daily injections of cocaine in rats caused behavioral sensitization. Withdrawal from cocaine in cocaine-sensitized rats induced an apparent time-dependent rebound of the MAOB protein level to about 200% over control on day 14 after withdrawal. Treatment of cocaine-withdrawn rats with the MAOB inhibitor deprenyl completely alleviated the behavioral sensitization to cocaine. Our results demonstrate a role of Sig-1R in transcriptional regulation and suggest cocaine may work through this newly discovered genomic action to achieve its addictive action. Results also suggest the MAOB inhibitor deprenyl as a therapeutic agent to block certain actions of cocaine during withdrawal. PMID:26554014

  6. Cynaropicrin from Cynara scolymus L. suppresses photoaging of skin by inhibiting the transcription activity of nuclear factor-kappa B.

    PubMed

    Tanaka, Yuka Tsuda; Tanaka, Kiyotaka; Kojima, Hiroyuki; Hamada, Tomoji; Masutani, Teruaki; Tsuboi, Makoto; Akao, Yukihiro

    2013-01-15

    Aging of skin is characterized by skin wrinkling, laxity, and pigmentation induced by several environmental stress factors. Histological changes during the photoaging of skin include hyperproliferation of keratinocytes and melanocytes causing skin wrinkles and pigmentation. Nuclear factor kappa B (NF-κB) is one of the representative transcription factors active in conjunction with inflammation. NF-κB is activated by stimulation such as ultraviolet rays and inflammatory cytokines and induces the expression of various genes such as those of basic fibroblast growth factor (bFGF) and matrix metalloprotease-1 (MMP-1). We screened several plant extracts for their possible inhibitory effect on the transcriptional activity of NF-κB. One of them, an extract from Cynara scolymus L., showed a greatest effect on the suppression of NF-κB transactivation. As a result, we found that cynaropicrin, which is a sesquiterpene lactone, inhibited the NF-κB-mediated transactivation of bFGF and MMP-1. Furthermore, it was confirmed that in an in vivo mouse model cynaropicrin prevented skin photoaging processes leading to the hyperproliferation of keratinocytes and melanocytes. These findings taken together indicate that cynaropicrin is an effective antiphotoaging agent that acts by inhibiting NF-κB-mediated transactivation.

  7. Transcriptional Regulation of CYP2B6 Expression by Hepatocyte Nuclear Factor 3β in Human Liver Cells.

    PubMed

    Li, Linhao; Li, Daochuan; Heyward, Scott; Wang, Hongbing

    2016-01-01

    CYP2B6 plays an increasingly important role in xenobiotic metabolism and detoxification. The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) have been established as predominant regulators for the inductive expression of CYP2B6 gene in human liver. However, there are dramatic interindividual variabilities in CYP2B6 expression that cannot be fully explained by the CAR/PXR-based modulation alone. Here, we show that expression level of CYP2B6 was correlated with that of hepatocyte nuclear factor 3β (HNF3β) in human primary hepatocytes prepared from 35 liver donors. Utilizing recombinant virus-mediated overexpression or knockdown of HNF3β in HepG2 cells, as well as constructs containing serial deletion and site-directed mutation of HNF3β binding motifs in CYP2B6 luciferase reporter assays, we demonstrated that the presence or lack of HNF3β expression markedly correlated with CYP2B6 gene expression and its promoter activity. Novel enhancer modules of HNF3β located upstream of the CYP2B6 gene transcription start site were identified and functionally validated as key elements governing HNF3β-mediated CYP2B6 expression. Chromatin immunoprecipitation assays in human primary hepatocytes and surface plasmon resonance binding affinity experiments confirmed the essential role of these enhancers in the recruitment of HNF3β to the promoter of CYP2B6 gene. Overall, these findings indicate that HNF3β represents a new liver enriched transcription factor that is involved in the transcription of CYP2B6 gene and contributes to the large interindividual variations of CYP2B6 expression in human population.

  8. Nuclear factors that bind two regions important to transcriptional activity of the simian immunodeficiency virus long terminal repeat.

    PubMed Central

    Winandy, S; Renjifo, B; Li, Y; Hopkins, N

    1992-01-01

    Previous studies identified two regions in the U3 region of a molecular clone of simian immunodeficiency virus, SIVmac142, that are important to transcriptional activity under conditions of induction as well as basal-level expression (B. Renjifo, N. A. Speck, S. Winandy, N. Hopkins, and Y. Li, J. Virol. 64:3130-3134, 1990). One region includes the NF-kappa B binding site, while the other lies just 5' of this site between nucleotides -162 and -114 (the -162 to -114 region). The fact that the NF-kappa B site mutation attenuated transcriptional activity in uninduced T cells and fibroblasts where activated NF-kappa B would not be present suggested that a factor(s) other than NF-kappa B could be acting through this site. In this study, we have identified a factor which binds to a cis element overlapping the NF-kappa B site. This factor, which we call simian factor 3 (SF3), would play a role in regulation under conditions of basal level expression, whereas under conditions of induction, NF-kappa B would act via this region. SF3 may also bind to an element in the -162 to -114 region. In addition, we have identified two other factors that bind the -162 to -114 region. One, which we designated SF1, is a ubiquitous basal factor, and the other, SF2, is a T-cell-predominant phorbol myristate acetate-inducible factor. Through identification of nuclear factors that interact with the U3 region of the SIVmac142 long terminal repeat, we can gain insight into how this virus is transcriptionally regulated under conditions of basal-level expression as well as conditions of T-cell activation. Images PMID:1501272

  9. Caffeic acid phenethyl ester inhibits T-cell activation by targeting both nuclear factor of activated T-cells and NF-kappaB transcription factors.

    PubMed

    Márquez, Nieves; Sancho, Rocío; Macho, Antonio; Calzado, Marco A; Fiebich, Bernd L; Muñoz, Eduardo

    2004-03-01

    Caffeic acid phenethyl ester (CAPE), which is derived from the propolis of honeybee hives, has been shown to reveal anti-inflammatory properties. Since T-cells play a key role in the onset of several inflammatory diseases, we have evaluated the immunosuppressive activity of CAPE in human T-cells, discovering that this phenolic compound is a potent inhibitor of early and late events in T-cell receptor-mediated T-cell activation. Moreover, we found that CAPE specifically inhibited both interleukin (IL)-2 gene transcription and IL-2 synthesis in stimulated T-cells. To further characterize the inhibitory mechanisms of CAPE at the transcriptional level, we examined the DNA binding and transcriptional activities of nuclear factor (NF)-kappaB, nuclear factor of activated cells (NFAT), and activator protein-1 (AP-1) transcription factors in Jurkat cells. We found that CAPE inhibited NF-kappaB-dependent transcriptional activity without affecting the degradation of the cytoplasmic NF-kappaB inhibitory protein, IkappaBalpha. However, both NF-kappaB binding to DNA and transcriptional activity of a Gal4-p65 hybrid protein were clearly prevented in CAPE-treated Jurkat cells. Moreover, CAPE inhibited both the DNA-binding and transcriptional activity of NFAT, a result that correlated with its ability to inhibit phorbol 12-myristate 13-acetate plus ionomycin-induced NFAT1 dephosphorylation. These findings provide new insights into the molecular mechanisms involved in the immunomodulatory and anti-inflammatory activities of this natural compound.

  10. The hypersensitive response to tomato leaf curl New Delhi virus nuclear shuttle protein is inhibited by transcriptional activator protein.

    PubMed

    Hussain, Mazhar; Mansoor, Shahid; Iram, Shazia; Zafar, Yusuf; Briddon, Rob W

    2007-12-01

    The hypersensitive response (HR) is a common feature of plant disease resistance reactions and a type of programmed cell death (PCD). Many pathogens are able to modulate pathways involved in cell death. In contrast to animal viruses, inhibitors of PCD activity have not been identified for plant-infecting viruses. Previously, we have reported that the nuclear shuttle protein (NSP) of Tomato leaf curl New Delhi virus (ToLCNDV) induces an HR in Nicotiana tabacum and Lycopersicon esculentum plants when expressed under the control of the Cauliflower mosaic virus 35S promoter. However, HR is not evident in plants infected with ToLCNDV, suggesting that the virus encodes a factor (or factors) that counters this response. Analysis of all ToLCNDV-encoded genes pinpointed the transcriptional activator protein (TrAP) as the factor mediating the anti-HR effect. Deletion mutagenesis showed the central region of TrAP, containing a zinc finger domain and nuclear localization signal, to be important in inhibiting the HR. These results demonstrate that TrAP counters HR-induced cell death, the first such activity identified for a plant-infecting virus.

  11. Interaction of Sp1 zinc finger with transport factor in the nuclear localization of transcription factor Sp1

    SciTech Connect

    Ito, Tatsuo; Kitamura, Haruka; Uwatoko, Chisana; Azumano, Makiko; Itoh, Kohji; Kuwahara, Jun

    2010-12-10

    Research highlights: {yields} Sp1 zinc fingers themselves interact with importin {alpha}. {yields} Sp1 zinc finger domains play an essential role as a nuclear localization signal. {yields} Sp1 can be transported into the nucleus in an importin-dependent manner. -- Abstract: Transcription factor Sp1 is localized in the nucleus and regulates the expression of many cellular genes, but the nuclear transport mechanism of Sp1 is not well understood. In this study, we revealed that GST-fused Sp1 protein bound to endogenous importin {alpha} in HeLa cells via the Sp1 zinc finger domains, which comprise the DNA binding domain of Sp1. It was found that the Sp1 zinc finger domains directly interacted with a wide range of importin {alpha} including the armadillo (arm) repeat domain and the C-terminal acidic domain. Furthermore, it turned out that all three zinc fingers of Sp1 are essential for binding to importin {alpha}. Taken together, these results suggest that the Sp1 zinc finger domains play an essential role as a NLS and Sp1 can be transported into the nucleus in an importin-dependent manner even though it possesses no classical NLSs.

  12. The enhancement of nuclear receptor transcriptional activation by a mouse actin-binding protein, alpha actinin 2.

    PubMed

    Huang, S M; Huang, C J; Wang, W M; Kang, J C; Hsu, W C

    2004-04-01

    The p160 coactivators, steroid receptor coactivator 1, glucocorticoid receptor interacting protein 1 (GRIP1) and the activator of thyroid and retinoic acid receptor, have two activation domains, AD1 and AD2, which transmit the activation signal from the DNA-bound nuclear receptor to the chromatin and/or transcription machinery. In screening for mammalian proteins that bind the AD2 of GRIP1, we identified a mouse actin-binding protein, alpha actinin 2 (mACTN2). mACTN2 was expressed in the heart, skeletal muscle, lung, brain and testis, but there was no expression in the spleen, liver or kidney. Interestingly, the expression level of mACTN2 in the developing embryo depended on the embryonic stage. We further demonstrated that mACTN2 could enhance two transactivation activities of GRIP1, which in turn could enhance the homodimerization of mACTN2. Importantly, mACTN2 not only served as a primary coactivator for androgen receptor, estrogen receptor and thyroid receptor activities, but also acted synergistically with GRIP1 to enhance these nuclear receptor (NR) functions. However, the NR binding motif, LXXLL, conserved in mACTN2 and other actinin family proteins, might be a dispensable domain for its coactivator roles in NRs. These findings suggested that mACTN2 might play an important role in GRIP1-induced NR coactivator functions.

  13. Dual function of a nuclear factor I binding site in MMTV transcription regulation.

    PubMed Central

    Buetti, E; Kühnel, B; Diggelmann, H

    1989-01-01

    Using linker-scanning mutagenesis we had previously identified four elements within the MMTV LTR which are necessary for transcriptional stimulation by glucocorticoid hormones. Two of them overlapped with regions to which the glucocorticoid receptor binds in vitro. The third element contained a NF-I binding site, and the fourth the TATA box. Here we show that mutations that abolish in vitro binding of NF-I had a negative effect also on the basal activity of the MMTV promoter of LTR-containing plasmids stably integrated in Ltk- fibroblasts. The analysis of double mutants altered in the NF-I plus either one of the receptor binding elements further demonstrated that the NF-I site functionally cooperated with the proximal (-120) element, which alone was extremely inefficient in stimulation. The stronger distal (-181/-172) element was independent of NF-I and showed functional cooperativity with the proximal hormone-binding element. Images PMID:2542892

  14. Nuclear phosphoproteome analysis of 3T3-L1 preadipocyte differentiation reveals system-wide phosphorylation of transcriptional regulators.

    PubMed

    Rabiee, Atefeh; Schwämmle, Veit; Sidoli, Simone; Dai, Jie; Rogowska-Wrzesinska, Adelina; Mandrup, Susanne; Jensen, Ole N

    2017-03-01

    Adipocytes (fat cells) are important endocrine and metabolic cells critical for systemic insulin sensitivity. Both adipose excess and insufficiency are associated with adverse metabolic function. Adipogenesis is the process whereby preadipocyte precursor cells differentiate into lipid-laden mature adipocytes. This process is driven by a network of transcriptional regulators (TRs). We hypothesized that protein PTMs, in particular phosphorylation, play a major role in activating and propagating signals within TR networks upon induction of adipogenesis by extracellular stimulus. We applied MS-based quantitative proteomics and phosphoproteomics to monitor the alteration of nuclear proteins during the early stages (4 h) of preadipocyte differentiation. We identified a total of 4072 proteins including 2434 phosphorylated proteins, a majority of which were assigned as regulators of gene expression. Our results demonstrate that adipogenic stimuli increase the nuclear abundance and/or the phosphorylation levels of proteins involved in gene expression, cell organization, and oxidation-reduction pathways. Furthermore, proteins acting as negative modulators involved in negative regulation of gene expression, insulin stimulated glucose uptake, and cytoskeletal organization showed a decrease in their nuclear abundance and/or phosphorylation levels during the first 4 h of adipogenesis. Among 288 identified TRs, 49 were regulated within 4 h of adipogenic stimulation including several known and many novel potential adipogenic regulators. We created a kinase-substrate database for 3T3-L1 preadipocytes by investigating the relationship between protein kinases and protein phosphorylation sites identified in our dataset. A majority of the putative protein kinases belong to the cyclin-dependent kinase family and the mitogen-activated protein kinase family including P38 and c-Jun N-terminal kinases, suggesting that these kinases act as orchestrators of early adipogenesis.

  15. Brain-derived neurotrophic factor activation of NFAT (nuclear factor of activated T-cells)-dependent transcription: a role for the transcription factor NFATc4 in neurotrophin-mediated gene expression.

    PubMed

    Groth, Rachel D; Mermelstein, Paul G

    2003-09-03

    A member of the neurotrophin family, brain-derived neurotrophic factor (BDNF) regulates neuronal survival and differentiation during development. Within the adult brain, BDNF is also important in neuronal adaptive processes, such as the activity-dependent plasticity that underlies learning and memory. These long-term changes in synaptic strength are mediated through alterations in gene expression. However, many of the mechanisms by which BDNF is linked to transcriptional and translational regulation remain unknown. Recently, the transcription factor NFATc4 (nuclear factor of activated T-cells isoform 4) was discovered in neurons, where it is believed to play an important role in long-term changes in neuronal function. Interestingly, NFATc4 is particularly sensitive to the second messenger systems activated by BDNF. Thus, we hypothesized that NFAT-dependent transcription may be an important mediator of BDNF-induced plasticity. In cultured rat CA3-CA1 hippocampal neurons, BDNF activated NFAT-dependent transcription via TrkB receptors. Inhibition of calcineurin blocked BDNF-induced nuclear translocation of NFATc4, thus preventing transcription. Further, phospholipase C was a critical signaling intermediate between BDNF activation of TrkB and the initiation of NFAT-dependent transcription. Both inositol 1,4,5-triphosphate (IP3)-mediated release of calcium from intracellular stores and activation of protein kinase C were required for BDNF-induced NFAT-dependent transcription. Finally, increased expression of IP3 receptor 1 and BDNF after neuronal exposure to BDNF was linked to NFAT-dependent transcription. These results suggest that NFATc4 plays a crucial role in neurotrophin-mediated synaptic plasticity.

  16. Increased AICD generation does not result in increased nuclear translocation or activation of target gene transcription

    SciTech Connect

    Waldron, Elaine; Isbert, Simone; Kern, Andreas; Jaeger, Sebastian; Martin, Anne M.; Hebert, Sebastien S.; Behl, Christian; Weggen, Sascha; De Strooper, Bart; Pietrzik, Claus U.

    2008-08-01

    A sequence of amyloid precursor protein (APP) cleavages culminates in the sequential release of the APP intracellular domain (AICD) and the amyloid {beta} peptide (A{beta}) and/or p3 fragment. One of the environmental factors favouring the accumulation of AICD appears to be a rise in intracellular pH. Here we further identified the metabolism and subcellular localization of artificially expressed constructs under such conditions. We also co-examined the mechanistic lead up to the AICD accumulation and explored possible significances for its increased expression. We found that most of the AICD generated under pH neutralized conditions is likely cleaved from C83. While the AICD surplus was unable to further activate transcription of a luciferase reporter via a Gal4-DNA-binding domain, it failed entirely via the endogenous promoter regions of proposed target genes, APP and KAI1. The lack of a specific transactivation potential was also demonstrated by the unchanged levels of target gene mRNA. However, rather than translocating to the nucleus, the AICD surplus remains membrane tethered or free in the cytosol where it interacts with Fe65. Therefore we provide strong evidence that an increase in AICD generation does not directly promote gene activation of previously proposed target 0011gen.

  17. PML-associated repressor of transcription (PAROT), a novel KRAB-zinc finger repressor, is regulated through association with PML nuclear bodies

    SciTech Connect

    Fleischer, Sandra; Wiemann, Stefan; Will, Hans; Hofmann, Thomas G. . E-mail: t.hofmann@dkfz.de

    2006-04-01

    Promyelocytic leukemia nuclear bodies (PML-NBs) are implicated in transcriptional regulation. Here we identify a novel transcriptional repressor, PML-associated repressor of transcription (PAROT), which is regulated in its repressor activity through recruitment to PML-NBs. PAROT is a Krueppel-associated box ( KRAB) zinc-finger (ZNF) protein, which comprises an amino terminal KRAB-A and KRAB-B box, a linker domain and 8 tandemly repeated C{sub 2}H{sub 2}-ZNF motifs at its carboxy terminus. Consistent with its domain structure, when tethered to DNA, PAROT represses transcription, and this is partially released by the HDAC inhibitor trichostatin A. PAROT colocalizes with members of the heterochromatin protein 1 (HP1) family and with transcriptional intermediary factor-1{beta}/KRAB-associated protein 1 (TIF-1{beta}/KAP1), a transcriptional corepressor for the KRAB-ZNF family. Interestingly, PML isoform IV, in contrast to PML-III, efficiently recruits PAROT and TIF-1{beta} from heterochromatin to PML-NBs. PML-NB recruitment of PAROT partially releases its transcriptional repressor activity, indicating that PAROT can be regulated through subnuclear compartmentalization. Taken together, our data identify a novel transcriptional repressor and provide evidence for its regulation through association with PML-NBs.

  18. PGC-1-Related Coactivator, a Novel, Serum-Inducible Coactivator of Nuclear Respiratory Factor 1-Dependent Transcription in Mammalian Cells

    PubMed Central

    Andersson, Ulf; Scarpulla, Richard C.

    2001-01-01

    The thermogenic peroxisome proliferator-activated receptor γ (PPAR-γ) coactivator 1 (PGC-1) has previously been shown to activate mitochondrial biogenesis in part through a direct interaction with nuclear respiratory factor 1 (NRF-1). In order to identify related coactivators that act through NRF-1, we searched the databases for sequences with similarities to PGC-1. Here, we describe the first characterization of a 177-kDa transcriptional coactivator, designated PGC-1-related coactivator (PRC). PRC is ubiquitously expressed in murine and human tissues and cell lines; but unlike PGC-1, PRC was not dramatically up-regulated during thermogenesis in brown fat. However, its expression was down-regulated in quiescent BALB/3T3 cells and was rapidly induced by reintroduction of serum, conditions where PGC-1 was not detected. PRC activated NRF-1-dependent promoters in a manner similar to that observed for PGC-1. Moreover, NRF-1 was immunoprecipitated from cell extracts by antibodies directed against PRC, and both proteins were colocalized to the nucleoplasm by confocal laser scanning microscopy. PRC interacts in vitro with the NRF-1 DNA binding domain through two distinct recognition motifs that are separated by an unstructured proline-rich region. PRC also contains a potent transcriptional activation domain in its amino terminus adjacent to an LXXLL motif. The spatial arrangement of these functional domains coincides with those found in PGC-1, supporting the conclusion that PRC and PGC-1 are structurally and functionally related. We conclude that PRC is a functional relative of PGC-1 that operates through NRF-1 and possibly other activators in response to proliferative signals. PMID:11340167

  19. The structural basis of gas-responsive transcription by the human nuclear hormone receptor REV-ERBbeta.

    PubMed

    Pardee, Keith I; Xu, Xiaohui; Reinking, Jeff; Schuetz, Anja; Dong, Aiping; Liu, Suya; Zhang, Rongguang; Tiefenbach, Jens; Lajoie, Gilles; Plotnikov, Alexander N; Botchkarev, Alexey; Krause, Henry M; Edwards, Aled

    2009-02-24

    Heme is a ligand for the human nuclear receptors (NR) REV-ERBalpha and REV-ERBbeta, which are transcriptional repressors that play important roles in circadian rhythm, lipid and glucose metabolism, and diseases such as diabetes, atherosclerosis, inflammation, and cancer. Here we show that transcription repression mediated by heme-bound REV-ERBs is reversed by the addition of nitric oxide (NO), and that the heme and NO effects are mediated by the C-terminal ligand-binding domain (LBD). A 1.9 A crystal structure of the REV-ERBbeta LBD, in complex with the oxidized Fe(III) form of heme, shows that heme binds in a prototypical NR ligand-binding pocket, where the heme iron is coordinately bound by histidine 568 and cysteine 384. Under reducing conditions, spectroscopic studies of the heme-REV-ERBbeta complex reveal that the Fe(II) form of the LBD transitions between penta-coordinated and hexa-coordinated structural states, neither of which possess the Cys384 bond observed in the oxidized state. In addition, the Fe(II) LBD is also able to bind either NO or CO, revealing a total of at least six structural states of the protein. The binding of known co-repressors is shown to be highly dependent upon these various liganded states. REV-ERBs are thus highly dynamic receptors that are responsive not only to heme, but also to redox and gas. Taken together, these findings suggest new mechanisms for the systemic coordination of molecular clocks and metabolism. They also raise the possibility for gas-based therapies for the many disorders associated with REV-ERB biological functions.

  20. The Structural Basis of Gas-Responsive Transcription by the Human Nuclear Hormone Receptor REV-ERBβ

    PubMed Central

    Pardee, Keith I; Xu, Xiaohui; Reinking, Jeff; Schuetz, Anja; Dong, Aiping; Liu, Suya; Zhang, Rongguang; Tiefenbach, Jens; Lajoie, Gilles; Plotnikov, Alexander N; Botchkarev, Alexey; Krause, Henry M; Edwards, Aled

    2009-01-01

    Heme is a ligand for the human nuclear receptors (NR) REV-ERBα and REV-ERBβ, which are transcriptional repressors that play important roles in circadian rhythm, lipid and glucose metabolism, and diseases such as diabetes, atherosclerosis, inflammation, and cancer. Here we show that transcription repression mediated by heme-bound REV-ERBs is reversed by the addition of nitric oxide (NO), and that the heme and NO effects are mediated by the C-terminal ligand-binding domain (LBD). A 1.9 Å crystal structure of the REV-ERBβ LBD, in complex with the oxidized Fe(III) form of heme, shows that heme binds in a prototypical NR ligand-binding pocket, where the heme iron is coordinately bound by histidine 568 and cysteine 384. Under reducing conditions, spectroscopic studies of the heme-REV-ERBβ complex reveal that the Fe(II) form of the LBD transitions between penta-coordinated and hexa-coordinated structural states, neither of which possess the Cys384 bond observed in the oxidized state. In addition, the Fe(II) LBD is also able to bind either NO or CO, revealing a total of at least six structural states of the protein. The binding of known co-repressors is shown to be highly dependent upon these various liganded states. REV-ERBs are thus highly dynamic receptors that are responsive not only to heme, but also to redox and gas. Taken together, these findings suggest new mechanisms for the systemic coordination of molecular clocks and metabolism. They also raise the possibility for gas-based therapies for the many disorders associated with REV-ERB biological functions. PMID:19243223

  1. Trafficking of the Transcription Factor Nrf2 to Promyelocytic Leukemia-Nuclear Bodies

    PubMed Central

    Malloy, Melanie Theodore; McIntosh, Deneshia J.; Walters, Treniqka S.; Flores, Andrea; Goodwin, J. Shawn; Arinze, Ifeanyi J.

    2013-01-01

    Ubiquitylation of Nrf2 by the Keap1-Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase complex targets Nrf2 for proteasomal degradation in the cytoplasm and is an extensively studied mechanism for regulating the cellular level of Nrf2. Although mechanistic details are lacking, reports abound that Nrf2 can also be degraded in the nucleus. Here, we demonstrate that Nrf2 is a target for sumoylation by both SUMO-1 and SUMO-2. HepG2 cells treated with As2O3, which enhances attachment of SUMO-2/3 to target proteins, increased SUMO-2/3-modification (polysumoylation) of Nrf2. We show that Nrf2 traffics, in part, to promyelocytic leukemia-nuclear bodies (PML-NBs). Cell fractions harboring key components of PML-NBs did not contain biologically active Keap1 but contained modified Nrf2 as well as RING finger protein 4 (RNF4), a poly-SUMO-specific E3 ubiquitin ligase. Overexpression of wild-type RNF4, but not the catalytically inactive mutant, decreased the steady-state levels of Nrf2, measured in the PML-NB-enriched cell fraction. The proteasome inhibitor MG-132 interfered with this decrease, resulting in elevated levels of polysumoylated Nrf2 that was also ubiquitylated. Wild-type RNF4 accelerated the half-life (t½) of Nrf2, measured in PML-NB-enriched cell fractions. These results suggest that RNF4 mediates polyubiquitylation of polysumoylated Nrf2, leading to its subsequent degradation in PML-NBs. Overall, this work identifies Nrf2 as a target for sumoylation and provides a novel mechanism for its degradation in the nucleus, independent of Keap1. PMID:23543742

  2. Signal Transducer and Activator of Transcription (STAT)-3 Activates Nuclear Factor (NF)-κB in Chronic Lymphocytic Leukemia Cells

    PubMed Central

    Liu, Zhiming; Hazan-Halevy, Inbal; Harris, David M.; Li, Ping; Ferrajoli, Alessandra; Faderl, Stefan; Keating, Michael J.; Estrov, Zeev

    2014-01-01

    Nuclear factor (NF)-κB plays a major role in the pathogenesis of B-cell neoplasms. A broad array of mostly extracellular stimuli has been reported to activate NF-κB, to various degrees, in chronic lymphocytic leukemia (CLL) cells. Because CLL cells harbor high levels of unphosphorylated (U) signal transducer and activator of transcription (STAT)-3 protein and U-STAT3 was reported to activate NF-κB, we sought to determine whether U-STAT3 activates NF-κB in CLL. Using the electrophoretic mobility shift assay (EMSA) we studied peripheral blood low-density cells from 15 patients with CLL and found that CLL cell nuclear extracts from all the samples bound to an NF-κB DNA probe, suggesting that NF-κB is constitutively activated in CLL. Immunoprecipitation studies showed that STAT3 bound NF-κB p65, and confocal microscopy studies detected U-STAT3/NF-κB complexes in the nuclei of CLL cells, thereby confirming these findings. Furthermore, infection of CLL cells with retroviral STAT3-shRNA attenuated the binding of NF-κB to DNA, as assessed by EMSA, and downregulated mRNA levels of NF-κB-regulated genes, as assessed by quantitative polymerase chain reaction. Taken together, our data suggest that U-STAT3 binds to the NF-κB p50/p65 dimers and that the U-STAT3/NF-κB complexes bind to DNA and activate NF-κB-regulated genes in CLL cells. PMID:21364020

  3. Post-fusion treatment with MG132 increases transcription factor expression in somatic cell nuclear transfer embryos in pigs.

    PubMed

    You, Jinyoung; Lee, Joohyeong; Kim, Jinyoung; Park, Junhong; Lee, Eunsong

    2010-02-01

    The objective of this study was to examine the effect of post-fusion treatment of somatic cell nuclear transfer (SCNT) oocytes with the proteasomal inhibitor MG132 on maturation promoting factor (MPF) activity, nuclear remodeling, embryonic development, and gene expression of cloned pig embryos. Immediately after electrofusion, SCNT oocytes were treated with MG132 and/or caffeine for 2 hr, vanadate for 0.5 hr, or vanadate for 0.5 hr followed by MG132 for 1.5 hr. Of the MG132 concentrations tested (0-5 microM), the 1 microM concentration showed a higher rate of blastocyst formation (25.9%) than 0 (14.2%), 0.5 (16.9%), and 5 microM (16.9%). Post-fusion treatment with MG132, caffeine, and both MG132 and caffeine improved blastocyst formation (22.1%, 21.4%, and 24.4%, respectively), whereas vanadate treatment inhibited blastocyst formation (6.5%) compared to the control (11.1%). When examined 2 hr after fusion and 1 hr after activation, MPF activity remained at a higher (P < 0.05) level in SCNT oocytes that were treated post-fusion with caffeine and/or MG132, but it was decreased by vanadate. The rate of oocytes showing premature chromosome condensation was not altered by MG132 but was decreased by vanadate treatment. In addition, formation of single pronuclei was increased by MG132 compared to control and vanadate treatment. MG132-treated embryos showed increased expression of POU5F1, DPPA2, DPPA3, DPPA5, and NDP52l1 genes compared to control embryos. Our results demonstrate that post-fusion treatment of SCNT oocytes with MG132 prevents MPF degradation and increases expression of transcription factors in SCNT embryos, which are necessary for normal development of SCNT embryos.

  4. FBI-1 enhances transcription of the nuclear factor-kappaB (NF-kappaB)-responsive E-selectin gene by nuclear localization of the p65 subunit of NF-kappaB.

    PubMed

    Lee, Dong-Kee; Kang, Jae-Eun; Park, Hye-Jin; Kim, Myung-Hwa; Yim, Tae-Hee; Kim, Jung-Min; Heo, Min-Kyu; Kim, Kyu-Yeun; Kwon, Ho Jeong; Hur, Man-Wook

    2005-07-29

    The POZ domain is a highly conserved protein-protein interaction motif found in many regulatory proteins. Nuclear factor-kappaB (NF-kappaB) plays a key role in the expression of a variety of genes in response to infection, inflammation, and stressful conditions. We found that the POZ domain of FBI-1 (factor that binds to the inducer of short transcripts of human immunodeficiency virus-1) interacted with the Rel homology domain of the p65 subunit of NF-kappaB in both in vivo and in vitro protein-protein interaction assays. FBI-1 enhanced NF-kappaB-mediated transcription of E-selectin genes in HeLa cells upon phorbol 12-myristate 13-acetate stimulation and overcame gene repression by IkappaB alpha or IkappaB beta. In contrast, the POZ domain of FBI-1, which is a dominant-negative form of FBI-1, repressed NF-kappaB-mediated transcription, and the repression was cooperative with IkappaB alpha or IkappaB beta. In contrast, the POZ domain tagged with a nuclear localization sequence polypeptide of FBI-1 enhanced NF-kappaB-responsive gene transcription, suggesting that the molecular interaction between the POZ domain and the Rel homology domain of p65 and the nuclear localization by the nuclear localization sequence are important in the transcription enhancement mediated by FBI-1. Confocal microscopy showed that FBI-1 increased NF-kappaB movement into the nucleus and increased the stability of NF-kappaB in the nucleus, which enhanced NF-kappaB-mediated transcription of the E-selectin gene. FBI-1 also interacted with IkappaB alpha and IkappaB beta.

  5. Activation of nuclear transcription factor-kappaB in mouse brain induced by a simulated microgravity environment

    NASA Technical Reports Server (NTRS)

    Wise, Kimberly C.; Manna, Sunil K.; Yamauchi, Keiko; Ramesh, Vani; Wilson, Bobby L.; Thomas, Renard L.; Sarkar, Shubhashish; Kulkarni, Anil D.; Pellis, Neil R.; Ramesh, Govindarajan T.

    2005-01-01

    Microgravity induces inflammatory responses and modulates immune functions that may increase oxidative stress. Exposure to a microgravity environment induces adverse neurological effects; however, there is little research exploring the etiology of these effects resulting from exposure to such an environment. It is also known that spaceflight is associated with increase in oxidative stress; however, this phenomenon has not been reproduced in land-based simulated microgravity models. In this study, an attempt has been made to show the induction of reactive oxygen species (ROS) in mice brain, using ground-based microgravity simulator. Increased ROS was observed in brain stem and frontal cortex with concomitant decrease in glutathione, on exposing mice to simulated microgravity for 7 d. Oxidative stress-induced activation of nuclear factor-kappaB was observed in all the regions of the brain. Moreover, mitogen-activated protein kinase kinase was phosphorylated equally in all regions of the brain exposed to simulated microgravity. These results suggest that exposure of brain to simulated microgravity can induce expression of certain transcription factors, and these have been earlier argued to be oxidative stress dependent.

  6. Heterogeneous nuclear ribonucleoprotein A/B and G inhibits the transcription of gonadotropin-releasing-hormone 1

    PubMed Central

    Zhao, Sheng; Korzan, Wayne J.; Chen, Chun-Chun; Fernald, Russell D.

    2008-01-01

    Gonadotropin releasing hormone 1 (GnRH1) causes the release of gonadotropins from the pituitary to control reproduction. Here we report that two heterogeneous nuclear ribonucleoproteins (hnRNP-A/B and hnRNP-G) bind to the GnRH-I upstream promoter region in a cichlid fish, Astatotilapia burtoni. We identified these binding proteins using a newly developed homology based method of mass spectrometric peptide mapping. We show that both hnRNP-A/B and hnRNP-G co-localize with GnRH1 in the pre-optic area of the hypothalamus in the brain. We also demonstrated that these ribonucleoproteins exhibit similar binding capacity in vivo, using immortalized mouse GT1-7 cells where overexpression of either hnRNP-A/B or hnRNP-G significantly down-regulate GnRH1 mRNA levels in GT1-7 cells, suggesting that both act as repressors in GnRH1 transcriptional regulation. PMID:17920292

  7. Definition and prediction of the full range of transcription factor binding sites—the hepatocyte nuclear factor 1 dimeric site

    PubMed Central

    Locker, Joseph; Ghosh, David; Luc, Phuong-Van; Zheng, Jianhua

    2002-01-01

    In animals, transcription factor binding sites are hard to recognize because of their extensive variation. We therefore characterized the general relationship between a specific protein-binding site and its DNA sequence and used this relationship to generate a predictive algorithm for searching other DNA sequences. The experimental process was defined by studying hepatocyte nuclear factor 1 (HNF1), which binds DNA as a dimer on two inverted-repeat 7-bp half sites separated by one base. The binding model was based on the equivalence of the two half sites, which was confirmed in examples where specific modified sites were compared. Binding competition analysis was used to determine the effects of substitution of all four bases at each position in the half site. From these data, a weighted half-site matrix was generated and the full site was evaluated as the sum of two half-site scores. This process accurately predicted even weak binding sites that were significantly different from the consensus sequence. The predictions also showed a direct correlation with measured protein binding. PMID:12202766

  8. Meiotic nuclear movements in fission yeast are regulated by the transcription factor Mei4 downstream of a Cds1-dependent replication checkpoint pathway.

    PubMed

    Ruan, Kun; Yamamoto, Takaharu G; Asakawa, Haruhiko; Chikashige, Yuji; Masukata, Hisao; Haraguchi, Tokuko; Hiraoka, Yasushi

    2015-03-01

    In meiosis, the fission yeast nucleus displays an elongated morphology, moving back and forth within the cell; these nuclear movements continue for approximately 2 h before meiotic nuclear divisions. Meiotic DNA replication occurs in an early phase of the nuclear movements and is followed by meiotic prophase. Here we report that in mutants deficient in meiotic DNA replication, the duration of nuclear movements is strikingly prolonged to four to 5 h. We found that this prolongation was caused by the Cds1-dependent replication checkpoint, which represses expression of the mei4(+) gene encoding a meiosis-specific transcription factor. In the absence of Mei4, nuclear movements persisted for more than 8 h. In contrast, overproduction of Mei4 accelerated termination of nuclear movements to approximately 30 min. These results show that Mei4 is involved in the termination of nuclear movements and that Mei4-mediated regulatory pathways link a DNA replication checkpoint to the termination of nuclear movements.

  9. Interaction of Yna1 and Yna2 Is Required for Nuclear Accumulation and Transcriptional Activation of the Nitrate Assimilation Pathway in the Yeast Hansenula polymorpha.

    PubMed

    Silvestrini, Lucia; Rossi, Beatrice; Gallmetzer, Andreas; Mathieu, Martine; Scazzocchio, Claudio; Berardi, Enrico; Strauss, Joseph

    2015-01-01

    A few yeasts, including Hansenula polymorpha are able to assimilate nitrate and use it as nitrogen source. The genes necessary for nitrate assimilation are organised in this organism as a cluster comprising those encoding nitrate reductase (YNR1), nitrite reductase (YNI1), a high affinity transporter (YNT1), as well as the two pathway specific Zn(II)2Cys2 transcriptional activators (YNA1, YNA2). Yna1p and Yna2p mediate induction of the system and here we show that their functions are interdependent. Yna1p activates YNA2 as well as its own (YNA1) transcription thus forming a nitrate-dependent autoactivation loop. Using a split-YFP approach we demonstrate here that Yna1p and Yna2p form a heterodimer independently of the inducer and despite both Yna1p and Yna2p can occupy the target promoter as mono- or homodimer individually, these proteins are transcriptionally incompetent. Subsequently, the transcription factors target genes containing a conserved DNA motif (termed nitrate-UAS) determined in this work by in vitro and in vivo protein-DNA interaction studies. These events lead to a rearrangement of the chromatin landscape on the target promoters and are associated with the onset of transcription of these target genes. In contrast to other fungi and plants, in which nuclear accumulation of the pathway-specific transcription factors only occur in the presence of nitrate, Yna1p and Yna2p are constitutively nuclear in H. polymorpha. Yna2p is needed for this nuclear accumulation and Yna1p is incapable of strictly positioning in the nucleus without Yna2p. In vivo DNA footprinting and ChIP analyses revealed that the permanently nuclear Yna1p/Yna2p heterodimer only binds to the nitrate-UAS when the inducer is present. The nitrate-dependent up-regulation of one partner protein in the heterodimeric complex is functionally similar to the nitrate-dependent activation of nuclear accumulation in other systems.

  10. Interaction of Yna1 and Yna2 Is Required for Nuclear Accumulation and Transcriptional Activation of the Nitrate Assimilation Pathway in the Yeast Hansenula polymorpha

    PubMed Central

    Silvestrini, Lucia; Rossi, Beatrice; Gallmetzer, Andreas; Mathieu, Martine; Scazzocchio, Claudio; Berardi, Enrico; Strauss, Joseph

    2015-01-01

    A few yeasts, including Hansenula polymorpha are able to assimilate nitrate and use it as nitrogen source. The genes necessary for nitrate assimilation are organised in this organism as a cluster comprising those encoding nitrate reductase (YNR1), nitrite reductase (YNI1), a high affinity transporter (YNT1), as well as the two pathway specific Zn(II)2Cys2 transcriptional activators (YNA1, YNA2). Yna1p and Yna2p mediate induction of the system and here we show that their functions are interdependent. Yna1p activates YNA2 as well as its own (YNA1) transcription thus forming a nitrate-dependent autoactivation loop. Using a split-YFP approach we demonstrate here that Yna1p and Yna2p form a heterodimer independently of the inducer and despite both Yna1p and Yna2p can occupy the target promoter as mono- or homodimer individually, these proteins are transcriptionally incompetent. Subsequently, the transcription factors target genes containing a conserved DNA motif (termed nitrate-UAS) determined in this work by in vitro and in vivo protein-DNA interaction studies. These events lead to a rearrangement of the chromatin landscape on the target promoters and are associated with the onset of transcription of these target genes. In contrast to other fungi and plants, in which nuclear accumulation of the pathway-specific transcription factors only occur in the presence of nitrate, Yna1p and Yna2p are constitutively nuclear in H. polymorpha. Yna2p is needed for this nuclear accumulation and Yna1p is incapable of strictly positioning in the nucleus without Yna2p. In vivo DNA footprinting and ChIP analyses revealed that the permanently nuclear Yna1p/Yna2p heterodimer only binds to the nitrate-UAS when the inducer is present. The nitrate-dependent up-regulation of one partner protein in the heterodimeric complex is functionally similar to the nitrate-dependent activation of nuclear accumulation in other systems. PMID:26335797

  11. BZLF1, an Epstein-Barr virus immediate-early protein, induces p65 nuclear translocation while inhibiting p65 transcriptional function

    SciTech Connect

    Morrison, Thomas E.; Kenney, Shannon C. . E-mail: shann@med.unc.edu

    2004-10-25

    We have previously demonstrated that the Epstein-Barr virus immediate-early BZLF1 protein interacts with, and is inhibited by, the NF-{kappa}B family member p65. However, the effects of BZLF1 on NF-{kappa}B activity have not been intensively studied. Here we show that BZLF1 inhibits p65-dependent gene expression. BZLF1 inhibited the ability of IL-1, as well as transfected p65, to activate the expression of two different NF-{kappa}B-responsive genes, ICAM-1 and I{kappa}B-{alpha}. BZLF1 also reduced the constitutive level of I{kappa}B-{alpha} protein in HeLa and A549 cells, and increased the amount of nuclear NF-{kappa}B to a similar extent as tumor necrosis factor-alpha (TNF-{alpha}) treatment. In spite of this BZLF1-associated increase in the nuclear form of NF-{kappa}B, BZLF1 did not induce binding of NF-{kappa}B to NF-{kappa}B responsive promoters (as determined by chromatin immunoprecipitation assay) in vivo, although TNF-{alpha} treatment induced NF-{kappa}B binding as expected. Overexpression of p65 dramatically inhibited the lytic replication cycle of EBV in 293-EBV cells, confirming that NF-{kappa}B also inhibits BZLF1 transcriptional function. Our results are consistent with a model in which BZLF1 inhibits the transcriptional function of p65, resulting in decreased transcription of I{kappa}B-{alpha}, decreased expression of I{kappa}B-{alpha} protein, and subsequent translocation of NF-{kappa}B to the nucleus. This nuclear translocation of NF-{kappa}B may promote viral latency by negatively regulating BZLF1 transcriptional activity. In situations where p65 activity is limiting in comparison to BZLF1, the ability of BZLF1 to inhibit p65 transcriptional function may protect the virus from the host immune system during the lytic form of infection.

  12. Role of Calcineurin, hnRNPA2 and Akt in Mitochondrial Respiratory Stress-Mediated Transcription Activation of Nuclear Gene Targets

    PubMed Central

    Guha, Manti; Tang, Weigang; Sondheimer, Neal; Avadhani, Narayan G.

    2010-01-01

    Pathophysiological conditions causing mitochondrial dysfunction and altered transmembrane potential (Δψm) initiate a mitochondrial respiratory stress response, also known as mitochondrial retrograde response, in a variety of mammalian cells. An increase in the cytosolic Ca2+ [Ca2+]c as part of this signaling cascade activates Ca2+ responsive phosphatase, Calcineurin (Cn). Activation of IGF1R accompanied by increased glycolysis, invasiveness, and resistance to apoptosis are phenotypic hallmarks of C2C12 rhabdomyoblast cells subjected to this stress. The signaling is associated with activation and increased nuclear translocation of a number of transcription factors including a novel NFκB (cRel: p50) pathway, NFAT, CREB and C/EBPδ. This culminates in the upregulation of a number of nuclear genes including Cathepsin L, RyR1, Glut4 and Akt1. We observed that stress regulated transcription activation of nuclear genes involves a cooperative interplay between NFκB (cRel:p50), C/EBPδ, CREB, NFAT. Our results show that the functional synergy of these factors requires the stress-activated heterogeneous nuclear ribonucleoprotein, hnRNPA2 as a transcriptional co-activator. We report here that mitochondrial stress leads to induced expression and activation of serine threonine kinase Akt1. Interestingly, we observe that Akt1 phosphorylates hnRNPA2 under mitochondrial stress conditions, which is a crucial step for the recruitment of this coactivator to the stress target promoters and culmination in mitochondrial stress-mediated transcription activation of target genes. We propose that mitochondrial stress plays an important role in tumor progression and emergence of invasive phenotypes. PMID:20153290

  13. Regulation of the nuclear export of the transcription factor NFATc1 by protein kinases after slow fibre type electrical stimulation of adult mouse skeletal muscle fibres.

    PubMed

    Shen, Tiansheng; Cseresnyés, Zoltán; Liu, Yewei; Randall, William R; Schneider, Martin F

    2007-03-01

    The transcription factor nuclear factor of activated T cells (NFAT)c1 has been shown to be involved in turning on slow skeletal muscle fibre gene expression. Previous studies from our laboratory have characterized the stimulation pattern-dependent nuclear import and resting shuttling of NFATc1-green fluorescent protein (GFP) in flexor digitorum brevis (FDB) muscle fibres from adult mouse. In this study, we use viral expression of the transcription factor NFATc1-GFP fusion protein to investigate the mechanisms underlying the nuclear export of the NFATc1-GFP that accumulated in the nuclei of cultured dissociated adult mouse FDB muscle fibres during slow-twitch fibre type electrical stimulation. In these studies, we found that inhibition of either glycogen synthase kinase 3beta (GSK3beta) or casein kinase 1 or 2 (CK1/2) markedly slowed the decay of nuclear NFATc1-GFP after cessation of muscle fibre electrical stimulation, whereas inhibition of casein kinase 1delta, p38 mitogen-activated protein kinase, c-Jun N-terminal kinase and protein kinase A had little effect. Simultaneous inhibition of GSK3beta and CK1/2 completely blocked the nuclear export of NFATc1-GFP after muscle activity. We also developed a simplified model of NFATc1 phosphorylation/dephosphorylation and nuclear fluxes, and used this model to simulate the observed time courses of nuclear NFATc1-GFP with and without NFATc1 kinase inhibition. Our results suggest that GSK3beta and CK1/2 are the major protein kinases that contribute to the removal of NFATc1 that accumulates in muscle fibre nuclei during muscle activity, and that GSK3beta and CK1/2 are responsible for phosphorylating NFATc1 in muscle nuclei in a complementary or synergistic fashion.

  14. A Phylogenetically Conserved Group of Nuclear Factor-Y Transcription Factors Interact to Control Nodulation in Legumes.

    PubMed

    Baudin, Maël; Laloum, Tom; Lepage, Agnès; Rípodas, Carolina; Ariel, Federico; Frances, Lisa; Crespi, Martin; Gamas, Pascal; Blanco, Flavio Antonio; Zanetti, Maria Eugenia; de Carvalho-Niebel, Fernanda; Niebel, Andreas

    2015-12-01

    The endosymbiotic association between legumes and soil bacteria called rhizobia leads to the formation of a new root-derived organ called the nodule in which differentiated bacteria convert atmospheric nitrogen into a form that can be assimilated by the host plant. Successful root infection by rhizobia and nodule organogenesis require the activation of symbiotic genes that are controlled by a set of transcription factors (TFs). We recently identified Medicago truncatula nuclear factor-YA1 (MtNF-YA1) and MtNF-YA2 as two M. truncatula TFs playing a central role during key steps of the Sinorhizobium meliloti-M. truncatula symbiotic interaction. NF-YA TFs interact with NF-YB and NF-YC subunits to regulate target genes containing the CCAAT box consensus sequence. In this study, using a yeast two-hybrid screen approach, we identified the NF-YB and NF-YC subunits able to interact with MtNF-YA1 and MtNF-YA2. In yeast (Saccharomyces cerevisiae) and in planta, we further demonstrated by both coimmunoprecipitation and bimolecular fluorescence complementation that these NF-YA, -B, and -C subunits interact and form a stable NF-Y heterotrimeric complex. Reverse genetic and chromatin immunoprecipitation-PCR approaches revealed the importance of these newly identified NF-YB and NF-YC subunits for rhizobial symbiosis and binding to the promoter of MtERN1 (for Ethylene Responsive factor required for Nodulation), a direct target gene of MtNF-YA1 and MtNF-YA2. Finally, we verified that a similar trimer is formed in planta by the common bean (Phaseolus vulgaris) NF-Y subunits, revealing the existence of evolutionary conserved NF-Y protein complexes to control nodulation in leguminous plants. This sheds light on the process whereby an ancient heterotrimeric TF mainly controlling cell division in animals has acquired specialized functions in plants.

  15. Genetic reprogramming of transcription factor ap-2gamma in bovine somatic cell nuclear transfer preimplantation embryos and placentomes.

    PubMed

    Aston, Kenneth I; Li, Gugan-Peng; Hicks, Brady A; Winger, Quinton A; White, Kenneth L

    2009-03-01

    Bovine somatic cell nuclear transfer (SCNT) efficiency remains very low despite a tremendous amount of research devoted to its improvement over the past decade. Frequent early and mid-gestational losses are commonly accompanied by placental abnormalities. A transcription factor, activating protein AP-2gamma, has been shown to be necessary for proper placental development in the mouse. We first evaluated the expression of the gene coding for AP-2gamma (Tfap2c) in several bovine fibroblast donor cell lines and found it was not expressed. Subsequently we determined the expression profile of Tfap2c in oocytes and various stages of preimplantation in vitro fertilized (IVF) embryos. Tfap2c was undetectable in oocytes and early embryos, and was detectable at relatively high levels in morula and blastocyst IVF embryos. The lack of expression in oocytes and donor cells means Tfap2c must be induced in the zygote at the morula stage in properly reprogrammed embryos. SCNT embryos expressed Tfap2c at the eight-cell stage, 2 days earlier than control embryos. Control embryos first expressed Tfap2c at the morula stage, and at this stage Tfap2c was significantly lower in the SCNT embryos. No differences in expression were detected at the blastocyst stage. To determine whether Tfap2c was properly reprogrammed in the placenta of SCNT pregnancies, we evaluated its expression in cotyledons and caruncles of SCNT and control pregnancies between days 55 and 90 gestation. Expression of Tfap2c in caruncles significantly increased between days 55 and 90, while expression in cotyledons was relatively consistent over that same period. Expression levels in SCNT tissues were not different from controls. This data indicates Tfap2c expression is altered in early preimplantation SCNT embryos, which may have developmental consequences resulting from genes influenced by Tfap2c, but expression was not different at the blastocyst stage and in placentomes.

  16. Dephosphorylation of the nuclear factor of activated T cells (NFAT) transcription factor is regulated by an RNA-protein scaffold complex.

    PubMed

    Sharma, Sonia; Findlay, Gregory M; Bandukwala, Hozefa S; Oberdoerffer, Shalini; Baust, Beate; Li, Zhigang; Schmidt, Valentina; Hogan, Patrick G; Sacks, David B; Rao, Anjana

    2011-07-12

    Nuclear factor of activated T cells (NFAT) proteins are Ca(2+)-regulated transcription factors that control gene expression in many cell types. NFAT proteins are heavily phosphorylated and reside in the cytoplasm of resting cells; when cells are stimulated by a rise in intracellular Ca(2+), NFAT proteins are dephosphorylated by the Ca(2+)/calmodulin-dependent phosphatase calcineurin and translocate to the nucleus to activate target gene expression. Here we show that phosphorylated NFAT1 is present in a large cytoplasmic RNA-protein scaffold complex that contains a long intergenic noncoding RNA (lincRNA), NRON [noncoding (RNA) repressor of NFAT]; a scaffold protein, IQ motif containing GTPase activating protein (IQGAP); and three NFAT kinases, casein kinase 1, glycogen synthase kinase 3, and dual specificity tyrosine phosphorylation regulated kinase. Combined knockdown of NRON and IQGAP1 increased NFAT dephosphorylation and nuclear import exclusively after stimulation, without affecting the rate of NFAT rephosphorylation and nuclear export; and both NRON-depleted T cells and T cells from IQGAP1-deficient mice showed increased production of NFAT-dependent cytokines. Our results provide evidence that a complex of lincRNA and protein forms a scaffold for a latent transcription factor and its regulatory kinases, and support an emerging consensus that lincRNAs that bind transcriptional regulators have a similar scaffold function.

  17. Dephosphorylation of the nuclear factor of activated T cells (NFAT) transcription factor is regulated by an RNA-protein scaffold complex

    PubMed Central

    Sharma, Sonia; Findlay, Gregory M.; Bandukwala, Hozefa S.; Oberdoerffer, Shalini; Baust, Beate; Li, Zhigang; Schmidt, Valentina; Hogan, Patrick G.; Sacks, David B.; Rao, Anjana

    2011-01-01

    Nuclear factor of activated T cells (NFAT) proteins are Ca2+-regulated transcription factors that control gene expression in many cell types. NFAT proteins are heavily phosphorylated and reside in the cytoplasm of resting cells; when cells are stimulated by a rise in intracellular Ca2+, NFAT proteins are dephosphorylated by the Ca2+/calmodulin-dependent phosphatase calcineurin and translocate to the nucleus to activate target gene expression. Here we show that phosphorylated NFAT1 is present in a large cytoplasmic RNA-protein scaffold complex that contains a long intergenic noncoding RNA (lincRNA), NRON [noncoding (RNA) repressor of NFAT]; a scaffold protein, IQ motif containing GTPase activating protein (IQGAP); and three NFAT kinases, casein kinase 1, glycogen synthase kinase 3, and dual specificity tyrosine phosphorylation regulated kinase. Combined knockdown of NRON and IQGAP1 increased NFAT dephosphorylation and nuclear import exclusively after stimulation, without affecting the rate of NFAT rephosphorylation and nuclear export; and both NRON-depleted T cells and T cells from IQGAP1-deficient mice showed increased production of NFAT-dependent cytokines. Our results provide evidence that a complex of lincRNA and protein forms a scaffold for a latent transcription factor and its regulatory kinases, and support an emerging consensus that lincRNAs that bind transcriptional regulators have a similar scaffold function. PMID:21709260

  18. Transcription of the Tollip gene is elevated in intestinal epithelial cells through impaired O-GlcNAcylation-dependent nuclear translocation of the negative regulator Elf-1

    SciTech Connect

    Sugi, Yutaka; Takahashi, Kyoko; Nakano, Kou; Hosono, Akira; Kaminogawa, Shuichi

    2011-09-09

    Highlights: {yields} Transcriptional activation of the Tollitip gene is higher in IECs than in monocytes. {yields} Nt -194/-186 region acts as a cis-element and is recognized by Elf-1. {yields} Elf-1 suppresses Tollip gene transcription in monocytes but not in IECs. {yields} O-GlcNAc modification is necessary for nuclear translocation of Elf-1. {yields} O-GlcNAcylation-dependent nuclear translocation of Elf-1 is impaired in IECs. -- Abstract: Intestinal epithelial cells (IECs) must be tolerant of the large number of commensal bacteria inhabiting the intestinal tract to avoid excessive inflammatory reactions. Toll-interacting protein (Tollip), a negative regulator of Toll-like receptor signaling, is known to be expressed at high levels in IECs, and to thereby contribute to the hyporesponsiveness of IECs to commensals. In this study, we analyzed the underlying mechanisms for elevated transcription of the Tollip gene in IECs using a human IEC line, Caco-2, and a human monocyte line, THP-1, as a control. Elf-1 was identified as a transcription factor that negatively regulates Tollip gene expression. The transcription factor Elf-1 was localized in the nucleus by O-linked N-acetylglucosamine (O-GlcNAc) modification, whereas the unmodified form was detected only in the cytoplasm. Comparison of Caco-2 and THP-1 cells revealed that O-GlcNAc modification of Elf-1 was significantly lower in IECs than in monocytes. Collectively, the results indicate that insufficient O-GlcNAc modification prevents Elf-1-mediated transcriptional repression and thereby upregulates Tollip gene expression in IECs.

  19. On involvement of transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells, activator protein-1 and signal transducer and activator of transcription-3 in photodynamic therapy-induced death of crayfish neurons and satellite glial cells

    NASA Astrophysics Data System (ADS)

    Berezhnaya, Elena; Neginskaya, Marya; Kovaleva, Vera; Sharifulina, Svetlana; Ischenko, Irina; Komandirov, Maxim; Rudkovskii, Mikhail; Uzdensky, Anatoly B.

    2015-07-01

    Photodynamic therapy (PDT) is currently used in the treatment of brain tumors. However, not only malignant cells but also neighboring normal neurons and glial cells are damaged during PDT. In order to study the potential role of transcription factors-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activator protein (AP-1), and signal transducer and activator of transcription-3 (STAT-3)-in photodynamic injury of normal neurons and glia, we photosensitized the isolated crayfish mechanoreceptor consisting of a single sensory neuron enveloped by glial cells. Application of different inhibitors and activators showed that transcription factors NF-κB (inhibitors caffeic acid phenethyl ester and parthenolide, activator betulinic acid), AP-1 (inhibitor SR11302), and STAT-3 (inhibitors stattic and cucurbitacine) influenced PDT-induced death and survival of neurons and glial cells in different ways. These experiments indicated involvement of NF-κB in PDT-induced necrosis of neurons and apoptosis of glial cells. However, in glial cells, it played the antinecrotic role. AP-1 was not involved in PDT-induced necrosis of neurons and glia, but mediated glial apoptosis. STAT-3 was involved in PDT-induced apoptosis of glial cells and necrosis of neurons and glia. Therefore, signaling pathways that regulate cell death and survival in neurons and glial cells are different. Using various inhibitors or activators of transcription factors, one can differently influence the sensitivity and resistance of neurons and glial cells to PDT.

  20. Heat shock factor 1 upregulates transcription of Epstein-Barr Virus nuclear antigen 1 by binding to a heat shock element within the BamHI-Q promoter

    SciTech Connect

    Wang, Feng-Wei; Wu, Xian-Rui; Liu, Wen-Ju; Liao, Yi-Ji; Lin, Sheng; Zong, Yong-Sheng; Zeng, Mu-Sheng; Zeng, Yi-Xin; Mai, Shi-Juan; Xie, Dan

    2011-12-20

    Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is essential for maintenance of the episome and establishment of latency. In this study, we observed that heat treatment effectively induced EBNA1 transcription in EBV-transformed B95-8 and human LCL cell lines. Although Cp is considered as the sole promoter used for the expression of EBNA1 transcripts in the lymphoblastoid cell lines, the RT-PCR results showed that the EBNA1 transcripts induced by heat treatment arise from Qp-initiated transcripts. Using bioinformatics, a high affinity and functional heat shock factor 1 (HSF1)-binding element within the - 17/+4 oligonucleotide of the Qp was found, and was determined by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Moreover, heat shock and exogenous HSF1 expression induced Qp activity in reporter assays. Further, RNA interference-mediated HSF1 gene silencing attenuated heat-induced EBNA1 expression in B95-8 cells. These results provide evidence that EBNA1 is a new target for the transcription factor HSF1.

  1. The mitochondrial fatty acid synthesis (mtFASII) pathway is capable of mediating nuclear-mitochondrial cross talk through the PPAR system of transcriptional activation

    SciTech Connect

    Parl, Angelika; Mitchell, Sabrina L.; Clay, Hayley B.; Reiss, Sara; Li, Zhen; Murdock, Deborah G.

    2013-11-15

    Highlights: •The function of the mitochondria fatty acid synthesis pathway is partially unknown. •Overexpression of the pathway causes transcriptional activation through PPARs. •Knock down of the pathway attenuates that activation. •The last enzyme in the pathway regulates its own transcription. •Products of the mtFASII pathway are able to drive nuclear transcription. -- Abstract: Mammalian cells contain two fatty acid synthesis pathways, the cytosolic FASI pathway, and the mitochondrial FASII pathway. The selection behind the conservation of the mitochondrial pathway is not completely understood, given the presence of the cytosolic FAS pathway. In this study, we show through heterologous gene reporter systems and PCR-based arrays that overexpression of MECR, the last step in the mtFASII pathway, causes modulation of gene expression through the PPAR pathway. Electromobility shift assays (EMSAs) demonstrate that overexpression of MECR causes increased binding of PPARs to DNA, while cell fractionation and imaging studies show that MECR remains localized to the mitochondria. Interestingly, knock down of the mtFASII pathway lessens the effect of MECR on this transcriptional modulation. Our data are most consistent with MECR-mediated transcriptional activation through products of the mtFASII pathway, although we cannot rule out MECR acting as a coactivator. Further investigation into the physiological relevance of this communication will be necessary to better understand some of the phenotypic consequences of deficits in this pathway observed in animal models and human disease.

  2. Interaction of Heat Shock Protein Cpn10 with the Cyclin E/Cdk2 Substrate Nuclear Protein Ataxia-Telangiectasia (NPAT) Is Involved in Regulating Histone Transcription.

    PubMed

    Ling Zheng, Li; Wang, Fei Ya; Cong, Xiao Xia; Shen, Yue; Rao, Xi Sheng; Huang, Dao Sheng; Fan, Wei; Yi, Peng; Wang, Xin Bao; Zheng, Lei; Zhou, Yi Ting; Luo, Yan

    2015-12-04

    Precise modulation of histone gene transcription is critical for cell cycle progression. As a direct substrate of Cyclin E/CDK2, nuclear protein ataxia-telangiectasia (NPAT) is a crucial factor in regulating histone transcription and cell cycle progression. Here we identified that Cpn10/HSPE, a 10-kDa heat shock protein, is a novel interacting partner of NPAT. A pool of Cpn10 is colocalized with NPAT foci during G1 and S phases in nuclei. Gain- and loss-of-function experiments unraveled an essential role of Cpn10 in histone transcription. A conserved DLFD motif within Cpn10 was critical for targeting NPAT and modulating histone transcription. More importantly, knockdown of Cpn10 disrupted the focus formation of both NPAT and FADD-like interleukin-1β-converting enzyme-associated huge protein without affecting Coilin-positive Cajal bodies. Finally, Cpn10 is important for S phase progression and cell proliferation. Taken together, our finding revealed a novel role of Cpn10 in the spatial regulation of NPAT signaling and disclosed a previously unappreciated link between the heat shock protein and histone transcription regulation.

  3. Interaction of Heat Shock Protein Cpn10 with the Cyclin E/Cdk2 Substrate Nuclear Protein Ataxia-Telangiectasia (NPAT) Is Involved in Regulating Histone Transcription*

    PubMed Central

    Ling Zheng, Li; Wang, Fei Ya; Cong, Xiao Xia; Shen, Yue; Rao, Xi Sheng; Huang, Dao Sheng; Fan, Wei; Yi, Peng; Wang, Xin Bao; Zheng, Lei; Zhou, Yi Ting; Luo, Yan

    2015-01-01

    Precise modulation of histone gene transcription is critical for cell cycle progression. As a direct substrate of Cyclin E/CDK2, nuclear protein ataxia-telangiectasia (NPAT) is a crucial factor in regulating histone transcription and cell cycle progression. Here we identified that Cpn10/HSPE, a 10-kDa heat shock protein, is a novel interacting partner of NPAT. A pool of Cpn10 is colocalized with NPAT foci during G1 and S phases in nuclei. Gain- and loss-of-function experiments unraveled an essential role of Cpn10 in histone transcription. A conserved DLFD motif within Cpn10 was critical for targeting NPAT and modulating histone transcription. More importantly, knockdown of Cpn10 disrupted the focus formation of both NPAT and FADD-like interleukin-1β-converting enzyme-associated huge protein without affecting Coilin-positive Cajal bodies. Finally, Cpn10 is important for S phase progression and cell proliferation. Taken together, our finding revealed a novel role of Cpn10 in the spatial regulation of NPAT signaling and disclosed a previously unappreciated link between the heat shock protein and histone transcription regulation. PMID:26429916

  4. Nuclear Localization of the Autism Candidate Gene Neurobeachin and Functional Interaction with the NOTCH1 Intracellular Domain Indicate a Role in Regulating Transcription

    PubMed Central

    Tuand, Krizia; Stijnen, Pieter; Volders, Karolien; Declercq, Jeroen; Nuytens, Kim; Meulemans, Sandra; Creemers, John

    2016-01-01

    Background Neurobeachin (NBEA) is an autism spectrum disorders (ASD) candidate gene. NBEA deficiency affects regulated secretion, receptor trafficking, synaptic architecture and protein kinase A (PKA)-mediated phosphorylation. NBEA is a large multidomain scaffolding protein. From N- to C-terminus, NBEA has a concanavalin A-like lectin domain flanked by armadillo repeats (ACA), an A-kinase anchoring protein domain that can bind to PKA, a domain of unknown function (DUF1088) and a BEACH domain, preceded by a pleckstrin homology-like domain and followed by WD40 repeats (PBW). Although most of these domains mediate protein-protein interactions, no interaction screen has yet been performed. Methods Yeast two-hybrid screens with the ACA and PBW domain modules of NBEA gave a list of interaction partners, which were analyzed for Gene Ontology (GO) enrichment. Neuro-2a cells were used for confocal microscopy and nuclear extraction analysis. NOTCH-mediated transcription was studied with luciferase reporter assays and qRT-PCR, combined with NBEA knockdown or overexpression. Results Both domain modules showed a GO enrichment for the nucleus. PBW almost exclusively interacted with transcription regulators, while ACA interacted with a number of PKA substrates. NBEA was partially localized in the nucleus of Neuro-2a cells, albeit much less than in the cytoplasm. A nuclear localization signal was found in the DUF1088 domain, which was shown to contribute to the nuclear localization of an EGFP-DPBW fusion protein. Yeast two-hybrid identified the Notch1 intracellular domain as a physical interactor of the PBW domain and a role for NBEA as a negative regulator in Notch-mediated transcription was demonstrated. Conclusion Defining novel interaction partners of conserved NBEA domain modules identified a role for NBEA as transcriptional regulator in the nucleus. The physical interaction of NBEA with NOTCH1 is most relevant for ASD pathogenesis because NOTCH signaling is essential for

  5. Nuclear cathepsin D enhances TRPS1 transcriptional repressor function to regulate cell cycle progression and transformation in human breast cancer cells.

    PubMed

    Bach, Anne-Sophie; Derocq, Danielle; Laurent-Matha, Valérie; Montcourrier, Philippe; Sebti, Salwa; Orsetti, Béatrice; Theillet, Charles; Gongora, Céline; Pattingre, Sophie; Ibing, Eva; Roger, Pascal; Linares, Laetitia K; Reinheckel, Thomas; Meurice, Guillaume; Kaiser, Frank J; Gespach, Christian; Liaudet-Coopman, Emmanuelle

    2015-09-29

    The lysosomal protease cathepsin D (Cath-D) is overproduced in breast cancer cells (BCC) and supports tumor growth and metastasis formation. Here, we describe the mechanism whereby Cath-D is accumulated in the nucleus of ERα-positive (ER+) BCC. We identified TRPS1 (tricho-rhino-phalangeal-syndrome 1), a repressor of GATA-mediated transcription, and BAT3 (Scythe/BAG6), a nucleo-cytoplasmic shuttling chaperone protein, as new Cath-D-interacting nuclear proteins. Cath-D binds to BAT3 in ER+ BCC and they partially co-localize at the surface of lysosomes and in the nucleus. BAT3 silencing inhibits Cath-D accumulation in the nucleus, indicating that Cath-D nuclear targeting is controlled by BAT3. Fully mature Cath-D also binds to full-length TRPS1 and they co-localize in the nucleus of ER+ BCC where they are associated with chromatin. Using the LexA-VP16 fusion co-activator reporter assay, we then show that Cath-D acts as a transcriptional repressor, independently of its catalytic activity. Moreover, microarray analysis of BCC in which Cath-D and/or TRPS1 expression were silenced indicated that Cath-D enhances TRPS1-mediated repression of several TRPS1-regulated genes implicated in carcinogenesis, including PTHrP, a canonical TRPS1 gene target. In addition, co-silencing of TRPS1 and Cath-D in BCC affects the transcription of cell cycle, proliferation and transformation genes, and impairs cell cycle progression and soft agar colony formation. These findings indicate that Cath-D acts as a nuclear transcriptional cofactor of TRPS1 to regulate ER+ BCC proliferation and transformation in a non-proteolytic manner.

  6. Src subfamily kinases regulate nuclear export and degradation of transcription factor Nrf2 to switch off Nrf2-mediated antioxidant activation of cytoprotective gene expression.

    PubMed

    Niture, Suryakant K; Jain, Abhinav K; Shelton, Phillip M; Jaiswal, Anil K

    2011-08-19

    Nrf2 (NF-E2-related factor 2) is a nuclear transcription factor that in response to chemical and radiation stress regulates coordinated induction of a battery of cytoprotective gene expressions leading to cellular protection. In this study, we investigated the role of Src kinases in the regulation of Nrf2 and downstream signaling. siRNA-mediated inhibition of Fyn, Src, Yes, and Fgr, but not Lyn, in mouse hepatoma Hepa-1 cells, led to nuclear accumulation of Nrf2 and up-regulation of Nrf2 downstream gene expression. Mouse embryonic fibroblasts with combined deficiency of Fyn/Src/Yes/Fgr supported results from siRNA. In addition, steady-state overexpression of Fyn, Src, and Yes phosphorylated Nrf2Tyr568 that triggered nuclear export and degradation of Nrf2 and down-regulation of Nrf2 downstream gene expression. Exposure of cells to antioxidant, oxidant, or UV radiation increased nuclear import of Fyn, Src, and Yes kinases, which phosphorylated Nrf2Tyr568 resulting in nuclear export and degradation of Nrf2. Further analysis revealed that stress-activated GSK3β acted upstream to the Src kinases and phosphorylated the Src kinases, leading to their nuclear localization and Nrf2 phosphorylation. The overexpression of Src kinases in Hepa-1 cells led to decreased Nrf2, increased apoptosis, and decreased cell survival. Mouse embryonic fibroblasts deficient in Src kinases showed nuclear accumulation of Nrf2, induction of Nrf2 and downstream gene expression, reduced apoptosis, and increased cell survival. The studies together demonstrate that Src kinases play a critical role in nuclear export and degradation of Nrf2, thereby providing a negative feedback mechanism to switch off Nrf2 activation and restore normal cellular homeostasis.

  7. Phosphorylation of Nrf2 in the transcription activation domain by casein kinase 2 (CK2) is critical for the nuclear translocation and transcription activation function of Nrf2 in IMR-32 neuroblastoma cells.

    PubMed

    Apopa, Patrick L; He, Xiaoqing; Ma, Qiang

    2008-02-01

    The antioxidant-activated transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the induction of cytoprotective genes against chemical toxicity and oxidative injuries. The role of phosphorylation in Nrf2 activation has been suggested but remains elusive. We report that phenolic antioxidant/pro-oxidant tert-butylhydroquinone (tBHQ) induced two forms of the Nrf2 protein in neuroblastoma cells (IMR-32), which migrated as distinctive bands on SDS-PAGE. In vitro treatment with lambda phosphatase eliminated the slower migrating form and increased the amount of the faster migrating form of Nrf2. In vivo (32)Pi-phosphorylation resulted in (32)Pi-labeling of the Nrf2 protein in the presence of tBHQ that can be dephosphorylated by lambda phosphotase, indicating that the slower migrating form is a phosphorylated Nrf2 protein and the faster form an unphosphorylated Nrf2. Unphosphorylated Nrf2 predominated in the cytoplasm, whereas the phosphorylated form preferentially localized in the nucleus. Nuclear Nrf2 can be dephosphorylated by lambda phosphotase in vitro and be converted to the faster migrating form, implicating phosphorylation of Nrf2 in the cytoplasmic-nuclear translocation of the protein. Deletional analyses from both the carboxyl- and amino-ends revealed the transcription activation (TA) domains Neh4 (Nrf2-ECH homology 4) and Neh5 (Nrf2-ECH homology 5) as a major region necessary for the phosphorylation. The TA domains are characterized by the presence of multiple phosphorylation sites of casein kinase 2 (CK2). Moreover, CK2 phosphorylated the TA domains in vitro. Treatment with CK2 inhibitor 2-dimethylamino-4,5,6,7,-tetrabromo-1H-benzimidazole (DMAT) blocked the induction of endogenous target genes of Nrf2 in cells and inhibited the TA activities of both the full length and the TA domains of Nrf2 to a large extent. Finally, phosphorylation of the TA domains correlated with the nuclear translocation of Nrf2 that was inhibited by DMAT in a

  8. A Conserved Nuclear Cyclophilin Is Required for Both RNA Polymerase II Elongation and Co-transcriptional Splicing in Caenorhabditis elegans

    PubMed Central

    Ahn, Jeong H.; Rechsteiner, Andreas; Strome, Susan; Kelly, William G.

    2016-01-01

    The elongation phase of transcription by RNA Polymerase II (Pol II) involves numerous events that are tightly coordinated, including RNA processing, histone modification, and chromatin remodeling. RNA splicing factors are associated with elongating Pol II, and the interdependent coupling of splicing and elongation has been documented in several systems. Here we identify a conserved, multi-domain cyclophilin family member, SIG-7, as an essential factor for both normal transcription elongation and co-transcriptional splicing. In embryos depleted for SIG-7, RNA levels for over a thousand zygotically expressed genes are substantially reduced, Pol II becomes significantly reduced at the 3’ end of genes, marks of transcription elongation are reduced, and unspliced mRNAs accumulate. Our findings suggest that SIG-7 plays a central role in both Pol II elongation and co-transcriptional splicing and may provide an important link for their coordination and regulation. PMID:27541139

  9. Chromium histidinate protects against heat stress by modulating the expression of hepatic nuclear transcription factors in quail.

    PubMed

    Orhan, C; Akdemir, F; Sahin, N; Tuzcu, M; Komorowski, J R; Hayirli, A; Sahin, K

    2012-01-01

    1. This experiment was conducted to evaluate the effects of supplemental chromium histidinate (CrHis) on performance and expressions of hepatic nuclear factors kappaB, an enhancer (NF-κB) and an inhibitor (IκBα) of activated B cells in heat-stressed Japanese quail (Coturnix coturnix japonica). 2. A total of 180, 10-d-old Japanese quail were allocated randomly into 6 groups in a 2 × 3 factorial arrangement. Birds were reared either at 22°C for 24 h/d (thermoneutral, TN) or 34°C for 8 h/d (heat stress, HS) for 32 d and fed on one of three diets supplemented with 0, 400 or 800 µg of CrHis per kg of diet. Each group consisted of 10 cages, each containing three quail. Data (performance variables and hepatic NF-κB and IκBα) were analysed using 2-way ANOVA. 3. Heat stress caused reductions in cumulative feed intake (FI) by 5·7%, weight gain (WG) by 13·0%, final body weight (FBW) by 10·3%, carcase weight by 12·6% and carcase efficiency by 2·3% and an increase in feed conversion ratio (FCR, feed consumed, g:weight gained, g) by 8·4%. As supplemental CrHis level increased up to 800 µg/kg, there were linear increases in cumulative FI (from 602 to 609 g), WG (from 134 to 138 g), FBW (from 167 to 171 g), cold carcase weight (from 110 to 114 g) and cold carcase efficiency (from 65·5 to 66·4%) and a decrease in FE (from 4·51 to 4·42). The environmental temperature by CrHis level interaction effect on performance parameters was insignificant. Hepatic NF-κB p65 concentration was higher and hepatic IκBα concentration was lower in quail exposed to HS than in quail kept at TN temperature. Increasing supplemental CrHis level linearly inhibited hepatic NF-κB p65 expression from 134·4 to 105·3% and linearly enhanced hepatic IκBα expression from 73·4 to 99·6%. The decrease in hepatic NF-κB expression and the increase in hepatic IκB expression were more notable in the TN environment than in the HS environment. 4. In conclusion, heat

  10. 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

  11. Nuclear import of the transcription factor SHOOT MERISTEMLESS depends on heterodimerization with BLH proteins expressed in discrete sub-domains of the shoot apical meristem of Arabidopsis thaliana.

    PubMed

    Cole, Melanie; Nolte, Carolin; Werr, Wolfgang

    2006-01-01

    The gene SHOOT MERISTEMLESS (STM) is required for the initiation and the maintenance of the shoot apical meristem (SAM) in Arabidopsis and encodes a MEINOX/three amino acid loop extension (TALE)-HD-type transcription factor. Translational fusions with the green fluorescent protein showed that STM is not nuclear by default. In a yeast two-hybrid screen performed with a meristem-enriched cDNA library, three interacting BLH (Bel1-like homeodomain) transcription factors were identified. According to bimolecular fluorescence complementation, STM is targeted into the nuclear compartment through heterodimerization with BLH partner proteins, which are expressed in distinct SAM domains from the center to the periphery. On a functional level, overexpression experiments in transgenic Arabidopsis plants suggest that individual heterodimers provide distinct contributions. These results contribute to our understanding of the STM transcription factor function in the SAM and also shed new light on the evolution of the TALE-HD super gene family in animal and plant lineages.

  12. Rho-kinase signaling controls nucleocytoplasmic shuttling of class IIa Histone Deacetylase (HDAC7) and transcriptional activation of orphan nuclear receptor NR4A1

    SciTech Connect

    Compagnucci, Claudia; Barresi, Sabina; Petrini, Stefania; Bertini, Enrico; Zanni, Ginevra

    2015-04-03

    Rho-kinase (ROCK) has been well documented to play a key role in RhoA-induced actin remodeling. ROCK activation results in myosin light chain (MLC) phosphorylation either by direct action on MLC kinase (MLCK) or by inhibition of MLC phosphatase (MLCP), modulating actin–myosin contraction. We found that inhibition of the ROCK pathway in induced pluripotent stem cells, leads to nuclear export of HDAC7 and transcriptional activation of the orphan nuclear receptor NR4A1 while in cells with constitutive ROCK hyperactivity due to loss of function of the RhoGTPase activating protein Oligophrenin-1 (OPHN1), the orphan nuclear receptor NR4A1 is downregulated. Our study identify a new target of ROCK signaling via myosin phosphatase subunit (MYPT1) and Histone Deacetylase (HDAC7) at the nuclear level and provide new insights in the cellular functions of ROCK. - Highlights: • ROCK regulates nucleocytoplasmic shuttling of HDAC7 via phosphorylation of MYPT1. • Nuclear export of HDAC7 and upregulation of NR4A1 occurs with low ROCK activity. • High levels of ROCK activity due to OPHN1 loss of function downregulate NR4A1.

  13. Nuclear receptor Rev-erb alpha (Nr1d1) functions in concert with Nr2e3 to regulate transcriptional networks in the retina.

    PubMed

    Mollema, Nissa J; Yuan, Yang; Jelcick, Austin S; Sachs, Andrew J; von Alpen, Désirée; Schorderet, Daniel; Escher, Pascal; Haider, Neena B

    2011-03-08

    The majority of diseases in the retina are caused by genetic mutations affecting the development and function of photoreceptor cells. The transcriptional networks directing these processes are regulated by genes such as nuclear hormone receptors. The nuclear hormone receptor gene Rev-erb alpha/Nr1d1 has been widely studied for its role in the circadian cycle and cell metabolism, however its role in the retina is unknown. In order to understand the role of Rev-erb alpha/Nr1d1 in the retina, we evaluated the effects of loss of Nr1d1 to the developing retina and its co-regulation with the photoreceptor-specific nuclear receptor gene Nr2e3 in the developing and mature retina. Knock-down of Nr1d1 expression in the developing retina results in pan-retinal spotting and reduced retinal function by electroretinogram. Our studies show that NR1D1 protein is co-expressed with NR2E3 in the outer neuroblastic layer of the developing mouse retina. In the adult retina, NR1D1 is expressed in the ganglion cell layer and is co-expressed with NR2E3 in the outer nuclear layer, within rods and cones. Several genes co-targeted by NR2E3 and NR1D1 were identified that include: Nr2c1, Recoverin, Rgr, Rarres2, Pde8a, and Nupr1. We examined the cyclic expression of Nr1d1 and Nr2e3 over a twenty-four hour period and observed that both nuclear receptors cycle in a similar manner. Taken together, these studies reveal a novel role for Nr1d1, in conjunction with its cofactor Nr2e3, in regulating transcriptional networks critical for photoreceptor development and function.

  14. Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4α dimerisation and transcriptional activity

    PubMed Central

    Eeckhoute, Jérôme; Oxombre, Bénédicte; Formstecher, Pierre; Lefebvre, Philippe; Laine, Bernard

    2003-01-01

    Hepatocyte Nuclear Factor 4α (HNF4α, NR2A1) is central to hepatocyte and pancreatic β-cell functions. Along with retinoid X receptor α (RXRα), HNF4α belongs to the nuclear receptor subfamily 2 (NR2), characterised by a conserved arginyl residue and a glutamate residue insert in helix 7 (H7) of the ligand binding domain (LBD). Crystallographic studies indicate that R348 and E352 residues in RXRα H7 are involved in charge-driven interactions that improve dimerisation. Consistent with these findings, we showed that removing the charge of the corresponding residues in HNF4α H7, R258 and E262, impaired dimerisation in solution. Moreover, our results provide a new concept according to which helices of the HNF4α LBD dimerisation interface contribute differently to dimerisation required for DNA binding; unlike H9 and H10, H7 is not involved in DNA binding. Substitutions of E262 decreased the repression of HNF4α transcriptional activity by a dominant-negative HNF4α mutant, highlighting the importance of this residue for dimerisation in the cell context. The E262 insert is crucial for HNF4α function since its deletion abolished HNF4α transcriptional activity and coactivator recruitment. The glutamate residue insert and the conserved arginyl residue in H7 most probably represent a signature of the NR2 subfamily of nuclear receptors. PMID:14602925

  15. Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4alpha dimerisation and transcriptional activity.

    PubMed

    Eeckhoute, Jérôme; Oxombre, Bénédicte; Formstecher, Pierre; Lefebvre, Philippe; Laine, Bernard

    2003-11-15

    Hepatocyte Nuclear Factor 4alpha (HNF4alpha, NR2A1) is central to hepatocyte and pancreatic beta-cell functions. Along with retinoid X receptor alpha (RXRalpha), HNF4alpha belongs to the nuclear receptor subfamily 2 (NR2), characterised by a conserved arginyl residue and a glutamate residue insert in helix 7 (H7) of the ligand binding domain (LBD). Crystallographic studies indicate that R348 and E352 residues in RXRalpha H7 are involved in charge-driven interactions that improve dimerisation. Consistent with these findings, we showed that removing the charge of the corresponding residues in HNF4alpha H7, R258 and E262, impaired dimerisation in solution. Moreover, our results provide a new concept according to which helices of the HNF4alpha LBD dimerisation interface contribute differently to dimerisation required for DNA binding; unlike H9 and H10, H7 is not involved in DNA binding. Substitutions of E262 decreased the repression of HNF4alpha transcriptional activity by a dominant-negative HNF4alpha mutant, highlighting the importance of this residue for dimerisation in the cell context. The E262 insert is crucial for HNF4alpha function since its deletion abolished HNF4alpha transcriptional activity and coactivator recruitment. The glutamate residue insert and the conserved arginyl residue in H7 most probably represent a signature of the NR2 subfamily of nuclear receptors.

  16. Altered RNA processing and export lead to retention of mRNAs near transcription sites and nuclear pore complexes or within the nucleolus

    PubMed Central

    Paul, Biplab; Montpetit, Ben

    2016-01-01

    Many protein factors are required for mRNA biogenesis and nuclear export, which are central to the eukaryotic gene expression program. It is unclear, however, whether all factors have been identified. Here we report on a screen of >1000 essential gene mutants in Saccharomyces cerevisiae for defects in mRNA processing and export, identifying 26 mutants with defects in this process. Single-molecule FISH data showed that the majority of these mutants accumulated mRNA within specific regions of the nucleus, which included 1) mRNAs within the nucleolus when nucleocytoplasmic transport, rRNA biogenesis, or RNA processing and surveillance was disrupted, 2) the buildup of mRNAs near transcription sites in 3′-end processing and chromosome segregation mutants, and 3) transcripts being enriched near nuclear pore complexes when components of the mRNA export machinery were mutated. These data show that alterations to various nuclear processes lead to the retention of mRNAs at discrete locations within the nucleus. PMID:27385342

  17. Nuclear respiratory factor 2 induces the expression of many but not all human proteins acting in mitochondrial DNA transcription and replication.

    PubMed

    Bruni, Francesco; Polosa, Paola Loguercio; Gadaleta, Maria Nicola; Cantatore, Palmiro; Roberti, Marina

    2010-02-05

    In mammals, NRF-2 (nuclear respiratory factor 2), also named GA-binding protein, is an Ets family transcription factor that controls many genes involved in cell cycle progression and protein synthesis as well as in mitochondrial biogenesis. In this paper, we analyzed the role of NRF-2 in the regulation of human genes involved in mitochondrial DNA transcription and replication. By a combination of bioinformatic and biochemical approaches, we found that the factor binds in vitro and in vivo to the proximal promoter region of the genes coding for the transcription termination factor mTERF, the RNA polymerase POLRMT, the B subunit of the DNA polymerase-gamma, the DNA helicase TWINKLE, and the single-stranded DNA-binding protein mtSSB. The role of NRF-2 in modulating the expression of those genes was further established by RNA interference and overexpression strategies. On the contrary, we found that NRF-2 does not control the genes for the subunit A of DNA polymerase-gamma and for the transcription repressor MTERF3; we suggest that these genes are under regulatory mechanisms that do not involve NRF proteins. Since NRFs are known to positively control the expression of transcription-activating proteins, the novelty emerging from our data is that proteins playing antithetical roles in mitochondrial DNA transcription, namely activators and repressors, are under different regulatory pathways. Finally, we developed a more stringent consensus with respect to the general consensus of NRF-2/GA-binding protein when searching for NRF-2 binding sites in the promoter of mitochondrial proteins.

  18. AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells

    SciTech Connect

    Lorenzato, Annalisa; Biolatti, Marta; Delogu, Giuseppe; Capobianco, Giampiero; Farace, Cristiano; Dessole, Salvatore; Cossu, Antonio; Tanda, Francesco; Madeddu, Roberto; Olivero, Martina; Di Renzo, Maria Flavia

    2013-10-15

    The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. - highlights: • CSE1L is a key player in nucleocytoplasmic traffic by forming complex with Ran. • AKT phosphorylates RanBP3 that regulates the nucleocytoplasmic gradient of Ran. • The activated oncogenic AKT drives the nuclear accumulation of CSE1L. • CSE1L in the nucleus up-regulates genes conveying pro-oncogenic signals. • CSE1L might contribute to tumor progression driven by the activated oncogenic AKT.

  19. Tamoxifen increases nuclear respiratory factor 1 transcription by activating estrogen receptor beta and AP-1 recruitment to adjacent promoter binding sites.

    PubMed

    Ivanova, Margarita M; Luken, Kristen H; Zimmer, Amber S; Lenzo, Felicia L; Smith, Ryan J; Arteel, Maia W; Kollenberg, Tara J; Mattingly, Kathleen A; Klinge, Carolyn M

    2011-04-01

    Little is known about endogenous estrogen receptor β (ERβ) gene targets in human breast cancer. We reported that estradiol (E(2)) induces nuclear respiratory factor-1 (NRF-1) transcription through ERα in MCF-7 breast cancer cells. Here we report that 4-hydroxytamoxifen (4-OHT), with an EC(50) of ~1.7 nM, increases NRF-1 expression by recruiting ERβ, cJun, cFos, CBP, and RNA polymerase II to and dismissing NCoR from the NRF1 promoter. Promoter deletion and transient transfection studies showed that the estrogen response element (ERE) is essential and that an adjacent AP-1 site contributes to maximal 4-OHT-induced NRF-1 transcription. siRNA knockdown of ERβ revealed that ERβ inhibits basal NRF-1 expression and is required for 4-OHT-induced NRF-1 transcription. An AP-1 inhibitor blocked 4-OHT-induced NRF-1 expression. The 4-OHT-induced increase in NRF-1 resulted in increased transcription of NRF-1 target CAPNS1 but not CYC1, CYC2, or TFAM despite increased NRF-1 coactivator PGC-1α protein. The absence of TFAM induction corresponds to a lack of Akt-dependent phosphorylation of NRF-1 with 4-OHT treatment. Overexpression of NRF-1 inhibited 4-OHT-induced apoptosis and siRNA knockdown of NRF-1 increased apoptosis, indicating an antiapoptotic role for NRF-1. Overall, NRF-1 expression and activity is regulated by 4-OHT via endogenous ERβ in MCF-7 cells.

  20. Evidence for the Involvement of Xenobiotic-responsive Nuclear Receptors in Transcriptional Effects Upon Perfluoroalkyl Acid Exposure in Diverse Species.

    EPA Science Inventory

    Humans and other species have detectable body burdens of a number of perfluorinated alkyl acids (PFAA) including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). In mouse and rat liver these compounds elicit transcriptional and phenotypic effects similar to pe...

  1. Evidence for the Involvement of Xenobiotic-response Nuclear Receptors in Transcriptional Effects Upon Perfluroroalkyl Acid Exposure in Diverse Species

    EPA Science Inventory

    Humans and ecological species have been found to have detectable body burdens of a number of perfluorinated alkyl acids (PFAA) including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). In mouse and rat liver these compounds elicit transcriptional and phenotyp...

  2. Hepatocyte Nuclear Factor 4α (HNF4α) Is a Transcription Factor of Vertebrate Fatty Acyl Desaturase Gene as Identified in Marine Teleost Siganus canaliculatus

    PubMed Central

    Dong, Yewei; Wang, Shuqi; Chen, Junliang; Zhang, Qinghao; Liu, Yang; You, Cuihong; Monroig, Óscar; Tocher, Douglas R.; Li, Yuanyou

    2016-01-01

    Rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the capability of biosynthesizing long-chain polyunsaturated fatty acids (LC-PUFA) from C18 precursors, and to possess a Δ4 fatty acyl desaturase (Δ4 Fad) which was the first report in vertebrates, and is a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. In order to understand regulatory mechanisms of transcription of Δ4 Fad, the gene promoter was cloned and characterized in the present study. An upstream sequence of 1859 bp from the initiation codon ATG was cloned as the promoter candidate. On the basis of bioinformatic analysis, several binding sites of transcription factors (TF) including GATA binding protein 2 (GATA-2), CCAAT enhancer binding protein (C/EBP), nuclear factor 1 (NF-1), nuclear factor Y (NF-Y), hepatocyte nuclear factor 4α (HNF4α) and sterol regulatory element (SRE), were identified in the promoter by site-directed mutation and functional assays. HNF4α and NF-1 were confirmed to interact with the core promoter of Δ4 Fad by gel shift assay and mass spectrometry. Moreover, over-expression of HNF4α increased promoter activity in HEK 293T cells and mRNA level of Δ4 Fad in rabbitfish primary hepatocytes, respectively. The results indicated that HNF4α is a TF of rabbitfish Δ4 Fad. To our knowledge, this is the first report on promoter structure of a Δ4 Fad, and also the first demonstration of HNF4α as a TF of vertebrate Fad gene involved in transcription regulation of LC-PUFA biosynthesis. PMID:27472219

  3. Ciliary Entry of the Hedgehog Transcriptional Activator Gli2 Is Mediated by the Nuclear Import Machinery but Differs from Nuclear Transport in Being Imp-α/β1-Independent.

    PubMed

    Torrado, Belén; Graña, Martín; Badano, José L; Irigoín, Florencia

    2016-01-01

    Gli2 is the primary transcriptional activator of Hedgehog signalling in mammals. Upon stimulation of the pathway, Gli2 moves into the cilium before reaching the nucleus. However, the mechanisms underlying its entry into the cilium are not completely understood. Since several similarities have been reported between nuclear and ciliary import, we investigated if the nuclear import machinery participates in Gli2 ciliary entry. Here we show that while two conserved classical nuclear localization signals mediate Gli2 nuclear localization via importin (Imp)-α/β1, these sequences are not required for Gli2 ciliary import. However, blocking Imp-mediated transport through overexpression of GTP-locked Ran reduced the percentage of Gli2 positive cilia, an effect that was not explained by increased CRM1-dependent export of Gli2 from the cilium. We explored the participation of Imp-β2 in Gli2 ciliary traffic and observed that this transporter is involved in moving Gli2 into the cilium, as has been described for other ciliary proteins. In addition, our data indicate that Imp-β2 might also collaborate in Gli2 nuclear entry. How does Imp-β2 determine the final destination of a protein that can localize to two distinct subcellular compartments remains an open question. Therefore, our data shows that the nuclear-cytoplasmic shuttling machinery plays a critical role mediating the subcellular distribution of Gli2 and the activation of the pathway, but distinct importins likely play a differential role mediating its ciliary and nuclear translocation.

  4. Ciliary Entry of the Hedgehog Transcriptional Activator Gli2 Is Mediated by the Nuclear Import Machinery but Differs from Nuclear Transport in Being Imp-α/β1-Independent

    PubMed Central

    Torrado, Belén; Graña, Martín; Badano, José L.; Irigoín, Florencia

    2016-01-01

    Gli2 is the primary transcriptional activator of Hedgehog signalling in mammals. Upon stimulation of the pathway, Gli2 moves into the cilium before reaching the nucleus. However, the mechanisms underlying its entry into the cilium are not completely understood. Since several similarities have been reported between nuclear and ciliary import, we investigated if the nuclear import machinery participates in Gli2 ciliary entry. Here we show that while two conserved classical nuclear localization signals mediate Gli2 nuclear localization via importin (Imp)-α/β1, these sequences are not required for Gli2 ciliary import. However, blocking Imp-mediated transport through overexpression of GTP-locked Ran reduced the percentage of Gli2 positive cilia, an effect that was not explained by increased CRM1-dependent export of Gli2 from the cilium. We explored the participation of Imp-β2 in Gli2 ciliary traffic and observed that this transporter is involved in moving Gli2 into the cilium, as has been described for other ciliary proteins. In addition, our data indicate that Imp-β2 might also collaborate in Gli2 nuclear entry. How does Imp-β2 determine the final destination of a protein that can localize to two distinct subcellular compartments remains an open question. Therefore, our data shows that the nuclear-cytoplasmic shuttling machinery plays a critical role mediating the subcellular distribution of Gli2 and the activation of the pathway, but distinct importins likely play a differential role mediating its ciliary and nuclear translocation. PMID:27579771

  5. Death-domain associated protein-6 (DAXX) mediated apoptosis in hantavirus infection is counter-balanced by activation of interferon-stimulated nuclear transcription factors

    SciTech Connect

    Khaiboullina, Svetlana F.; Morzunov, Sergey P.; Boichuk, Sergei V.; Palotás, András; Jeor, Stephen St.; Lombardi, Vincent C.; Rizvanov, Albert A.

    2013-09-01

    Hantaviruses are negative strand RNA species that replicate predominantly in the cytoplasm. They also activate numerous cellular responses, but their involvement in nuclear processes is yet to be established. Using human umbilical vein endothelial cells (HUVECs), this study investigates the molecular finger-print of nuclear transcription factors during hantavirus infection. The viral-replication-dependent activation of pro-myelocytic leukemia protein (PML) was followed by subsequent localization in nuclear bodies (NBs). PML was also found in close proximity to activated Sp100 nuclear antigen and interferon-stimulated gene 20 kDa protein (ISG-20), but co-localization with death-domain associated protein-6 (DAXX) was not observed. These data demonstrate that hantavirus triggers PML activation and localization in NBs in the absence of DAXX-PLM-NB co-localization. The results suggest that viral infection interferes with DAXX-mediated apoptosis, and expression of interferon-activated Sp100 and ISG-20 proteins may indicate intracellular intrinsic antiviral attempts.

  6. Transcriptional activation of LON Gene by a new form of mitochondrial stress: A role for the nuclear respiratory factor 2 in StAR overload response (SOR).

    PubMed

    Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Isaac, Sara; Eden, Amir; Lauria, Ines; Langer, Thomas; Orly, Joseph

    2015-06-15

    High output of steroid hormone synthesis in steroidogenic cells of the adrenal cortex and the gonads requires the expression of the steroidogenic acute regulatory protein (StAR) that facilitates cholesterol mobilization to the mitochondrial inner membrane where the CYP11A1/P450scc enzyme complex converts the sterol to the first steroid. Earlier studies have shown that StAR is active while pausing on the cytosolic face of the outer mitochondrial membrane while subsequent import of the protein into the matrix terminates the cholesterol mobilization activity. Consequently, during repeated activity cycles, high level of post-active StAR accumulates in the mitochondrial matrix. To prevent functional damage due to such protein overload effect, StAR is degraded by a sequence of three to four ATP-dependent proteases of the mitochondria protein quality control system, including LON and the m-AAA membranous proteases AFG3L2 and SPG7/paraplegin. Furthermore, StAR expression in both peri-ovulatory ovarian cells, or under ectopic expression in cell line models, results in up to 3-fold enrichment of the mitochondrial proteases and their transcripts. We named this novel form of mitochondrial stress as StAR overload response (SOR). To better understand the SOR mechanism at the transcriptional level we analyzed first the unexplored properties of the proximal promoter of the LON gene. Our findings suggest that the human nuclear respiratory factor 2 (NRF-2), also known as GA binding protein (GABP), is responsible for 88% of the proximal promoter activity, including the observed increase of transcription in the presence of StAR. Further studies are expected to reveal if common transcriptional determinants coordinate the SOR induced transcription of all the genes encoding the SOR proteases.

  7. GSK-3 mediated phosphorylation couples ER-Golgi transport and nuclear stabilisation of the CREB-H transcription factor to mediate Apolipoprotein secretion.

    PubMed

    Barbosa, Sónia; Carreira, Suzanne; O'Hare, Peter

    2017-04-05

    CREB-H, an ER-anchored transcription factor plays a key role in regulating secretion in metabolic pathways, particularly triglyceride homeostasis. It controls the production both of secretory pathway components and cargoes including apolipoproteins ApoA-IV and ApoC-II, contributing to VLDL/HDL distribution and lipolysis. The key mechanism controlling CREB-H activity involves its ER retention and forward transport to the Golgi, where it is cleaved by Golgi-resident proteases releasing the N-terminal product which traffics to the nucleus to effect transcriptional responses. Here we show that a serine-rich motif, termed the P-motif located in the N-terminus between serines 73 to 90, controls release of the precursor transmembrane form from the ER and its forward transport to the Golgi. This motif is subject to GSK-3 phosphorylation promoting ER-retention while mutation of target serines or drug inhibition of GSK-3 activity, co-ordinately induces both forward transport of the precursor and cleavage, resulting in nuclear import. We previously showed that for the nuclear product, the P-motif is subject to multiple phosphorylations which regulate stability by targeting the protein to the SCF(Fbw1a) E3 ubiquitin ligase. Thus phosphorylation at the P-motif provides integrated control of CREB-H function, coupling intercompartmental transport in the cytoplasm with stabilisation of the active form in the nucleus.

  8. Enhanced Cardiac Akt/Protein Kinase B Signaling Contributes to Pathological Cardiac Hypertrophy in Part by Impairing Mitochondrial Function via Transcriptional Repression of Mitochondrion-Targeted Nuclear Genes

    PubMed Central

    Wende, Adam R.; O'Neill, Brian T.; Bugger, Heiko; Riehle, Christian; Tuinei, Joseph; Buchanan, Jonathan; Tsushima, Kensuke; Wang, Li; Caro, Pilar; Guo, Aili; Sloan, Crystal; Kim, Bum Jun; Wang, Xiaohui; Pereira, Renata O.; McCrory, Mark A.; Nye, Brenna G.; Benavides, Gloria A.; Darley-Usmar, Victor M.; Shioi, Tetsuo; Weimer, Bart C.

    2014-01-01

    Sustained Akt activation induces cardiac hypertrophy (LVH), which may lead to heart failure. This study tested the hypothesis that Akt activation contributes to mitochondrial dysfunction in pathological LVH. Akt activation induced LVH and progressive repression of mitochondrial fatty acid oxidation (FAO) pathways. Preventing LVH by inhibiting mTOR failed to prevent the decline in mitochondrial function, but glucose utilization was maintained. Akt activation represses expression of mitochondrial regulatory, FAO, and oxidative phosphorylation genes in vivo that correlate with the duration of Akt activation in part by reducing FOXO-mediated transcriptional activation of mitochondrion-targeted nuclear genes in concert with reduced signaling via peroxisome proliferator-activated receptor α (PPARα)/PGC-1α and other transcriptional regulators. In cultured myocytes, Akt activation disrupted mitochondrial bioenergetics, which could be partially reversed by maintaining nuclear FOXO but not by increasing PGC-1α. Thus, although short-term Akt activation may be cardioprotective during ischemia by reducing mitochondrial metabolism and increasing glycolysis, long-term Akt activation in the adult heart contributes to pathological LVH in part by reducing mitochondrial oxidative capacity. PMID:25535334

  9. Equal impact of diffusion and DNA binding rates on the potential spatial distribution of nuclear factor κB transcription factor inside the nucleus.

    PubMed

    Sycheva, A M; Kel, A; Nikolaev, E N; Moshkovskii, S A

    2014-06-01

    There are two physical processes that influence the spatial distribution of transcription factor molecules entering the nucleus of a eukaryotic cell, the binding to genomic DNA and the diffusion throughout the nuclear volume. Comparison of the DNA-protein association rate constant and the protein diffusion constant may determine which one is the limiting factor. If the process is diffusion-limited, transcription factor molecules are captured by DNA before their even distribution in the nuclear volume. Otherwise, if the reaction rate is limiting, these molecules diffuse evenly and then find their binding sites. Using well-studied human NF-κB dimer as an example, we calculated its diffusion constant using the Debye-Smoluchowski equation. The value of diffusion constant was about 10(-15) cm(3)/s, and it was comparable to the NF-κB association rate constant for DNA binding known from previous studies. Thus, both diffusion and DNA binding play an equally important role in NF-κB spatial distribution. The importance of genome 3D-structure in gene expression regulation and possible dependence of gene expression on the local concentration of open chromatin can be hypothesized from our theoretical estimate.

  10. IL6 Inhibits HBV Transcription by Targeting the Epigenetic Control of the Nuclear cccDNA Minichromosome

    PubMed Central

    Palumbo, Gianna Aurora; Scisciani, Cecilia; Pediconi, Natalia; Lupacchini, Leonardo; Alfalate, Dulce; Guerrieri, Francesca; Calvo, Ludovica; Salerno, Debora; Di Cocco, Silvia; Levrero, Massimo; Belloni, Laura

    2015-01-01

    The HBV covalently closed circular DNA (cccDNA) is organized as a mini-chromosome in the nuclei of infected hepatocytes by histone and non-histone proteins. Transcription from the cccDNA of the RNA replicative intermediate termed pre-genome (pgRNA), is the critical step for genome amplification and ultimately determines the rate of HBV replication. Multiple evidences suggest that cccDNA epigenetic modifications, such as histone modifications and DNA methylation, participate in regulating the transcriptional activity of the HBV cccDNA. Inflammatory cytokines (TNFα, LTβ) and the pleiotropic cytokine interleukin-6 (IL6) inhibit hepatitis B virus (HBV) replication and transcription. Here we show, in HepG2 cells transfected with linear HBV monomers and HBV-infected NTCP-HepG2 cells, that IL6 treatment leads to a reduction of cccDNA-bound histone acetylation paralleled by a rapid decrease in 3.5kb/pgRNA and subgenomic HBV RNAs transcription without affecting cccDNA chromatinization or cccDNA levels. IL6 repressive effect on HBV replication is mediated by a loss of HNF1α and HNF4α binding to the cccDNA and a redistribution of STAT3 binding from the cccDNA to IL6 cellular target genes. PMID:26580974

  11. NUCLEAR FACTOR Y, Subunit C (NF-YC) Transcription Factors Are Positive Regulators of Photomorphogenesis in Arabidopsis thaliana

    PubMed Central

    Siriwardana, Chamindika L.; Holt III, Ben F.

    2016-01-01

    Recent reports suggest that NF-Y transcription factors are positive regulators of skotomorphogenesis in Arabidopsis thaliana. Three NF-YC genes (NF-YC3, NF-YC4, and NF-YC9) are known to have overlapping functions in photoperiod dependent flowering and previous studies demonstrated that they interact with basic leucine zipper (bZIP) transcription factors. This included ELONGATED HYPOCOTYL 5 (HY5), which has well-demonstrated roles in photomorphogenesis. Similar to hy5 mutants, we report that nf-yc3 nf-yc4 nf-yc9 triple mutants failed to inhibit hypocotyl elongation in all tested light wavelengths. Surprisingly, nf-yc3 nf-yc4 nf-yc9 hy5 mutants had synergistic defects in light perception, suggesting that NF-Ys represent a parallel light signaling pathway. As with other photomorphogenic transcription factors, nf-yc3 nf-yc4 nf-yc9 triple mutants also partially suppressed the short hypocotyl and dwarf rosette phenotypes of CONSTITUTIVE PHOTOMORPHOGENIC 1 (cop1) mutants. Thus, our data strongly suggest that NF-Y transcription factors have important roles as positive regulators of photomorphogenesis, and in conjunction with other recent reports, implies that the NF-Y are multifaceted regulators of early seedling development. PMID:27685091

  12. Adaptive and specialised transcriptional responses to xenobiotic stress in Caenorhabditis elegans are regulated by nuclear hormone receptors.

    PubMed

    Jones, Laura M; Rayson, Samantha J; Flemming, Anthony J; Urwin, Peter E

    2013-01-01

    Characterisation of the pathways by which xenobiotics are metabolised and excreted in both target and non-target organisms is crucial for the rational design of effective and specific novel bioactive molecules. Consequently, we have investigated the induced responses of the model nematode Caenorhabditis elegans to a variety of xenobiotics which represent a range of putative modes of action. The majority of genes that were specifically induced in preliminary microarray analyses encoded enzymes from Phase I and II metabolism, including cytochrome P450s, short chain dehydrogenases, UDP-glucuronosyl transferases and glutathione transferases. Changes in gene expression were confirmed by quantitative PCR and GFP induction in reporter strains driven by promoters for transcription of twelve induced enzymes was investigated. The particular complement of metabolic genes induced was found to be highly contingent on the xenobiotic applied. The known regulators of responses to applied chemicals ahr-1, hif-1, mdt-15 and nhr-8 were not required for any of these inducible responses and skn-1 regulated GFP expression from only two of the promoters. Reporter strains were used in conjunction with systematic RNAi screens to identify transcription factors which drive expression of these genes under xenobiotic exposure. These transcription factors appeared to regulate specific xenobiotic responses and have no reported phenotypes under standard conditions. Focussing on nhr-176 we demonstrate the role of this transcription factor in mediating the resistance to thiabendazole.

  13. Dissecting nuclear Wingless signalling: recruitment of the transcriptional co-activator Pygopus by a chain of adaptor proteins.

    PubMed

    Städeli, Reto; Basler, Konrad

    2005-11-01

    Members of the Wingless (Wg)/Wnt family of secreted glycoproteins control cell fate during embryonic development and adult homeostasis. Wnt signals regulate the expression of target genes by activating a conserved signal transduction pathway. Upon receptor activation, the signal is transmitted intracellularly by stabilization of Armadillo (Arm)/beta-catenin. Arm/beta-catenin translocates to the nucleus, interacts with DNA-binding factors of the Pangolin (Pan)/TCF/LEF class and activates transcription of target genes in cooperation with the recently identified proteins Legless/BCL9 (Lgs) and Pygopus (Pygo). Here, we analyse the mode of action of Pan, Arm, Lgs, and Pygo in Drosophila cultured cells. We provide evidence that together these four proteins form a 'chain of adaptors' linking the NH2-terminal homology domain (NHD) of Pygo to the DNA-binding domain of Pan. We show that the NHD has potent transcriptional activation capacity, which differs from that of acidic activator domains and depends on a conserved NPF tripeptide. A single point mutation within this NPF motif abolishes the transcriptional activity of the Pygo NHD in vitro and strongly reduces Wg signalling in vivo. Together, our results suggest that the transcriptional output of Wg pathway activity largely relies on a 'chain of adaptors' design to direct the Pygo NHD to Wg target promoters in an Arm-dependent manner.

  14. CA150, a nuclear protein associated with the RNA polymerase II holoenzyme, is involved in Tat-activated human immunodeficiency virus type 1 transcription.

    PubMed Central

    Suñé, C; Hayashi, T; Liu, Y; Lane, W S; Young, R A; Garcia-Blanco, M A

    1997-01-01

    Maximal human immunodeficiency virus type 1 (HIV-1) gene expression requires specific cellular factors in addition to the virus-encoded trans-activator protein Tat and the RNA element TAR. We developed a functional assay, based on transcriptional activation in vitro, to identify these cellular factors. Here, we describe the purification and molecular cloning of CA150, a nuclear protein that is associated with the human RNA polymerase II holoenzyme and is involved in Tat-dependent HIV-1 transcriptional activation. The sequence of CA150 contains an extensive glutamine- and alanine-rich repeat that is found in transcriptional modulators such as GAL11 and SSN6 in Saccharomyces cerevisiae and Zeste in Drosophila melanogaster. Immunodepletion of CA150 abolished Tat trans activation in vitro. Moreover, overexpression of a mutant CA150 protein specifically and dramatically decreased Tat-mediated activation of the HIV-1 promoter in vivo, strongly suggesting a role for CA150 in HIV-1 gene regulation. Immunoprecipitation experiments demonstrated that both CA150 and Tat associate with the RNA polymerase II holoenzyme. Furthermore, we found that functional Tat associates with the holoenzyme whereas activation-deficient Tat mutants do not. Thus, we propose that Tat action is transduced via an RNA polymerase II holoenzyme that contains CA150. PMID:9315662

  15. Exo70 is transcriptionally up-regulated by hepatic nuclear factor 4α and contributes to cell cycle control in hepatoma cells

    PubMed Central

    Zhao, Yujie; Hou, Jihuan; Mi, Panying; Mao, Liyuan; Xu, Liang; Zhang, Youyu; Xiao, Li; Cao, Hanwei; Zhang, Wenqing; Zhang, Bing; Song, Gang; Hu, Tianhui; Zhan, Yan-yan

    2016-01-01

    Exo70, a member of the exocyst complex, is involved in cell exocytosis, migration, invasion and autophagy. However, the expression regulation and function of Exo70 in hepatocellular carcinoma are still poorly understood. In this study, we found Exo70 expression in human hepatoma cells was greatly reduced after knocking down hepatic nuclear factor 4α (HNF4α), the most important and abundant transcription factor in liver. This regulation occurred at the transcriptional level but not post-translational level. HNF4α transactivated Exo70 promoter through directly binding to the HNF4α-response element in this promoter. Cell cycle analysis further revealed that down-regulation of HNF4α and Exo70 was essential to berberine-stimulated G2/M cell cycle arrest in hepatoma cells. Moreover, knocking down either Exo70 or HNF4α induced G2/M phase arrest of hepatoma cells. Exo70 acted downstream of HNF4α to stimulate G2/M transition via increasing Cdc2 expression. Together, our results identify Exo70 as a novel transcriptional target of HNF4α to promote cell cycle progression in hepatoma, thus provide a basis for the development of therapeutic strategies for hepatocellular carcinoma. PMID:26848864

  16. Down-regulation of the zinc-finger homeobox protein TSHZ2 releases GLI1 from the nuclear repressor complex to restore its transcriptional activity during mammary tumorigenesis

    PubMed Central

    Riku, Miho; Inaguma, Shingo; Ito, Hideaki; Tsunoda, Takumi; Ikeda, Hiroshi; Kasai, Kenji

    2016-01-01

    Although breast cancer is one of the most common malignancies, the molecular mechanisms underlying its development and progression are not fully understood. To identify key molecules involved, we screened publicly available microarray datasets for genes differentially expressed between breast cancers and normal mammary glands. We found that three of the genes predicted in this analysis were differentially expressed among human mammary tissues and cell lines. Of these genes, we focused on the role of the zinc-finger homeobox protein TSHZ2, which is down-regulated in breast cancer cells. We found that TSHZ2 is a nuclear protein harboring a bipartite nuclear localization signal, and we confirmed its function as a C-terminal binding protein (CtBP)-dependent transcriptional repressor. Through comprehensive screening, we identified TSHZ2-suppressing genes such as AEBP1 and CXCR4, which are conversely up-regulated by GLI1, the downstream transcription factor of Hedgehog signaling. We found that GLI1 forms a ternary complex with CtBP2 in the presence of TSHZ2 and that the transcriptional activity of GLI1 is suppressed by TSHZ2 in a CtBP-dependent manner. Indeed, knockdown of TSHZ2 increases the expression of AEBP1 and CXCR4 in TSHZ2-expressing immortalized mammary duct epithelium. Concordantly, immunohistochemical staining of mammary glands revealed that normal duct cells expresses GLI1 in the nucleus along with TSHZ2 and CtBP2, whereas invasive ductal carcinoma cells, which does not express TSHZ2, show the increase in the expression of AEBP1 and CXCR4 and in the cytoplasmic localization of GLI1. Thus, we propose that down-regulation of TSHZ2 is crucial for mammary tumorigenesis via the activation of GLI1. PMID:26744317

  17. Post-transcriptional regulation of cyclins D1, D3 and G1 and proliferation of human cancer cells depend on IMP-3 nuclear localization.

    PubMed

    Rivera Vargas, T; Boudoukha, S; Simon, A; Souidi, M; Cuvellier, S; Pinna, G; Polesskaya, A

    2014-05-29

    RNA-binding proteins of the IMP family (insulin-like growth factor 2 (IGF2) mRNA-binding proteins 1-3) are important post-transcriptional regulators of gene expression. Multiple studies have linked high expression of IMP proteins, and especially of IMP-3, to an unfavorable prognosis in numerous types of cancer. The specific importance of IMP-3 for cancer transformation remains poorly understood. We here show that all three IMPs can directly bind the mRNAs of cyclins D1, D3 and G1 (CCND1, D3 and G1) in vivo and in vitro, and yet only IMP-3 regulates the expression of these cyclins in a significant manner in six human cancer cell lines of different origins. In the absence of IMP-3, the levels of CCND1, D3 and G1 proteins fall dramatically, and the cells accumulate in the G1 phase of the cell cycle, leading to almost complete proliferation arrest. Our results show that, compared with IMP-1 and IMP-2, IMP-3 is enriched in the nucleus, where it binds the transcripts of CCND1, D3 and G1. The nuclear localization of IMP-3 depends on its protein partner HNRNPM and is indispensable for the post-transcriptional regulation of expression of the cyclins. Cytoplasmic retention of IMP-3 and HNRNPM in human cancer cells leads to significant drop in proliferation. In conclusion, a nuclear IMP-3-HNRNPM complex is important for the efficient synthesis of CCND1, D3 and G1 and for the proliferation of human cancer cells.

  18. Nuclear transcription factor Oct-1 binds to the 5'-upstream region of CYP1A1 and negatively regulates its expression.

    PubMed

    Bhat, R; Weaver, J A; Sterling, K M; Bresnick, E

    1996-02-01

    The cytochrome P450-dependent monooxygenases, which represent an extended superfamily, catalyze the biotransformation of many endogenous and exogenous substances. One of these hemoproteins, cytochrome P4501A1, is most closely associated with the bioactivation of polycyclic aromatic hydrocarbons such as benzo[a]pyrene, which may play a role in environmental carcinogenesis. A negative regulatory element (NRE) has been localized in the 5'-upstream region of the cytochrome P4501A1 gene (CYP1A1) at -843 to -746 base pairs from the site of transcription. The purpose of this research was to define any interactions of trans-acting proteins with this cis element. Rat liver nuclei were used as the source of trans-acting proteins and a biotinylated NRE-bearing fragment (-782 to -843 bp) from a plasmid which contained the CYP1A1 was prepared by the polymerase chain reaction technique. Gel mobility shift assays were used to demonstrate interactions between this NRE fragment and nuclear proteins. The specific binding to an octamer-containing motif in the 5'-upstream region of CYP1A1 was demonstrated; this was used as a step in the partial purification from rat liver of the transcription factor, Oct-1. Conventional chromatographic procedures and DNA recognition site affinity chromatography were also used. HepG2 human hepatoma cells were transfected with both pMCoLUC+ which contains the luciferase gene as a reporter gene driven by the CYP1A1 promoter (including the NRE), and an Oct-1 expression vector. Luciferase activity/mg protein in the doubly-transfected cells was significantly lower than in cells containing only pMCoLUC+. A nuclear transcription factor Oct-1 interacts with a portion of the NRE of the rat CYP1A1, suppressing the expression of this gene. These findings may help to explain the low level of basal expression of CYP1A1 in mammalian systems.

  19. Nuclear Localization of CD26 Induced by a Humanized Monoclonal Antibody Inhibits Tumor Cell Growth by Modulating of POLR2A Transcription

    PubMed Central

    Yamada, Kohji; Hayashi, Mutsumi; Madokoro, Hiroko; Nishida, Hiroko; Du, Wenlin; Ohnuma, Kei; Sakamoto, Michiie; Morimoto, Chikao; Yamada, Taketo

    2013-01-01

    CD26 is a type II glycoprotein known as dipeptidyl peptidase IV and has been identified as one of the cell surface markers associated with various types of cancers and a subset of cancer stem cells. Recent studies have suggested that CD26 expression is involved in tumor growth, tumor invasion, and metastasis. The CD26 is shown in an extensive intracellular distribution, ranging from the cell surface to the nucleus. We have previously showed that the humanized anti-CD26 monoclonal antibody (mAb), YS110, exhibits inhibitory effects on various cancers. However, functions of CD26 on cancer cells and molecular mechanisms of impaired tumor growth by YS110 treatment are not well understood. In this study, we demonstrated that the treatment with YS110 induced nuclear translocation of both cell-surface CD26 and YS110 in cancer cells and xenografted tumor. It was shown that the CD26 and YS110 were co-localized in nucleus by immunoelectron microscopic analysis. In response to YS110 treatment, CD26 was translocated into the nucleus via caveolin-dependent endocytosis. It was revealed that the nuclear CD26 interacted with a genomic flanking region of the gene for POLR2A, a subunit of RNA polymerase II, using a chromatin immunoprecipitation assay. This interaction with nuclear CD26 and POLR2A gene consequently led to transcriptional repression of the POLR2A gene, resulting in retarded cell proliferation of cancer cells. Furthermore, the impaired nuclear transport of CD26 by treatment with an endocytosis inhibitor or expressions of deletion mutants of CD26 reversed the POLR2A repression induced by YS110 treatment. These findings reveal that the nuclear CD26 functions in the regulation of gene expression and tumor growth, and provide a novel mechanism of mAb-therapy related to inducible translocation of cell-surface target molecule into the nucleus. PMID:23638030

  20. FUS interacts with nuclear matrix-associated protein SAFB1 as well as Matrin3 to regulate splicing and ligand-mediated transcription

    PubMed Central

    Yamaguchi, Atsushi; Takanashi, Keisuke

    2016-01-01

    FUS (Fused-in-Sarcoma) is a multifunctional DNA/RNA binding protein linked to familial amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). Since FUS is localized mainly in the nucleus with nucleo-cytoplasmic shuttling, it is critical to understand physiological functions in the nucleus to clarify pathogenesis. Here we report a yeast two-hybrid screening identified FUS interaction with nuclear matrix-associated protein SAFB1 (scaffold attachment factor B1). FUS and SAFB1, abundant in chromatin-bound fraction, interact in a DNA-dependent manner. N-terminal SAP domain of SAFB1, a DNA-binding motif, was required for its localization to chromatin-bound fraction and splicing regulation. In addition, depletion of SAFB1 reduced FUS’s localization to chromatin-bound fraction and splicing activity, suggesting SAFB1 could tether FUS to chromatin compartment thorough N-terminal DNA-binding motif. FUS and SAFB1 also interact with Androgen Receptor (AR) regulating ligand-dependent transcription. Moreover, FUS interacts with another nuclear matrix-associated protein Matrin3, which is muted in a subset of familial ALS cases and reportedly interacts with TDP-43. Interestingly, ectopic ALS-linked FUS mutant sequestered endogenous Matrin3 and SAFB1 in the cytoplasmic aggregates. These findings indicate SAFB1 could be a FUS’s functional platform in chromatin compartment to regulate RNA splicing and ligand-dependent transcription and shed light on the etiological significance of nuclear matrix-associated proteins in ALS pathogenesis. PMID:27731383

  1. Transcriptional suppression of CTP:phosphoethanolamine cytidylyltransferase by 25-hydroxycholesterol is mediated by nuclear factor-Y and Yin Yang 1.

    PubMed

    Ando, Hiromi; Aoyama, Chieko; Horibata, Yasuhiro; Satou, Motoyasu; Mitsuhashi, Satomi; Itoh, Masahiko; Hosaka, Kohei; Sugimoto, Hiroyuki

    2015-11-01

    Pcyt2 (CTP:phosphoethanolamine cytidylyltransferase) is the rate-limiting enzyme in mammalian PE (phosphatidylethanolamine) biosynthesis. Previously, we reported that Pcyt2 mRNA levels increased in several types of cells after serum starvation, an effect that could be suppressed by supplementation with low-density lipoprotein or 25-HC (25-hydroxycholesterol). Transcription of Hmgcr, which encodes 3-hydroxy-3-methylglutaryl-CoA reductase, is also suppressed by 25-HC in the same dose-dependent manner. Nevertheless, a sterol-regulatory element was not detected in the Pcyt2 promoter region. The important element for transcriptional control of Pcyt2 by 25-HC (1.25 μM) was determined to reside between -56 and -36 on the basis of analysis with several Pcyt2 promoter deletion-luciferase reporters in NIH 3T3 cells. Using the yeast one-hybrid system, we found that NF-Y (nuclear factor-Y) binds at C(-37)CAAT(-41) and YY1 (Yin Yang1) binds at C(-42)AT(-40) in the Pcyt2 promoter. Endogenous NF-Y and YY1 bind clearly and competitively to these sites and are important for basal Pcyt2 transcription. Moreover, NF-Y binds to the Hmgcr promoter at C(-14)CA(-12) in gel-shift analysis, and suppression of the basal luciferase activity of the Hmgcr promoter-reporter construct (-30/+61) by 25-HC was abolished when C(-14)CA(-12) was mutated. Furthermore, transcriptional suppression of Pcyt2 by 25-HC was reduced following knockdown targeting of NF-YA or YY1. ChIP analysis revealed that 25-HC inhibited the interaction between NF-Y and RNA polymerase II on the Pcyt2 and Hmgcr promoters. On the basis of these results, we conclude that NF-Y and YY1 are important for the basal transcription of Pcyt2 and that NF-Y is involved in the inhibitory effects of 25-HC on Pcyt2 transcription.

  2. Transcriptional Regulation of the Sodium-activated Potassium Channel SLICK (KCNT2) Promoter by Nuclear Factor-κB*

    PubMed Central

    Tomasello, Danielle L.; Gancarz-Kausch, Amy M.; Dietz, David M.; Bhattacharjee, Arin

    2015-01-01

    Although recent studies have shown the sodium-activated potassium channel SLACK (KCNT1) can contribute to neuronal excitability, there remains little information on the physiological role of the closely related SLICK (KCNT2) channel. Activation of SLICK channels may be important during pathological states such as ischemia, in which an increase in intracellular sodium and chloride can perturb membrane potential and ion homeostasis. We have identified two NFκB-binding sites within the promoter region of the human SLICK (KCNT2) and orthologous rat Slick (Kcnt2) genes, suggesting that conditions in which NFκB transcriptional activity is elevated promote expression of this channel. NFκB binding to the rat Slick promoter was confirmed in vivo by ChIP analyses, and NFκB was found differentially bound to the two sites. We verified NFκB transcriptional regulation of SLICK/Slick by mutational analyses and studying gene expression by luciferase assay in P19 cells, where NFκB is constitutively active. For the rat gene, activation of the Slick promoter was found to be additive in single NFκB mutations and synergistic in double mutations. Unexpectedly, for the human gene, NFκB exhibited cooperativity in activating the SLICK promoter. The human SLICK promoter constructs were then tested under hypoxic conditions in PC-12 cells, where NFκB is not active. Only under hypoxic conditions could luciferase activity be detected; the double NFκB mutant construct failed to exhibit activity. Transcriptional regulation of Slick by NFκB was verified in primary neurons. The Slick transcript decreased 24 h after NFκB inhibition. Our data show SLICK expression is predominantly under the control of NFκB. Because neuronal NFκB activation occurs during stressful stimuli such as hypoxia and injury, our findings suggest that SLICK is a neuroprotective gene. PMID:26100633

  3. Transcriptional Regulation of the Sodium-activated Potassium Channel SLICK (KCNT2) Promoter by Nuclear Factor-κB.

    PubMed

    Tomasello, Danielle L; Gancarz-Kausch, Amy M; Dietz, David M; Bhattacharjee, Arin

    2015-07-24

    Although recent studies have shown the sodium-activated potassium channel SLACK (KCNT1) can contribute to neuronal excitability, there remains little information on the physiological role of the closely related SLICK (KCNT2) channel. Activation of SLICK channels may be important during pathological states such as ischemia, in which an increase in intracellular sodium and chloride can perturb membrane potential and ion homeostasis. We have identified two NFκB-binding sites within the promoter region of the human SLICK (KCNT2) and orthologous rat Slick (Kcnt2) genes, suggesting that conditions in which NFκB transcriptional activity is elevated promote expression of this channel. NFκB binding to the rat Slick promoter was confirmed in vivo by ChIP analyses, and NFκB was found differentially bound to the two sites. We verified NFκB transcriptional regulation of SLICK/Slick by mutational analyses and studying gene expression by luciferase assay in P19 cells, where NFκB is constitutively active. For the rat gene, activation of the Slick promoter was found to be additive in single NFκB mutations and synergistic in double mutations. Unexpectedly, for the human gene, NFκB exhibited cooperativity in activating the SLICK promoter. The human SLICK promoter constructs were then tested under hypoxic conditions in PC-12 cells, where NFκB is not active. Only under hypoxic conditions could luciferase activity be detected; the double NFκB mutant construct failed to exhibit activity. Transcriptional regulation of Slick by NFκB was verified in primary neurons. The Slick transcript decreased 24 h after NFκB inhibition. Our data show SLICK expression is predominantly under the control of NFκB. Because neuronal NFκB activation occurs during stressful stimuli such as hypoxia and injury, our findings suggest that SLICK is a neuroprotective gene.

  4. Control of V(D)J Recombination through Transcriptional Elongation and Changes in Locus Chromatin Structure and Nuclear Organization.

    PubMed

    Del Blanco, Beatriz; García, Vanina; García-Mariscal, Alberto; Hernández-Munain, Cristina

    2011-01-01

    V(D)J recombination is the assembly of gene segments at the antigen receptor loci to generate antigen receptor diversity in T and B lymphocytes. This process is regulated, according to defined developmental programs, by the action of a single specific recombinase complex formed by the recombination antigen gene (RAG-1/2) proteins that are expressed in immature lymphocytes. V(D)J recombination is strictly controlled by RAG-1/2 accessibility to specific recombination signal sequences in chromatin at several levels: cellular lineage, temporal regulation, gene segment order, and allelic exclusion. DNA cleavage by RAG-1/2 is regulated by the chromatin structure, transcriptional elongation, and three-dimensional architecture and position of the antigen receptor loci in the nucleus. Cis-elements specifically direct transcription and V(D)J recombination at these loci through interactions with transacting factors that form molecular machines that mediate a sequence of structural events. These events open chromatin to activate transcriptional elongation and to permit the access of RAG-1/2 to their recombination signal sequences to drive the juxtaposition of the V, D, and J segments and the recombination reaction itself. This chapter summarizes the advances in this area and the important role of the structure and position of antigen receptor loci within the nucleus to control this process.

  5. Disturbed Cyclical Stretch of Endothelial Cells Promotes Nuclear Expression of the Pro-Atherogenic Transcription Factor NF-κB.

    PubMed

    Pedrigi, Ryan M; Papadimitriou, Konstantinos I; Kondiboyina, Avinash; Sidhu, Sukhjinder; Chau, James; Patel, Miten B; Baeriswyl, Daniel C; Drakakis, Emmanuel M; Krams, Rob

    2017-04-01

    Exposure of endothelial cells to low and multidirectional blood flow is known to promote a pro-atherogenic phenotype. The mechanics of the vessel wall is another important mechano-stimulus within the endothelial cell environment, but no study has examined whether changes in the magnitude and direction of cell stretch can be pro-atherogenic. Herein, we developed a custom cell stretching device to replicate the in vivo stretch environment of the endothelial cell and examined whether low and multidirectional stretch promote nuclear translocation of NF-κB. A fluid-structure interaction model of the device demonstrated a nearly uniform strain within the region of cell attachment and a negligible magnitude of shear stress due to cyclical stretching of the cells in media. Compared to normal cyclical stretch, a low magnitude of cyclical stretch or no stretch caused increased expression of nuclear NF-κB (p = 0.09 and p < 0.001, respectively). Multidirectional stretch also promoted significant nuclear NF-κB expression, comparable to the no stretch condition, which was statistically higher than the low (p < 0.001) and normal (p < 0.001) stretch conditions. This is the first study to show that stretch conditions analogous to atherogenic blood flow profiles can similarly promote a pro-atherogenic endothelial cell phenotype, which supports a role for disturbed vessel wall mechanics as a pathological cell stimulus in the development of advanced atherosclerotic plaques.

  6. Addition and correction: the NF-kappa B-like DNA binding activity observed in Dictyostelium nuclear extracts is due to the GBF transcription factor.

    PubMed

    Traincard, F; Ponte, E; Pun, J; Coukell, B; Veron, M

    2001-10-01

    We have previously reported that a NF-kappa B transduction pathway was likely to be present in the cellular slime mold Dictyostelium discoideum. This conclusion was based on several observations, including the detection of developmentally regulated DNA binding proteins in Dictyostelium nuclear extracts that bound to bona fide kappa B sequences. We have now performed additional experiments which demonstrate that the protein responsible for this NF-kappa B-like DNA binding activity is the Dictyostelium GBF (G box regulatory element binding factor) transcription factor. This result, along with the fact that no sequence with significant similarity to components of the mammalian NF-kappa B pathway can be found in Dictyostelium genome, now almost entirely sequenced, led us to reconsider our previous conclusion on the occurrence of a NF-kappa B signal transduction pathway in Dictyostelium.

  7. Transcription, translation, and cellular localization of three Autographa californica nuclear polyhedrosis virus structural proteins: ODV-E18, ODV-E35, and ODV-EC27.

    PubMed

    Braunagel, S C; He, H; Ramamurthy, P; Summers, M D

    1996-08-01

    This paper identifies two structural proteins of the occluded derived viral envelope of Autographa californica nuclear polyhedrosis virus (AcMNPV): ODV-E18 and ODV-E35. In addition, we identify a protein, ODV-EC27, that is incorporated into the capsid of occluded virus, which is not detected in budded virus. The genes for these proteins reside within the IE0 intron. The intron was sequenced, and five open reading frames (ORF) were identified. ORF 3 (genomic ORF 143) codes for the ODV envelope protein, ODV-E18. ORF 4 (genomic ORF 144) codes for ODV-EC27, and Western blot analyses locate this protein to both the ODV capsid and envelope. Transcripts for both ODV-E18 and ODV-EC27 initiate from conserved TAAG motifs, and transcripts are detected from 16 through 72 hr p.i. Antiserum to ODV-E18 recognizes a band of 18 kDa on Western blots of extracts from infected cells and bands of 18 and 35 kDa on Western blots of proteins from purified ODV envelope. N-terminal amino acid sequencing reveals that both ODV-E18 and ODV-E35 contain the same N-terminus. Antiserum to ODV-EC27 recognizes a protein of 27 kDa on Western blots of extracts from infected cells and bands of 27 and 35 kDa on Western blots of proteins from purified ODV. Using immunogold labeling techniques, ODV-E18 and/or ODV-E35 are detected in viral induced intranuclear microvesicles and are not detected in the plasma membrane, cytoplasmic membranes, or the nuclear envelope. Immunogold labeling using antisera to ODV-EC27 detects this protein on both the ODV envelope and capsid.

  8. Positive Regulation by γ-Aminobutyric Acid B Receptor Subunit-1 of Chondrogenesis through Acceleration of Nuclear Translocation of Activating Transcription Factor-4*

    PubMed Central

    Takahata, Yoshifumi; Hinoi, Eiichi; Takarada, Takeshi; Nakamura, Yukari; Ogawa, Shinya; Yoneda, Yukio

    2012-01-01

    A view that signaling machineries for the neurotransmitter γ-aminobutyric acid (GABA) are functionally expressed by cells outside the central nervous system is now prevailing. In this study, we attempted to demonstrate functional expression of GABAergic signaling molecules by chondrocytes. In cultured murine costal chondrocytes, mRNA was constitutively expressed for metabotropic GABAB receptor subunit-1 (GABABR1), but not for GABABR2. Immunohistochemical analysis revealed the predominant expression of GABABR1 by prehypertrophic to hypertrophic chondrocytes in tibial sections of newborn mice. The GABABR agonist baclofen failed to significantly affect chondrocytic differentiation determined by Alcian blue staining and alkaline phosphatase activity in cultured chondrocytes, whereas newborn mice knocked out of GABABR1 (KO) showed a decreased body size and delayed calcification in hyoid bone and forelimb and hindlimb digits. Delayed calcification was also seen in cultured metatarsals from KO mice with a marked reduction of Indian hedgehog gene (Ihh) expression. Introduction of GABABR1 led to synergistic promotion of the transcriptional activity of activating transcription factor-4 (ATF4) essential for normal chondrogenesis, in addition to facilitating ATF4-dependent Ihh promoter activation. Although immunoreactive ATF4 was negligibly detected in the nucleus of chondrocytes from KO mice, ATF4 expression was again seen in the nucleus and cytoplasm after the retroviral introduction of GABABR1 into cultured chondrocytes from KO mice. In nuclear extracts of KO chondrocytes, a marked decrease was seen in ATF4 DNA binding. These results suggest that GABABR1 positively regulates chondrogenesis through a mechanism relevant to the acceleration of nuclear translocation of ATF4 for Ihh expression in chondrocytes. PMID:22879594

  9. The transcriptional integrator CREB-binding protein mediates positive cross talk between nuclear hormone receptors and the hematopoietic bZip protein p45/NF-E2.

    PubMed Central

    Cheng, X; Reginato, M J; Andrews, N C; Lazar, M A

    1997-01-01

    Thyroid hormone (T3) and retinoic acid (RA) play important roles in erythropoiesis. We found that the hematopoietic cell-specific bZip protein p45/NF-E2 interacts with T3 receptor (TR) and RA receptor (RAR) but not retinoid X receptor. The interaction is between the DNA-binding domain of the nuclear receptor and the leucine zipper region of p45/NF-E2 but is markedly enhanced by cognate ligand. Remarkably, ligand-dependent transactivation by TR and RAR is markedly potentiated by p45/NF-E2. This effect of p45/NF-E2 is prevented by maf-like protein p18, which functions positively as a heterodimer with p45/NF-E2 on DNA. Potentiation of hormone action by p45/NF-E2 requires its activation domain, which interacts strongly with the multifaceted coactivator cyclic AMP response element protein-binding protein (CBP). The region of CBP which interacts with p45/NF-E2 is the same interaction domain that mediates inhibition of hormone-stimulated transcription by AP1 transcription factors. Overexpression of the bZip interaction domain of CBP specifically abolishes the positive cross talk between TR and p45/NF-E2. Thus, positive cross talk between p45/NF-E2 and nuclear hormone receptors requires direct protein-protein interactions between these factors and with CBP, whose integration of positive signals from two transactivation domains provides a novel mechanism for potentiation of hormone action in hematopoietic cells. PMID:9032267

  10. Cocaine induces cell death and activates the transcription nuclear factor kappa-b in pc12 cells

    PubMed Central

    Lepsch, Lucilia B; Munhoz, Carolina D; Kawamoto, Elisa M; Yshii, Lidia M; Lima, Larissa S; Curi-Boaventura, Maria F; Salgado, Thais ML; Curi, Rui; Planeta, Cleopatra S; Scavone, Cristoforo

    2009-01-01

    Cocaine is a worldwide used drug and its abuse is associated with physical, psychiatric and social problems. The mechanism by which cocaine causes neurological damage is very complex and involves several neurotransmitter systems. For example, cocaine increases extracellular levels of dopamine and free radicals, and modulates several transcription factors. NF-κB is a transcription factor that regulates gene expression involved in cellular death. Our aim was to investigate the toxicity and modulation of NF-κB activity by cocaine in PC 12 cells. Treatment with cocaine (1 mM) for 24 hours induced DNA fragmentation, cellular membrane rupture and reduction of mitochondrial activity. A decrease in Bcl-2 protein and mRNA levels, and an increase in caspase 3 activity and cleavage were also observed. In addition, cocaine (after 6 hours treatment) activated the p50/p65 subunit of NF-κB complex and the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, attenuated the NF-κB activation. Inhibition of NF-κB activity by using PDTC and Sodium Salicilate increased cell death caused by cocaine. These results suggest that cocaine induces cell death (apoptosis and necrosis) and activates NF-κB in PC12 cells. This activation occurs, at least partially, due to activation of D1 receptors and seems to have an anti-apoptotic effect on these cells. PMID:19183502

  11. Cocaine induces cell death and activates the transcription nuclear factor kappa-B in PC12 cells.

    PubMed

    Lepsch, Lucilia B; Munhoz, Carolina D; Kawamoto, Elisa M; Yshii, Lidia M; Lima, Larissa S; Curi-Boaventura, Maria F; Salgado, Thais M L; Curi, Rui; Planeta, Cleopatra S; Scavone, Cristoforo

    2009-02-01

    Cocaine is a worldwide used drug and its abuse is associated with physical, psychiatric and social problems. The mechanism by which cocaine causes neurological damage is very complex and involves several neurotransmitter systems. For example, cocaine increases extracellular levels of dopamine and free radicals, and modulates several transcription factors. NF-kappaB is a transcription factor that regulates gene expression involved in cellular death. Our aim was to investigate the toxicity and modulation of NF-kappaB activity by cocaine in PC 12 cells. Treatment with cocaine (1 mM) for 24 hours induced DNA fragmentation, cellular membrane rupture and reduction of mitochondrial activity. A decrease in Bcl-2 protein and mRNA levels, and an increase in caspase 3 activity and cleavage were also observed. In addition, cocaine (after 6 hours treatment) activated the p50/p65 subunit of NF-kappaB complex and the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, attenuated the NF-kappaB activation. Inhibition of NF-kappaB activity by using PDTC and Sodium Salicilate increased cell death caused by cocaine. These results suggest that cocaine induces cell death (apoptosis and necrosis) and activates NF-kappaB in PC12 cells. This activation occurs, at least partially, due to activation of D1 receptors and seems to have an anti-apoptotic effect on these cells.

  12. The Chromatin Regulator DMAP1 Modulates Activity of the Nuclear Factor κB (NF-κB) Transcription Factor Relish in the Drosophila Innate Immune Response*

    PubMed Central

    Goto, Akira; Fukuyama, Hidehiro; Imler, Jean-Luc; Hoffmann, Jules A.

    2014-01-01

    The host defense of the model organism Drosophila is under the control of two major signaling cascades controlling transcription factors of the NF-κB family, the Toll and the immune deficiency (IMD) pathways. The latter shares extensive similarities with the mammalian TNF-R pathway and was initially discovered for its role in anti-Gram-negative bacterial reactions. A previous interactome study from this laboratory reported that an unexpectedly large number of proteins are binding to the canonical components of the IMD pathway. Here, we focus on DNA methyltransferase-associated protein 1 (DMAP1), which this study identified as an interactant of Relish, a Drosophila transcription factor reminiscent of the mammalian p105 NF-κB protein. We show that silencing of DMAP1 expression both in S2 cells and in flies results in a significant reduction of Escherichia coli-induced expression of antimicrobial peptides. Epistatic analysis indicates that DMAP1 acts in parallel or downstream of Relish. Co-immunoprecipitation experiments further reveal that, in addition to Relish, DMAP1 also interacts with Akirin and the Brahma-associated protein 55 kDa (BAP55). Taken together, these results reveal that DMAP1 is a novel nuclear modulator of the IMD pathway, possibly acting at the level of chromatin remodeling. PMID:24947515

  13. In vitro transcription of a Drosophila U1 small nuclear RNA gene requires TATA box-binding protein and two proximal cis-acting elements with stringent spacing requirements.

    PubMed Central

    Zamrod, Z; Tyree, C M; Song, Y; Stumph, W E

    1993-01-01

    Transcription of a Drosophila U1 small nuclear RNA gene was functionally analyzed in cell extracts derived from 0- to 12-h embryos. Two promoter elements essential for efficient initiation of transcription in vitro by RNA polymerase II were identified. The first, termed PSEA, is located between positions -41 and -61 relative to the transcription start site, is crucial for promoter activity, and is the dominant element for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vertebrate small nuclear RNA genes. The second element, termed PSEB, is located at positions -25 to -32 and is required for an efficient level of transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduced transcriptional activity relative to that of the wild-type promoter. Although the PSEB sequence does not have any obvious sequence similarity to a TATA box, conversion of PSEB to the canonical TATA sequence dramatically increased the efficiency of the U1 promoter and simultaneously relieved the requirement for the upstream PSEA. Despite these effects, introduction of the TATA sequence into the U1 promoter had no effect on the choice of start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is required for transcription from the wild-type U1 promoter as well as from the TATA-containing U1 promoter. Images PMID:8355718

  14. Identification of cis-acting sequences responsible for phorbol ester induction of human serum amyloid A gene expression via a nuclear factor kB-like transcription factor

    SciTech Connect

    Edbrooke, M.R.; Cheshire, J.K.; Woo, P.; Burt, B.W.

    1989-05-01

    The authors have analyzed the 5'-flanking region of one of the genes coding for the human acute-phase protein, serum amyloid A (SAA). They found that SAA mRNA could be increased fivefold in transfected cells by treatment with phorbol 12-myristate 13-acetate (PMA). To analyze this observation further, they placed a 265-base-pair 5' SAA fragment upstream of the reporter chloramphenicol acetyltransferase (CAT) gene and transfected this construct into HeLa cells. PMA treatment of these transient transfectants resulted in increased CAT expression. Nuclear proteins from PMA-treated HeLa cells bound to this DNA fragment, and methylation interference analysis showed that the binding was specific to the sequence GGGACTTTCC (between -82 and -91), a sequence previously described by others as the binding site for the nuclear factor NF/kappa/B. In a cotransfection competition experiment, they could abolish PMA-induced CAT activity by using cloned human immunodeficiency virus long-terminal-repeat DNA containing the NF/kappa/B-binding sequence. The same long-terminal-repeat DNA containing mutant NF/kappa/B-binding sequences did not affect CAT expression, which suggested that binding by an NF/kappa/B-like factor is required for increased SAA transcription.

  15. An evolutionary conserved interaction between the Gcm transcription factor and the SF1 nuclear receptor in the female reproductive system.

    PubMed

    Cattenoz, Pierre B; Delaporte, Claude; Bazzi, Wael; Giangrande, Angela

    2016-11-25

    NR5A1 is essential for the development and for the function of steroid producing glands of the reproductive system. Moreover, its misregulation is associated with endometriosis, which is the first cause of infertility in women. Hr39, the Drosophila ortholog of NR5A1, is expressed and required in the secretory cells of the spermatheca, the female exocrine gland that ensures fertility by secreting substances that attract and capacitate the spermatozoids. We here identify a direct regulator of Hr39 in the spermatheca: the Gcm transcription factor. Furthermore, lack of Gcm prevents the production of the secretory cells and leads to female sterility in Drosophila. Hr39 regulation by Gcm seems conserved in mammals and involves the modification of the DNA methylation profile of mNr5a1. This study identifies a new molecular pathway in female reproductive system development and suggests a role for hGCM in the progression of reproductive tract diseases in humans.

  16. Keap1 silencing boosts lipopolysaccharide-induced transcription of interleukin 6 via activation of nuclear factor κB in macrophages

    SciTech Connect

    Lv, Peng; Xue, Peng; Dong, Jian; Peng, Hui; Clewell, Rebecca; Wang, Aiping; Wang, Yue; Peng, Shuangqing; Qu, Weidong; Zhang, Qiang; Andersen, Melvin E.; Pi, Jingbo

    2013-11-01

    Interleukin-6 (IL6) is a multifunctional cytokine that regulates immune and inflammatory responses. Multiple transcription factors, including nuclear factor κB (NF-κB) and nuclear factor E2-related factor 2 (Nrf2), regulate IL6 transcription. Kelch-like ECH-associated protein 1 (Keap1) is a substrate adaptor protein for the Cullin 3-dependent E3 ubiquitin ligase complex, which regulates the degradation of many proteins, including Nrf2 and IκB kinase β (IKKβ). Here, we found that stable knockdown of Keap1 (Keap1-KD) in RAW 264.7 (RAW) mouse macrophages and human monocyte THP-1 cells significantly increased expression of Il6, and Nrf2-target genes, under basal and lipopolysaccharide (LPS, 0.001–0.1 μg/ml)-challenged conditions. However, Nrf2 activation alone, by tert-butylhydroquinone treatment of RAW cells, did not increase expression of Il6. Compared to cells transduced with scrambled non-target negative control shRNA, Keap1-KD RAW cells showed enhanced protein levels of IKKβ and increased expression and phosphorylation of NF-κB p65 under non-stressed and LPS-treated conditions. Because the expression of Il6 in Keap1-KD RAW cells was significantly attenuated by silencing of Ikkβ, but not Nrf2, it appears that stabilized IKKβ is responsible for the enhanced transactivation of Il6 in Keap1-KD cells. This study demonstrated that silencing of Keap1 in macrophages boosts LPS-induced transcription of Il6 via NF-κB activation. Given the importance of IL6 in the inflammatory response, the Keap1–IKKβ–NF-κB pathway may be a novel target for treatment and prevention of inflammation and associated disorders. - Highlights: • Knockdown of Keap1 increases expression of Il6 in macrophages. • Silencing of Keap1 results in protein accumulation of IKKβ and NF-κB p65. • Induction of Il6 resulting from Keap1 silencing is attributed to NF-κB activation.

  17. Epigallocatechin-3-gallate prevents lipid peroxidation and enhances antioxidant defense system via modulating hepatic nuclear transcription factors in heat-stressed quails.

    PubMed

    Sahin, K; Orhan, C; Tuzcu, M; Ali, S; Sahin, N; Hayirli, A

    2010-10-01

    Epigallocatechin-3-gallate (EGCG), a polyphenol derived from green tea, exerts antioxidant effects. Oxidative stress is one of the consequences of heat stress (HS), which also depresses performance in poultry. This experiment was conducted to elucidate the action mode of EGCG in alleviation of oxidative stress in heat-stressed quail (Coturnix coturnix japonica). A total of 180 five-week-old female Japanese quails were reared either at 22°C for 24 h/d (thermoneutral, TN) or 34°C for 8 h/d (HS) for 12 wk. Birds in both environments were randomly fed 1 of 3 diets: basal diet and basal diet added with 200 or 400 mg of EGCG/kg of diet. Each of the 2×3 factorially arranged groups was replicated in 10 cages, each containing 3 quails. Performance variables [feed intake (FI) and egg production (EP)], oxidative stress biomarkers [malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px)] and hepatic transcription factors [nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2)] were analyzed using 2-way ANOVA. Exposure to HS caused reductions in FI by 9.7% and EP by 14.4%, increased hepatic MDA level by 84.8%, and decreased hepatic SOD, CAT, and GSH-Px activities by 25.8, 52.3, and 45.5%, respectively (P<0.0001 for all). The hepatic NF-κB expression was greater (156 vs. 82%) and Nrf2 expression was lower (84 vs. 118%) for quails reared under the HS environment than for those reared under the TN environment (P<0.0001 for both). In response to increasing supplemental EGCG level, there were linear increases in FI from 29.6 to 30.9 g/d and EP from 84.3 to 90.1%/d, linear decreases in hepatic MDA level from 2.82 to 1.72 nmol/g and Nrf2 expression from 77.5 to 123.3%, and linear increases in hepatic SOD (146.4 to 182.2), CAT (36.2 to 47.1), and GSH-Px (13.5 to 18.5) activities (U/mg of protein) and NF-κB expression (149.7 to 87.3%) (P<0.0001 for all). Two

  18. Food Components Modulate Obesity and Energy Metabolism via the Transcriptional Regulation of Lipid-Sensing Nuclear Receptors.

    PubMed

    Goto, Tsuyoshi; Takahashi, Nobuyuki; Kawada, Teruo

    2015-01-01

    Obesity is a major risk factor for chronic diseases such as diabetes, cardiovascular diseases, and hypertension. Many modern people have a tendency to overeat owing to stress and loosening of self-control. Moreover, energy expenditure varies greatly among individuals. Scientific reduction of obesity is important under these circumstances. Furthermore, recent research on molecular levels has clarified the differentiation of adipocytes, the level of subsequent fat accumulation, and the secretion of the biologically active adipokines by adipocytes. Adipose tissues and obesity have become the most important target for the prevention and treatment of many chronic diseases. We have identified various food-derived compounds modulating nuclear receptors, especially peroxisome proliferators-activated receptor(PPAR), in the regulation of energy metabolism and obesity. In this review, we discuss the PPARs that are most important in obesity and energy metabolism.

  19. Identification of the Flavonoid Luteolin as a Repressor of the Transcription Factor Hepatocyte Nuclear Factor 4α*

    PubMed Central

    Li, Juan; Inoue, Jun; Choi, Jung-Min; Nakamura, Shugo; Yan, Zhen; Fushinobu, Shinya; Kamada, Haruhiko; Kato, Hisanori; Hashidume, Tsutomu; Shimizu, Makoto; Sato, Ryuichiro

    2015-01-01

    Hepatocyte nuclear factor 4α (HNF4α) is a nuclear receptor that regulates the expression of genes involved in the secretion of apolipoprotein B (apoB)-containing lipoproteins and in glucose metabolism. In the present study, we identified a naturally occurring flavonoid, luteolin, as a repressor of HNF4α by screening for effectors of the human microsomal triglyceride transfer protein (MTP) promoter. Luciferase reporter gene assays revealed that the activity of the MTP gene promoter was suppressed by luteolin and that the mutation of HNF4α-binding element abolished luteolin responsiveness. Luteolin treatment caused a significant decrease in the mRNA levels of HNF4α target genes in HepG2 cells and inhibited apoB-containing lipoprotein secretion in HepG2 and differentiated Caco2 cells. The interaction between luteolin and HNF4α was demonstrated using absorption spectrum analysis and luteolin-immobilized beads. Luteolin did not affect the DNA binding of HNF4α to the promoter region of its target genes but suppressed the acetylation level of histone H3 in the promoter region of certain HNF4α target genes. Short term treatment of mice with luteolin significantly suppressed the expression of HNF4α target genes in the liver. In addition, long term treatment of mice with luteolin significantly suppressed their diet-induced obesity and improved their serum glucose and lipid parameters. Importantly, long term luteolin treatment lowered serum VLDL and LDL cholesterol and serum apoB protein levels, which was not accompanied by fat accumulation in the liver. These results suggest that the flavonoid luteolin ameliorates an atherogenic lipid profile in vivo that is likely to be mediated through the inactivation of HNF4α. PMID:26272613

  20. A quasi-lentiviral green fluorescent protein reporter exhibits nuclear export features of late human immunodeficiency virus type 1 transcripts

    SciTech Connect

    Graf, Marcus; Ludwig, Christine; Kehlenbeck, Sylvia; Jungert, Kerstin; Wagner, Ralf . E-mail: ralf.wagner@klinik.uni-regensburg.de

    2006-09-01

    We have previously shown that Rev-dependent expression of HIV-1 Gag from CMV immediate early promoter critically depends on the AU-rich codon bias of the gag gene. Here, we demonstrate that adaptation of the green fluorescent protein (GFP) reporter gene to HIV codon bias is sufficient to turn this hivGFP RNA into a quasi-lentiviral message following the rules of late lentiviral gene expression. Accordingly, GFP expression was significantly decreased in transfected cells strictly correlating with reduced RNA levels. In the presence of the HIV 5' major splice donor, the hivGFP RNAs were stabilized in the nucleus and efficiently exported to the cytoplasm following fusion of the 3' Rev-responsive element (RRE) and coexpression of HIV-1 Rev. This Rev-dependent translocation was specifically inhibited by leptomycin B suggesting export via the CRM1-dependent pathway used by late lentiviral transcripts. In conclusion, this quasi-lentiviral reporter system may provide a new platform for developing sensitive Rev screening assays.

  1. An evolutionary conserved interaction between the Gcm transcription factor and the SF1 nuclear receptor in the female reproductive system

    PubMed Central

    Cattenoz, Pierre B.; Delaporte, Claude; Bazzi, Wael; Giangrande, Angela

    2016-01-01

    NR5A1 is essential for the development and for the function of steroid producing glands of the reproductive system. Moreover, its misregulation is associated with endometriosis, which is the first cause of infertility in women. Hr39, the Drosophila ortholog of NR5A1, is expressed and required in the secretory cells of the spermatheca, the female exocrine gland that ensures fertility by secreting substances that attract and capacitate the spermatozoids. We here identify a direct regulator of Hr39 in the spermatheca: the Gcm transcription factor. Furthermore, lack of Gcm prevents the production of the secretory cells and leads to female sterility in Drosophila. Hr39 regulation by Gcm seems conserved in mammals and involves the modification of the DNA methylation profile of mNr5a1. This study identifies a new molecular pathway in female reproductive system development and suggests a role for hGCM in the progression of reproductive tract diseases in humans. PMID:27886257

  2. Epstein-Barr virus nuclear antigen 3C targets p53 and modulates its transcriptional and apoptotic activities

    SciTech Connect

    Yi Fuming; Saha, Abhik; Murakami, Masanao; Kumar, Pankaj; Knight, Jason S.; Cai Qiliang; Choudhuri, Tathagata; Robertson, Erle S.

    2009-06-05

    The p53 tumor suppressor gene is one of the most commonly mutated genes in human cancers and the corresponding encoded protein induces apoptosis or cell-cycle arrest at the G1/S checkpoint in response to DNA damage. To date, previous studies have shown that antigens encoded by human tumor viruses such as SV40 large T antigen, adenovirus E1A and HPV E6 interact with p53 and disrupt its functional activity. In a similar fashion, we now show that EBNA3C, one of the EBV latent antigens essential for the B-cell immortalization in vitro, interacts directly with p53. Additionally, we mapped the interaction of EBNA3C with p53 to the C-terminal DNA-binding and the tetramerization domain of p53, and the region of EBNA3C responsible for binding to p53 was mapped to the N-terminal domain of EBNA3C (residues 130-190), previously shown to interact with a number of important cell-cycle components, specifically SCF{sup Skp2}, cyclin A, and cMyc. Furthermore, we demonstrate that EBNA3C substantially represses the transcriptional activity of p53 in luciferase based reporter assays, and rescues apoptosis induced by ectopic p53 expression in SAOS-2 (p53{sup -/-}) cells. Interestingly, we also show that the DNA-binding ability of p53 is diminished in the presence of EBNA3C. Thus, the interaction between the p53 and EBNA3C provides new insights into the mechanism(s) by which the EBNA3C oncoprotein can alter cellular gene expression in EBV associated human cancers.

  3. Concerted effects of heterogeneous nuclear ribonucleoprotein C1/C2 to control vitamin D-directed gene transcription and RNA splicing in human bone cells

    PubMed Central

    Zhou, Rui; Park, Juw Won; Chun, Rene F.; Lisse, Thomas S.; Garcia, Alejandro J.; Zavala, Kathryn; Sea, Jessica L.; Lu, Zhi-xiang; Xu, Jianzhong; Adams, John S.; Xing, Yi; Hewison, Martin

    2017-01-01

    Traditionally recognized as an RNA splicing regulator, heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC1/C2) can also bind to double-stranded DNA and function in trans as a vitamin D response element (VDRE)-binding protein. As such, hnRNPC1/C2 may couple transcription induced by the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D) with subsequent RNA splicing. In MG63 osteoblastic cells, increased expression of the 1,25(OH)2D target gene CYP24A1 involved immunoprecipitation of hnRNPC1/C2 with CYP24A1 chromatin and RNA. Knockdown of hnRNPC1/C2 suppressed expression of CYP24A1, but also increased expression of an exon 10-skipped CYP24A1 splice variant; in a minigene model the latter was attenuated by a functional VDRE in the CYP24A1 promoter. In genome-wide analyses, knockdown of hnRNPC1/C2 resulted in 3500 differentially expressed genes and 2232 differentially spliced genes, with significant commonality between groups. 1,25(OH)2D induced 324 differentially expressed genes, with 187 also observed following hnRNPC1/C2 knockdown, and a further 168 unique to hnRNPC1/C2 knockdown. However, 1,25(OH)2D induced only 10 differentially spliced genes, with no overlap with differentially expressed genes. These data indicate that hnRNPC1/C2 binds to both DNA and RNA and influences both gene expression and RNA splicing, but these actions do not appear to be linked through 1,25(OH)2D-mediated induction of transcription. PMID:27672039

  4. Production of Viral mRNA in Adenovirus-Transformed Cells by the Post-Transcriptional Processing of Heterogeneous Nuclear RNA Containing Viral and Cell Sequences

    PubMed Central

    Wall, R.; Weber, J.; Gage, Z.; Darnell, J. E.

    1973-01-01

    Adenovirus 2-transformed cells contain virus-specific sequences which are covalently linked to cell-specific RNA sequences in heterogeneous nuclear RNA (HnRNA) molecules larger than 45S. Virus sequences are identified by hybridization to viral DNA, and the cell sequences are detected by hybridization to cellular DNA under conditions where hybridization only occurs to reiterated sites in cell DNA. Such large composite viral-cell HnRNA molecules presumably arise through the uninterrupted transcription of host sequences and integrated viral DNA. Adenovirus-specific polysomal RNA from these cells sediments as three discrete species at 16, 20, and 26S. These specific classes of viral mRNA do not contain rapidly hybridizing host-specific RNA sequences. Both virus-specific HnRNA and mRNA contain polyadenylic acid sequences since they bind to polyU columns at levels characteristics of other polyA-terminated HnRNA and mRNA. Thus, the discrete species of virus-specific mRNA in adenovirus 2 transformed cells appear to be derived from high-molecular-weight virus-specific HnRNA through a series of post-transcriptional modifications involving polyA addition. Subsequently the HnRNA is cleaved so that the cell-specific RNA sequences that originate from the reiterated sites in cell DNA do not accompany the adenovirus mRNA to the cytoplasm. These events for the adenovirus-specific mRNA appear, therefore, to be similar to the stages in the biogenesis of the majority of mRNA in eukaryotic cells. PMID:4736534

  5. Transcriptional Corepressor SMILE Recruits SIRT1 to Inhibit Nuclear Receptor Estrogen Receptor-related Receptor γ Transactivation*

    PubMed Central

    Xie, Yuan-Bin; Park, Jeong-Hoh; Kim, Don-Kyu; Hwang, Jung Hwan; Oh, Sangmi; Park, Seung Bum; Shong, Minho; Lee, In-Kyu; Choi, Hueng-Sik

    2009-01-01

    SMILE (small heterodimer partner interacting leucine zipper protein) has been identified as a corepressor of the glucocorticoid receptor, constitutive androstane receptor, and hepatocyte nuclear factor 4α. Here we show that SMILE also represses estrogen receptor-related receptor γ (ERRγ) transactivation. Knockdown of SMILE gene expression increases ERRγ activity. SMILE directly interacts with ERRγ in vitro and in vivo. Domain mapping analysis showed that SMILE binds to the AF2 domain of ERRγ. SMILE represses ERRγ transactivation partially through competition with coactivators PGC-1α, PGC-1β, and GRIP1. Interestingly, the repression of SMILE on ERRγ is released by SIRT1 inhibitors, a catalytically inactive SIRT1 mutant, and SIRT1 small interfering RNA but not by histone protein deacetylase inhibitor. In vivo glutathione S-transferase pulldown and coimmunoprecipitation assays validated that SMILE physically interacts with SIRT1. Furthermore, the ERRγ inverse agonist GSK5182 enhances the interaction of SMILE with ERRγ and SMILE-mediated repression. Knockdown of SMILE or SIRT1 blocks the repressive effect of GSK5182. Moreover, chromatin immunoprecipitation assays revealed that GSK5182 augments the association of SMILE and SIRT1 on the promoter of the ERRγ target PDK4. GSK5182 and adenoviral overexpression of SMILE cooperate to repress ERRγ-induced PDK4 gene expression, and this repression is released by overexpression of a catalytically defective SIRT1 mutant. Finally, we demonstrated that ERRγ regulates SMILE gene expression, which in turn inhibits ERRγ. Overall, these findings implicate SMILE as a novel corepressor of ERRγ and recruitment of SIRT1 as a novel repressive mechanism for SMILE and ERRγ inverse agonist. PMID:19690166

  6. Berberis vulgaris root extract alleviates the adverse effects of heat stress via modulating hepatic nuclear transcription factors in quails.

    PubMed

    Sahin, Kazim; Orhan, Cemal; Tuzcu, Mehmet; Borawska, Maria H; Jabłonski, Jakub; Guler, Osman; Sahin, Nurhan; Hayirli, Armagan

    2013-08-01

    To evaluate the action mode of Berberis vulgaris root extract in the alleviation of oxidative stress, female Japanese quails (n 180, aged 5 weeks) were reared, either at 22°C for 24 h/d (thermoneutral, TN) or 34°C for 8 h/d (heat stress, HS), and fed one of three diets: diets containing 0, 100 or 200 mg of B. vulgaris root extract per kg for 12 weeks. Exposure to HS depressed feed intake by 8·5% and egg production by 12·1%, increased hepatic malondialdehyde (MDA) level by 98·0% and decreased hepatic superoxide dismutase, catalase and glutathione peroxidase activities by 23·5, 35·4 and 55·7%, respectively (P<0·001 for all). There were also aggravations in expressions of hepatic NF-κB and heat-shock protein 70 (HSP70) by 42 and 43%, respectively and suppressions in expressions of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and haeme-oxygenase 1 (HO-1) by 57 and 61%, respectively, in heat-stressed quails (P<0·001 for all). As supplemental B. vulgaris extract increased, there were linear increases in performance parameters, activities of antioxidant enzymes and hepatic Nrf2 and HO-1 expressions (P<0·001 for all) and linear decreases in hepatic MDA level and NF-κB and HSP70 expressions at a greater extent in quails reared under TN condition and those reared under HS condition. In conclusion, dietary supplementation of B. vulgaris root extract to quails reduces the detrimental effects of oxidative stress and lipid peroxidation resulting from HS via activating the host defence system at the cellular level.

  7. Green tea component EGCG, insulin and IGF-1 promote nuclear efflux of atrophy-associated transcription factor Foxo1 in skeletal muscle fibers.

    PubMed

    Wimmer, Robert J; Russell, Sarah J; Schneider, Martin F

    2015-12-01

    Prevention and slowing of skeletal muscle atrophy with nutritional approaches offers the potential to provide far-reaching improvements in the quality of life for our increasingly aging population. Here we show that polyphenol flavonoid epigallocatechin 3-gallate (EGCG), found in the popular beverage green tea (Camellia sinensis), demonstrates similar effects to the endogenous hormones insulin-like growth factor 1 (IGF-1) and insulin in the ability to suppress action of the atrophy-promoting transcription factor Foxo1 through a net translocation of Foxo1 out of the nucleus as monitored by nucleo-cytoplasmic movement of Foxo1-green fluorescent protein (GFP) in live skeletal muscle fibers. Foxo1-GFP nuclear efflux is rapid in IGF-1 or insulin, but delayed by an additional 30 min for EGCG. Once activated, kinetic analysis with a simple mathematical model shows EGCG, IGF-1 and insulin all produce similar apparent rate constants for Foxo1-GFP unidirectional nuclear influx and efflux. Interestingly, EGCG appears to have its effect at least partially via parallel signaling pathways that are independent of IGF-1's (and insulin's) downstream PI3K/Akt/Foxo1 signaling axis. Using the live fiber model system, we also determine the dose-response curve for both IGF-1 and insulin on Foxo1 nucleo-cytoplasmic distribution. The continued understanding of the activation mechanisms of EGCG could allow for nutritional promotion of green tea's antiatrophy skeletal muscle benefits and have implications in the development of a clinically significant parallel pathway for new drugs to target muscle wasting and the reduced insulin receptor sensitivity which causes type II diabetes mellitus.

  8. Kinome-Wide Functional Genomics Screen Reveals a Novel Mechanism of TNFα-Induced Nuclear Accumulation of the HIF-1α Transcription Factor in Cancer Cells

    PubMed Central

    Schoolmeesters, Angela; Brown, Daniel D.; Fedorov, Yuriy

    2012-01-01

    Hypoxia-inducible factor-1 (HIF-1) and its most important subunit, HIF-1α, plays a central role in tumor progression by regulating genes involved in cancer cell survival, proliferation and metastasis. HIF-1α activity is associated with nuclear accumulation of the transcription factor and regulated by several mechanisms including modulation of protein stability and degradation. Among recent advances are the discoveries that inflammation-induced cytokines and growth factors affect protein accumulation of HIF-1α under normoxia conditions. TNFα, a major pro-inflammatory cytokine that promotes tumorigenesis is known as a stimulator of HIF-1α activity. To improve our understanding of TNFα-mediated regulation of HIF-1α nuclear accumulation we screened a kinase-specific siRNA library using a cell imaging–based HIF-1α-eGFP chimera reporter assay. Interestingly, this systematic analysis determined that depletion of kinases involved in conventional TNFα signaling (IKK/NFκB and JNK pathways) has no detrimental effect on HIF-1α accumulation. On the other hand, depletion of PRKAR2B, ADCK2, TRPM7, and TRIB2 significantly decreases the effect of TNFα on HIF-1α stability in osteosarcoma and prostate cancer cell lines. These newly discovered regulators conveyed their activity through a non-conventional RELB-depended NFκB signaling pathway and regulation of superoxide activity. Taken together our data allow us to conclude that TNFα uses a distinct and complex signaling mechanism to induce accumulation of HIF-1α in cancer cells. In summary, our results illuminate a novel mechanism through which cancer initiation and progression may be promoted by inflammatory cytokines, highlighting new potential avenues for fighting this disease. PMID:22355351

  9. Differential ligand-dependent interactions between the AF-2 activating domain of nuclear receptors and the putative transcriptional intermediary factors mSUG1 and TIF1.

    PubMed Central

    vom Baur, E; Zechel, C; Heery, D; Heine, M J; Garnier, J M; Vivat, V; Le Douarin, B; Gronemeyer, H; Chambon, P; Losson, R

    1996-01-01

    Using a yeast two-hybrid system we report the isolation of a novel mouse protein, mSUG1, that interacts with retinoic acid receptor alpha (RAR alpha) both in yeast cells and in vitro in a ligand- and AF-2 activating domain (AF-2 AD)-dependent manner and show that it is a structural and functional homologue of the essential yeast protein SUG1. mSUG1 also efficiently interacts with other nuclear receptors, including oestrogen (ER), thyroid hormone (TR), Vitamin D3 (VDR) and retinoid X (RXR) receptors. By comparing the interaction properties of these receptors with mSUG1 and TIF1, we demonstrate that: (i) RXR alpha efficiently interacts with TIF1, but not with mSUG1, whereas TR alpha interacts much more efficiently with mSUG1 than with TIF1, and RAR alpha, VDR and ER efficiently interact with mSUG1 and TIF1; (ii) the amphipathic alpha-helix core of the AF-2 AD is differentially involved in interactions of RAR alpha with mSUG1 and TIF1; (iii) the AF-2 AD cores of RAR alpha and ER are similarly involved in their interaction with TIF1, but not with mSUG1. Thus, the interaction interfaces between the different receptors and either mSUG1 or TIF1 may vary depending on the nature of the receptor and the putative mediator of its AF-2 function. We discuss the possibility that mSUG1 and TIF1 may mediate the transcriptional activity of the AF-2 of nuclear receptors through different mechanisms. Images PMID:8598193

  10. Calcium-Dependent Protein Kinase in Ginger Binds with Importin-α through Its Junction Domain for Nuclear Localization, and Further Interacts with NAC Transcription Factor

    PubMed Central

    Vivek, Padmanabhan Jayanthi; Resmi, Mohankumar Saraladevi; Sreekumar, Sweda; Sivakumar, K. C.; Tuteja, Narendra; Soniya, Eppurathu Vasudevan

    2017-01-01

    Calcium-dependent protein kinases (CDPKs) are important sensors of Ca2+ elevations in plant cells regulating the gene expression linked with various cellular processes like stress response, growth and development, metabolism, and cytoskeleton dynamics. Ginger is an extensively used spice due to its unique flavor and immense medicinal value. The two major threats that interfere with the large scale production of ginger are the salinity and drought stress. ZoCDPK1 (Zingiber officinale Calcium-dependent protein kinase 1) is a salinity and drought-inducible CDPK gene isolated from ginger and undergoes dynamic subcellular localization during stress conditions. ZoCDPK1, with signature features of a typical Ca2+ regulated kinase, also possesses a bipartite nuclear localization sequence (NLS) in its junction domain (JD). A striking feature in ZoCDPK1 is the rare occurrence of a coupling between the NLS in JD and consensus sequences in regulatory domain. Here, we further identified its nature of nuclear localization and its interaction partners. In the homology model generated for ZoCDPK1, the regulatory domain mimics the crystal structure of the regulatory domain in Arabidopsis CDPK1. Molecular docking simulation of importin (ZoIMPα), an important protein involved in nuclear translocation, into the NLS of ZoCDPK1 was well-visualized. Furthermore, the direct interaction of ZoCDPK1 and ZoIMPα proteins was studied by the yeast 2-hybrid (Y2H) system, which confirmed that junction domain (JD) is an important interaction module required for ZoCDPK1 and ZoIMPα binding. The probable interacting partners of ZoCDPK1 were also identified using Y2H experiment. Of the 10 different stress-related interacting partners identified for ZoCDPK1, NAC transcription factor (TF) needs special mention, especially in the context of ZoCDPK1 function. The interaction between ZoCDPK1 and NAC TF, in fact, corroborate with the results of gene expression and over-expression studies of ZoCDPK1. Hence

  11. Nuclear Factor of κB1 Is a Key Regulator for the Transcriptional Activation of Milk Synthesis in Bovine Mammary Epithelial Cells.

    PubMed

    Huang, Xin; Zang, Yanli; Zhang, Minghui; Yuan, Xiaohan; Li, Meng; Gao, Xuejun

    2017-02-03

    The nuclear factor of κB (NFκB) family has been well known for its significant role in regulating the expression of numerous genes that control many biological processes. However, it is unclear whether NFκB could regulate milk synthesis. In this study, we identified NFκB1 as a critical regulator for milk synthesis in bovine mammary epithelial cells (BMECs). Gene function study revealed that NFκB1 modulates the expression of mammalian target of rapamycin (mTOR), sterol response element-binding protein (SREBP)-1c, and β4Gal-T2 for milk synthesis. Furthermore, chromatin immunoprecipitation assays showed that both methionine (Met) and estrogen (E) triggered NFκB1 to bind to gene promoters of mTOR, SREBP-1c, and β4Gal-T2 in BMECs. In addition, we confirmed that Met and E triggered NFκB1 expression and phosphorylation via phosphatidylinositol-3-kinase (PI3K) but not mTOR signaling pathway. Taken together, our study reveals that NFκB1 acts as a PI3K but not mTOR-dependent critical mediator for the transcriptional activation of signaling molecules regulating milk synthesis in BMECs.

  12. Celastrol regulates multiple nuclear transcription factors belonging to HSP90's clients in a dose- and cell type-dependent way.

    PubMed

    Zhang, Denghai; Xu, Limin; Cao, Fanfan; Wei, Tingxuan; Yang, Chunxin; Uzan, Georges; Peng, Bin

    2010-11-01

    Celastrol, a novel HSP90 inhibitor, has recently attracted much attention due to its potential in multiple applications, such as anti-inflammation use, degenerative neuron disease relief, and tumor management. At present, the studies in celastrol's effects on HSP90's clients have focused on the kinase sub-population, while another key sub-population, nuclear transcription factors (TFs), is not being well-explored. In this study, we observe the effects of celastrol on 18 TFs (belonging to HSP90 clients) in three human cell lines: MCF-7 (breast cancer), HepG2 (hepatoma), and THP-1 (monocytic leukemia). The results show that at least half of the detectable TFs were affected by celastrol, though the effect patterns varied with cell type and dosage. Bi-directional regulations of some TFs were identified, a phenomenon not yet seen with other HSP90 inhibitors. Celastrol's capability to affect multiple TFs was consistent with its altering HSP90/TFs interactions and disrupting HSP90/Hop interaction, in addition to the reported damaging HSP90/Cdc37 interaction. This work confirms, for the first time, that celastrol has broad effects on TFs belonging to HSP90's clients, casts new light on understanding these reported actions, and suggests new possible applications for celastrol, such as diabetes management.

  13. Effect of fluoride on calcium ion concentration and expression of nuclear transcription factor kappa-B ρ65 in rat hippocampus.

    PubMed

    Zhang, Jing; Zhu, Wen-Jing; Xu, Xiao-Hong; Zhang, Zi-Gui

    2011-07-01

    The study investigated the neurotoxicity of drinking water fluorosis in rat hippocampus. Just weaning male Sprague-Dawley (SD) rats were given 15, 30, 60 mg/L NaF solution and tap water for 9 months. The calcium ion concentration ([Ca(2+)]) in synaptosomes was measured by double wavelength fluorescence spectrophotometer and the expression level of nuclear transcription factor kappa-B ρ65 (NF-κB ρ65) in hippocampal CA3 region was measured by immunohistochemistry. The results showed that [Ca(2+)] significantly increased (F = 33.218, P < 0.01) in moderate fluoride group compared with the control group, and the expression level of NF-κB ρ65 in CA3 region presented an increasing trend as fluoride concentration increased. These results indicate that increase of synaptosomes [Ca(2+)] and NF-κB ρ65 expression level may be the molecular basis of central nervous system damage caused by chronic fluoride intoxication. NF-κB ρ65 in CA3 region is probably a target molecule for fluorosis.

  14. In vivo footprinting of the human IL-2 gene reveals a nuclear factor bound to the transcription start site in T cells.

    PubMed Central

    Brunvand, M W; Krumm, A; Groudine, M

    1993-01-01

    The IL-2 gene is a T cell specific gene that is expressed early during the activation-specific T lymphocyte development program. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assays have defined DNA/protein interactions at the IL-2 promoter cis-elements in vitro. To determine if the trans-activators documented in T cell nuclear extracts actually bind the IL-2 promoter in vivo, ligation mediated PCR (LMPCR) genomic footprinting was performed on the IL-2 promoter in both activated and non-activated T cells and HL60 promyelocytes, which do not express the IL-2 gene. The in vivo footprints indicate that the IL-2 gene transcription start site and TATA sequence are protected in both activated and resting T cells, prior to the appearance of detectable IL-2 steady state message. The distal NF-AT and the NF kappa B sites are each footprinted and the Oct/OAP site contains hypersensitive residues in the unstimulated T lymphocytes. Additional residues are protected in each of these sites after T cell activation. The proximal NF-AT site (NF-IL-2B) and the AP-1 site at -150 are protected in activated Jurkat T lymphocytes, but these two sites are not protected in activated Jurkat lymphocytes stably transfected a gene construct containing multiple NFAT binding sites. Images PMID:8233832

  15. The nuclear-encoded sigma factor SIG4 directly activates transcription of chloroplast psbA and ycf17 genes in the unicellular red alga Cyanidioschyzon merolae.

    PubMed

    Fujii, Gaku; Imamura, Sousuke; Era, Atsuko; Miyagishima, Shin-ya; Hanaoka, Mitsumasa; Tanaka, Kan

    2015-05-01

    The plant organelle chloroplast originated from the endosymbiosis of a cyanobacterial-like photosynthetic bacterium, and still retains its own genome derived from this ancestor. We have been focusing on a unicellular red alga, Cyanidioschyzon merolae, as a model photosynthetic eukaryote. In this study, we analyzed the transcriptional specificity of SIG4, which is one of four nuclear-encoded chloroplast RNA polymerase sigma factors in this alga. Accumulation of the SIG4 protein was observed in response to nitrogen depletion or high light conditions. By comparing the chloroplast transcriptomes under nitrogen depletion and SIG4-overexpressing conditions, we identified several candidate genes as SIG4 targets. Together with the results of chromatin immunoprecipitation analysis, the promoters of the psbA (encoding the D1 protein of the photosystem II reaction center) and ycf17 (encoding a protein of the early light-inducible protein family) genes were shown to be direct activation targets. The phycobilisome (PBS) CpcB protein was decreased by SIG4 overexpression, which suggests the negative involvement of SIG4 in PBS accumulation.

  16. Glycogen synthase kinase 3 regulates expression of nuclear factor-erythroid-2 related transcription factor-1 (Nrf1) and inhibits pro-survival function of Nrf1

    SciTech Connect

    Biswas, Madhurima; Kwong, Erick K.; Park, Eujean; Nagra, Parminder; Chan, Jefferson Y.

    2013-08-01

    Nuclear factor E2-related factor-1 (Nrf1) is a basic leucine zipper transcription factor that is known to regulate antioxidant and cytoprotective gene expression. It was recently shown that Nrf1 is regulated by SCF–Fbw7 ubiquitin ligase. However our knowledge of upstream signals that targets Nrf1 for degradation by the UPS is not known. We report here that Nrf1 expression is negatively regulated by glycogen synthase kinase 3 (GSK3) in Fbw7-dependent manner. We show that GSK3 interacts with Nrf1 and phosphorylates the Cdc4 phosphodegron domain (CPD) in Nrf1. Mutation of serine residue in the CPD of Nrf1 to alanine (S350A), blocks Nrf1 from phosphorylation by GSK3, and stabilizes Nrf1. Knockdown of Nrf1 and expression of a constitutively active form of GSK3 results in increased apoptosis in neuronal cells in response to ER stress, while expression of the GSK3 phosphorylation resistant S350A–Nrf1 attenuates apoptotic cell death. Together these data suggest that GSK3 regulates Nrf1 expression and cell survival function in response to stress activation. Highlights: • The effect of GSK3 on Nrf1 expression was examined. • GSK3 destabilizes Nrf1 protein via Fbw7 ubiquitin ligase. • GSK3 binds and phosphorylates Nrf1. • Protection from stress-induced apoptosis by Nrf1 is inhibited by GSK3.

  17. Berry Phenolic Compounds Increase Expression of Hepatocyte Nuclear Factor-1α (HNF-1α) in Caco-2 and Normal Colon Cells Due to High Affinities with Transcription and Dimerization Domains of HNF-1α.

    PubMed

    Real Hernandez, Luis M; Fan, Junfeng; Johnson, Michelle H; Gonzalez de Mejia, Elvira

    2015-01-01

    Hepatocyte nuclear factor-1α (HNF-1α) is found in the kidneys, spleen, thymus, testis, skin, and throughout the digestive organs. It has been found to promote the transcription of various proteins involved in the management of type II diabetes, including dipeptidyl peptidase-IV (DPP-IV). Phenolic compounds from berries and citrus fruits are known to inhibit DPP-IV, but have not been tested for their interactions with wild-type HNF-1α. By studying the interactions of compounds from berries and citrus fruits have with HNF-1α, pre-transcriptional mechanisms that inhibit the expression of proteins such as DPP-IV may be elucidated. In this study, the interactions of berry phenolic compounds and citrus flavonoids with the dimerization and transcriptional domains of HNF-1α were characterized using the molecular docking program AutoDock Vina. The anthocyanin delphinidin-3-O-arabinoside had the highest binding affinity for the dimerization domain as a homodimer (-7.2 kcal/mol) and transcription domain (-8.3 kcal/mol) of HNF-1α. Anthocyanins and anthocyanidins had relatively higher affinities than resveratrol and citrus flavonoids for both, the transcription domain and the dimerization domain as a homodimer. The flavonoid flavone had the highest affinity for a single unit of the dimerization domain (-6.5 kcal/mol). Nuclear expression of HNF-1α was measured in Caco-2 and human normal colon cells treated with blueberry and blackberry anthocyanin extracts. All extracts tested increased significantly (P < 0.05) the nuclear expression of HNF-1α in Caco-2 cells by 85.2 to 260% compared to a control. The extracts tested increased significantly (P < 0.02) the nuclear expression of HNF-1α in normal colon cells by 48.6 to 243%. It was confirmed that delphinidin-3-O-glucoside increased by 3-fold nuclear HNF-1α expression in Caco-2 cells (P < 0.05). Anthocyanins significantly increased nuclear HNF-1α expression, suggesting that these compounds might regulate the genes HNF-1

  18. Berry Phenolic Compounds Increase Expression of Hepatocyte Nuclear Factor-1α (HNF-1α) in Caco-2 and Normal Colon Cells Due to High Affinities with Transcription and Dimerization Domains of HNF-1α

    PubMed Central

    Real Hernandez, Luis M.; Fan, Junfeng; Johnson, Michelle H.; Gonzalez de Mejia, Elvira

    2015-01-01

    Hepatocyte nuclear factor-1α (HNF-1α) is found in the kidneys, spleen, thymus, testis, skin, and throughout the digestive organs. It has been found to promote the transcription of various proteins involved in the management of type II diabetes, including dipeptidyl peptidase-IV (DPP-IV). Phenolic compounds from berries and citrus fruits are known to inhibit DPP-IV, but have not been tested for their interactions with wild-type HNF-1α. By studying the interactions of compounds from berries and citrus fruits have with HNF-1α, pre-transcriptional mechanisms that inhibit the expression of proteins such as DPP-IV may be elucidated. In this study, the interactions of berry phenolic compounds and citrus flavonoids with the dimerization and transcriptional domains of HNF-1α were characterized using the molecular docking program AutoDock Vina. The anthocyanin delphinidin-3-O-arabinoside had the highest binding affinity for the dimerization domain as a homodimer (-7.2 kcal/mol) and transcription domain (-8.3 kcal/mol) of HNF-1α. Anthocyanins and anthocyanidins had relatively higher affinities than resveratrol and citrus flavonoids for both, the transcription domain and the dimerization domain as a homodimer. The flavonoid flavone had the highest affinity for a single unit of the dimerization domain (-6.5 kcal/mol). Nuclear expression of HNF-1α was measured in Caco-2 and human normal colon cells treated with blueberry and blackberry anthocyanin extracts. All extracts tested increased significantly (P < 0.05) the nuclear expression of HNF-1α in Caco-2 cells by 85.2 to 260% compared to a control. The extracts tested increased significantly (P < 0.02) the nuclear expression of HNF-1α in normal colon cells by 48.6 to 243%. It was confirmed that delphinidin-3-O-glucoside increased by 3-fold nuclear HNF-1α expression in Caco-2 cells (P < 0.05). Anthocyanins significantly increased nuclear HNF-1α expression, suggesting that these compounds might regulate the genes HNF-1

  19. Transforming growth factor beta 1-responsive element: closely associated binding sites for USF and CCAAT-binding transcription factor-nuclear factor I in the type 1 plasminogen activator inhibitor gene.

    PubMed Central

    Riccio, A; Pedone, P V; Lund, L R; Olesen, T; Olsen, H S; Andreasen, P A

    1992-01-01

    Transforming growth factor beta (TGF-beta) is the name of a group of closely related polypeptides characterized by a multiplicity of effects, including regulation of extracellular proteolysis and turnover of the extracellular matrix. Its cellular mechanism of action is largely unknown. TGF-beta 1 is a strong and fast inducer of type 1 plasminogen activator inhibitor gene transcription. We have identified a TGF-beta 1-responsive element in the 5'-flanking region of the human type 1 plasminogen activator inhibitor gene and shown that it is functional both in its natural context and when fused to a heterologous nonresponsive promoter. Footprinting and gel retardation experiments showed that two different nuclear factors, present in extracts from both TGF-beta 1-treated and nontreated cells, bind to adjacent sequences contained in the responsive unit. A palindromic sequence binds a trans-acting factor(s) of the CCAAT-binding transcription factor-nuclear factor I family. A partially overlapping dyad symmetry interacts with a second protein that much evidence indicates to be USF. USF is a transactivator belonging to the basic helix-loop-helix family of transcription factors. Mutations which abolish the binding of either CCAAT-binding transcription factor-nuclear factor I or USF result in reduction of transcriptional activation upon exposure to TGF-beta 1, thus showing that both elements of the unit are necessary for the TGF-beta 1 response. We discuss the possible relationship of these findings to the complexity of the TGF-beta action. Images PMID:1549130

  20. Competition of nuclear factor-erythroid 2 factors related transcription factor isoforms, Nrf1 and Nrf2, in antioxidant enzyme induction.

    PubMed

    Chepelev, Nikolai L; Zhang, Hongqiao; Liu, Honglei; McBride, Skye; Seal, Andrew J; Morgan, Todd E; Finch, Caleb E; Willmore, William G; Davies, Kelvin J A; Forman, Henry Jay

    2013-01-01

    Although the Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2) regulated expression of multiple antioxidant and cytoprotective genes through the electrophile responsive element (EpRE) is well established, interaction of Nrf2/EpRE with Nrf1, a closely-related transcription factor, is less well understood. Due to either proteolysis or alternative translation, Nrf1 has been found as proteins of varying size, p120, p95, and p65, which have been described as either activators of EpRE or competitive inhibitors of Nrf2. We investigated the effect of Nrf1 on EpRE-regulated gene expression using the catalytic and modifier subunits of glutamate cysteine ligase (GCLC and GCLM) as models and explored the potential role of Nrf1 in altering their expression in aging and upon chronic exposure to airborne nano-sized particulate matter (nPM). Nrf1 knockout resulted in the increased expression of GCLC and GCLM in human bronchial epithelial (HBE1) cells. Overexpression Nrf2 in combination with either p120 or p65 diminished or failed to further increase the GCLC- and GLCM-EpRE luciferase activity. All known forms of Nrf1 protein, remained unchanged in the lungs of mice with age or in response to nPM. Our study shows that Nrf1 could inhibit EpRE activity in vitro, whereas the precise role of Nrf1 in vivo requires further investigations. We conclude that Nrf1 may not be directly responsible for the loss of Nrf2-dependent inducibility of antioxidant and cytoprotective genes observed in aged animals.

  1. Transcriptional regulation of NADPH oxidase isoforms, Nox1 and Nox4, by nuclear factor-{kappa}B in human aortic smooth muscle cells

    SciTech Connect

    Manea, Adrian; Tanase, Laurentia I.; Raicu, Monica; Simionescu, Maya

    2010-06-11

    Inflammation-induced changes in the activity and expression of NADPH oxidases (Nox) play a key role in atherogenesis. The molecular mechanisms of Nox regulation are scantily elucidated. Since nuclear factor-{kappa}B (NF-{kappa}B) controls the expression of many genes associated to inflammation-related diseases, in this study we have investigated the role of NF-{kappa}B signaling in the regulation of Nox1 and Nox4 transcription in human aortic smooth muscle cells (SMCs). Cultured cells were exposed to tumor necrosis factor-{alpha} (TNF{alpha}), a potent inducer of both Nox and NF-{kappa}B, up to 24 h. Lucigenin-enhanced chemiluminescence and dichlorofluorescein assays, real-time polymerase chain reaction, and Western blot analysis showed that inhibition of NF-{kappa}B pathway reduced significantly the TNF{alpha}-dependent up-regulation of Nox-derived reactive oxygen species production, Nox1 and Nox4 expression. In silico analysis indicated the existence of typical NF-{kappa}B elements in the promoters of Nox1 and Nox4. Transient overexpression of p65/NF-{kappa}B significantly increased the promoter activities of both isoforms. Physical interaction of p65/NF-{kappa}B proteins with the predicted sites was demonstrated by chromatin immunoprecipitation assay. These findings demonstrate that NF-{kappa}B is an essential regulator of Nox1- and Nox4-containing NADPH oxidase in SMCs. Elucidation of the complex relationships between NF-{kappa}B and Nox enzymes may lead to a novel pharmacological strategy to reduce both inflammation and oxidative stress in atherosclerosis and its associated complications.

  2. Transcriptional mechanism for the paired miR-433 and miR-127 genes by nuclear receptors SHP and ERRγ

    PubMed Central

    Song, Guisheng; Wang, Li

    2008-01-01

    MicroRNAs (miRNAs, miRs) are genomically encoded small ∼22 nt RNA molecules that have been shown to mediate translational repression of target mRNAs involved in cellular proliferation, differentiation and death. Despite intensive studies on their physiological and pathological functions, the molecular mechanism of how miRNA gene transcription is regulated remains largely unknown. Microarray profiling revealed 21 miRNAs clustered on chromosome 12, including miR-433 and miR-127, that were co-upregulated in small heterodimer partner (SHP, NR0B2) SHP knockouts (SHP–/–) liver. Gene cloning revealed that the 3′-coding region of pri-miR-433 served as the promoter region of pri-miR-127. Estrogen related receptor (ERRγ, NR3B3) robustly activated miR-433 and miR-127 promoter reporters through ERRE, which was transrepressed by SHP. The strong elevation of miR-433 and miR-127 in Hepa-1 cells correlated with the down-regulation of SHP and up-regulation of ERRγ. Ectopic expression of ERRγ induced miR-433 and miR-127 expression, which was repressed by SHP coexpression. In contrast, knockdown ERRγ decreased miR-433 and miR-127 expression. In addition, the ERRγ agonist GSK4716 induced miR-433 and miR-127 expression both in vitro and in vivo, respectively. In summary, the coupled miR-433 and miR-127 genes were transcribed from independent promoters regulated by nuclear receptors ERRγ/SHP in a compact space by using overlapping genomic regions. PMID:18776219

  3. Your Radiologist Explains Nuclear Medicine

    MedlinePlus

    ... Sponsored by Image/Video Gallery Your Radiologist Explains Nuclear Medicine Transcript Welcome to Radiology Info dot org ... I’d like to talk to you about nuclear medicine. Nuclear medicine offers the potential to identify ...

  4. Your Radiologist Explains Nuclear Medicine

    MedlinePlus Videos and Cool Tools

    ... by Image/Video Gallery Your Radiologist Explains Nuclear Medicine Transcript Welcome to Radiology Info dot org Hello! ... d like to talk to you about nuclear medicine. Nuclear medicine offers the potential to identify disease ...

  5. Transcription coactivator PRIP, the peroxisome proliferator-activated receptor (PPAR)-interacting protein, is redundant for the function of nuclear receptors PParalpha and CAR, the constitutive androstane receptor, in mouse liver.

    PubMed

    Sarkar, Joy; Qi, Chao; Guo, Dongsheng; Ahmed, Mohamed R; Jia, Yuzhi; Usuda, Nobuteru; Viswakarma, Navin; Rao, M Sambasiva; Reddy, Janardan K

    2007-01-01

    Disruption of the genes encoding for the transcription coactivators, peroxisome proliferator-activated receptor (PPAR)-interacting protein (PRIP/ASC-2/RAP250/TRBP/NRC) and PPAR-binding protein (PBP/TRAP220/DRIP205/MED1), results in embryonic lethality by affecting placental and multiorgan development. Targeted deletion of coactivator PBP gene in liver parenchymal cells (PBP(LIV-/-)) results in the near abrogation of the induction of PPARalpha and CAR (constitutive androstane receptor)-regulated genes in liver. Here, we show that targeted deletion of coactivator PRIP gene in liver (PRIP(LIV-/-)) does not affect the induction of PPARalpha-regulated pleiotropic responses, including hepatomegaly, hepatic peroxisome proliferation, and induction of mRNAs of genes involved in fatty acid oxidation system, indicating that PRIP is not essential for PPARalpha-mediated transcriptional activity. We also provide additional data to show that liver-specific deletion of PRIP gene does not interfere with the induction of genes regulated by nuclear receptor CAR. Furthermore, disruption of PRIP gene in liver did not alter zoxazolamine-induced paralysis, and acetaminophen-induced hepatotoxicity. Studies with adenovirally driven EGFP-CAR expression in liver demonstrated that, unlike PBP, the absence of PRIP does not prevent phenobarbital-mediated nuclear translocation/retention of the receptor CAR in liver in vivo and cultured hepatocytes in vitro. These results show that PRIP deficiency in liver does not interfere with the function of nuclear receptors PPARalpha and CAR. The dependence of PPARalpha- and CAR-regulated gene transcription on coactivator PBP but not on PRIP attests to the existence of coactivator selectivity in nuclear receptor function.

  6. Activated nuclear transcription factor {kappa}B in patients with myocarditis and dilated cardiomyopathy-relation to inflammation and cardiac function

    SciTech Connect

    Alter, Peter . E-mail: palter@med.uni-marburg.de; Rupp, Heinz; Maisch, Bernhard

    2006-01-06

    Objectives and background: Myocarditis is caused by various agents and autoimmune processes. It is unknown whether viral genome persistence represents inactive remnants of previous infections or whether it is attributed to ongoing adverse processes. The latter also applies to the course of autoimmune myocarditis. One principal candidate for an adverse remodeling is nuclear factor-{kappa}B (NF{kappa}B). Methods: A total of 93 patients with suspected myocarditis/cardiomyopathy was examined. Hemodynamics were assessed by echocardiography as well as right and left heart catheterization. Endomyocardial biopsies were taken from the left ventricle. Biopsies were examined by immunohistochemistry and PCR for viral genomes. Selective immunostaining of activated NF{kappa}B was performed. Results: NF{kappa}B was increased in patients with myocarditis when compared with controls (11.1 {+-} 7.1% vs. 5.0 {+-} 5.3%, P < 0.005) whereas dilated cardiomyopathy showed no significant increase. Patients with myocarditis and preserved left ventricular function exhibited increased activated NF{kappa}B when compared with reduced function (r {sup 2} = 0.72, P < 0.001). In parallel, inverse correlation of NF{kappa}B and left ventricular enddiasstolic volume was found (r {sup 2} = 0.43, P < 0.02). Increased activated NF{kappa}B was found in adenovirus persistence when compared with controls (P = 0.001). Only a trend of increased NF{kappa}B activation was seen in cytomegalovirus persistence. Parvovirus B19 persistence did not affect NF{kappa}B activation. Conclusions: Increased activation of NF{kappa}B is related to inflammatory processes in myocarditis. Since activated NF{kappa}B correlates with left ventricular function, it could be assumed that NF{kappa}B activation occurs at early stages of inflammation. Potentially, NF{kappa}B could inhibit loss of cardiomyocytes by apoptosis and protect from cardiac dilation. Since NF{kappa}B is a crucial key transcription factor of inflammation, its

  7. Glucocorticoids facilitate the transcription from the human cytomegalovirus major immediate early promoter in glucocorticoid receptor- and nuclear factor-I-like protein-dependent manner.

    PubMed

    Inoue-Toyoda, Maki; Kato, Kohsuke; Nagata, Kyosuke; Yoshikawa, Hiroyuki

    2015-02-27

    Human cytomegalovirus (HCMV) is a common and usually asymptomatic virus agent in healthy individuals. Initiation of HCMV productive infection depends on expression of the major immediate early (MIE) genes. The transcription of HCMV MIE genes is regulated by a diverse set of transcription factors. It was previously reported that productive HCMV infection is triggered probably by elevation of the plasma hydroxycorticoid level. However, it is poorly understood whether the transcription of MIE genes is directly regulated by glucocorticoid. Here, we found that the dexamethasone (DEX), a synthetic glucocorticoid, facilitates the transcription of HCMV MIE genes through the MIE promoter and enhancer in a glucocorticoid receptor (GR)-dependent manner. By competitive EMSA and reporter assays, we revealed that an NF-I like protein is involved in DEX-mediated transcriptional activation of the MIE promoter. Thus, this study supports a notion that the increased level of hydroxycorticoid in the third trimester of pregnancy reactivates HCMV virus production from the latent state.

  8. Nuclear dualism.

    PubMed

    Karrer, Kathleen M

    2012-01-01

    Nuclear dualism is a characteristic feature of the ciliated protozoa. Tetrahymena have two different nuclei in each cell. The larger, polyploid, somatic macronucleus (MAC) is the site of transcriptional activity in the vegetatively growing cell. The smaller, diploid micronucleus (MIC) is transcriptionally inactive in vegetative cells, but is transcriptionally active in mating cells and responsible for the genetic continuity during sexual reproduction. Although the MICs and MACs develop from mitotic products of a common progenitor and reside in a common cytoplasm, they are different from one another in almost every respect.

  9. Glucocorticoids facilitate the transcription from the human cytomegalovirus major immediate early promoter in glucocorticoid receptor- and nuclear factor-I-like protein-dependent manner

    SciTech Connect

    Inoue-Toyoda, Maki; Kato, Kohsuke; Nagata, Kyosuke; Yoshikawa, Hiroyuki

    2015-02-27

    Human cytomegalovirus (HCMV) is a common and usually asymptomatic virus agent in healthy individuals. Initiation of HCMV productive infection depends on expression of the major immediate early (MIE) genes. The transcription of HCMV MIE genes is regulated by a diverse set of transcription factors. It was previously reported that productive HCMV infection is triggered probably by elevation of the plasma hydroxycorticoid level. However, it is poorly understood whether the transcription of MIE genes is directly regulated by glucocorticoid. Here, we found that the dexamethasone (DEX), a synthetic glucocorticoid, facilitates the transcription of HCMV MIE genes through the MIE promoter and enhancer in a glucocorticoid receptor (GR)-dependent manner. By competitive EMSA and reporter assays, we revealed that an NF-I like protein is involved in DEX-mediated transcriptional activation of the MIE promoter. Thus, this study supports a notion that the increased level of hydroxycorticoid in the third trimester of pregnancy reactivates HCMV virus production from the latent state. - Highlights: • DEX facilitates the transcription from the HCMV MIE promoter. • GR is involved in DEX-dependent transcription from the HCMV MIE promoter. • A 17 bp repeat is responsible for the HCMV MIE promoter activation by DEX. • An NF-I-like protein is involved in the HCMV MIE promoter activation by DEX.

  10. A nuclear mutant of Arabidopsis with impaired stability on distinct transcripts of the plastid psbB, psbD/C, ndhH, and ndhC operons.

    PubMed Central

    Meurer, J; Berger, A; Westhoff, P

    1996-01-01

    The high-chlorophyll fluorescence photosynthesis mutant hcf109 of Arabidopsis was characterized in detail to gain insights into the regulatory mechanism of RNA processing in higher plants. By using electron transport, chlorophyll fluorescence, and immunoblot studies, we assigned the mutational lesion to photosystems I and II and the plastid NAD(P)H dehydrogenase complex. The functional pleiotropy was reflected in RNA deficiencies. Although all nuclear-encoded photosynthetic RNAs analyzed revealed no difference in size or steady state level between mutant and wild type, the RNA patterns of the plastome-encoded psbB-psbT-psbH-petB-petD, psbD-psbC-ycf9, ndhC-ndhK-ndhJ, and ndhH-ndhA-ndhI-ndhG-ndhE-psaC-ndh D transcription units were severely disturbed. These operons encode subunits of photosystems I (psa) and II (psb), the cytochrome bGf complex (pet), the plastid NAD(P)H dehydrogenase (ndh), and the unidentified open reading frame ycf9. With the exception of the ndhC operon, the RNA deficiencies observed were specific and restricted to particular segments of the psbB, psbD/C, and ndhH operons, that is, the psbB-psbT, ycf9, and psaC regions. Run-on transcription studies with isolated chloroplasts showed that the failure of these transcripts to accumulate was due to RNA stability and not transcription. Other polycistronic transcription units analyzed were not affected by the mutation. This result indicates that the trans-regulatory factor encoded by the hcf109 gene is not a general RNA stability factor but that it specifically controls the stability of only these distinct transcripts. Because the hcf109 locus was mapped at a distance < 0.1 centimorgans from the phytochrome C gene, its molecular characterization by positional cloning is possible. PMID:8768377

  11. Cloning and functional analysis of spliced isoforms of human nuclear factor I-X: interference with transcriptional activation by NFI/CTF in a cell-type specific manner.

    PubMed Central

    Apt, D; Liu, Y; Bernard, H U

    1994-01-01

    Previous studies of the epithelial specificity of the human papillomavirus type 16 (HPV-16) enhancer pointed out an important role of nuclear factor I (NFI). In epithelial cells, NFI proteins are derived from the NFI-C gene and referred to as NFI/CTF. In contrast, fibroblasts, where the enhancer is inactive, express high levels of NFI from the NFI-X gene. To compare NFI-C and NFI-X derived transcription factors, we cloned and functionally investigated two differentially spliced forms of NFI-X from human fibroblasts. NFI-X1 has 95% homology with a transcript previously identified in hamster liver cells. NFI-X2, a spliced variant, misses 41 amino acids of the proline-rich activation domain. NFI-X expression, examined by Northern blots, shows strong cell-type specific variation in comparison with NFI/CTF. While the transcriptional activation domain of NFI-X2, functionally tested as GAL4-fusion protein in epithelial and fibroblast cells, activates transcription from promoter as well as enhancer position similar to NFI/CTF-1, the activation domain of NFI-X1 fails to activate transcription from enhancer position. In Drosophila cells, void of endogenous NFI proteins, full length NFI/CTF-1 and NFI-X2 activate a reporter construct containing only NFI sites as well as the NFI dependent HPV-16 enhancer. In contrast, NFI-X1 fails to activate the HPV-16 enhancer. Furthermore, overexpression of NFI-X1 in epithelial cells down-regulates the HPV-16 enhancer. Our findings suggest that the family of NFI transcription factors should not be viewed as constitutive activators, but rather, that NFI-C and NFI-X have divergent functions after binding in promoter or enhancer position. This property, combined with the differential expression of NFI-X, can achieve cell-type specificity of NFI dependent promoters and enhancers. Images PMID:7937100

  12. Transcriptional coupling of synaptic transmission and energy metabolism: role of nuclear respiratory factor 1 in co-regulating neuronal nitric oxide synthase and cytochrome c oxidase genes in neurons.

    PubMed

    Dhar, Shilpa S; Liang, Huan Ling; Wong-Riley, Margaret T T

    2009-10-01

    Neuronal activity is highly dependent on energy metabolism; yet, the two processes have traditionally been regarded as independently regulated at the transcriptional level. Recently, we found that the same transcription factor, nuclear respiratory factor 1 (NRF-1) co-regulates an important energy-generating enzyme, cytochrome c oxidase, as well as critical subunits of glutamatergic receptors. The present study tests our hypothesis that the co-regulation extends to the next level of glutamatergic synapses, namely, neuronal nitric oxide synthase, which generates nitric oxide as a downstream signaling molecule. Using in silico analysis, electrophoretic mobility shift assay, chromatin immunoprecipitation, promoter mutations, and NRF-1 silencing, we documented that NRF-1 functionally bound to Nos1, but not Nos2 (inducible) and Nos3 (endothelial) gene promoters. Both COX and Nos1 transcripts were up-regulated by depolarizing KCl treatment and down-regulated by TTX-mediated impulse blockade in neurons. However, NRF-1 silencing blocked the up-regulation of both Nos1 and COX induced by KCl depolarization, and over-expression of NRF-1 rescued both Nos1 and COX transcripts down-regulated by TTX. These findings are consistent with our hypothesis that synaptic neuronal transmission and energy metabolism are tightly coupled at the molecular level.

  13. The direct interaction between ASH2, a Drosophila trithorax group protein, and SKTL, a nuclear phosphatidylinositol 4-phosphate 5-kinase, implies a role for phosphatidylinositol 4,5-bisphosphate in maintaining transcriptionally active chromatin.

    PubMed Central

    Cheng, Mimi K; Shearn, Allen

    2004-01-01

    The products of trithorax group (trxG) genes maintain active transcription of many important developmental regulatory genes, including homeotic genes. Several trxG proteins have been shown to act in multimeric protein complexes that modify chromatin structure. ASH2, the product of the Drosophila trxG gene absent, small, or homeotic discs 2 (ash2) is a component of a 500-kD complex. In this article, we provide biochemical evidence that ASH2 binds directly to Skittles (SKTL), a predicted phosphatidylinositol 4-phosphate 5-kinase, and genetic evidence that the association of these proteins is functionally significant. We also show that histone H1 hyperphosphorylation is dramatically increased in both ash2 and sktl mutant polytene chromosomes. These results suggest that ASH2 maintains active transcription by binding a producer of nuclear phosphoinositides and downregulating histone H1 hyperphosphorylation. PMID:15280236

  14. Nuclear Speckles

    PubMed Central

    Spector, David L.; Lamond, Angus I.

    2011-01-01

    Nuclear speckles, also known as interchromatin granule clusters, are nuclear domains enriched in pre-mRNA splicing factors, located in the interchromatin regions of the nucleoplasm of mammalian cells. When observed by immunofluorescence microscopy, they usually appear as 20–50 irregularly shaped structures that vary in size. Speckles are dynamic structures, and their constituents can exchange continuously with the nucleoplasm and other nuclear locations, including active transcription sites. Studies on the composition, structure, and dynamics of speckles have provided an important paradigm for understanding the functional organization of the nucleus and the dynamics of the gene expression machinery. PMID:20926517

  15. Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4+ T cells through p65 and H2B phosphorylation

    PubMed Central

    Li, Jasmine; Hardy, Kristine; Phetsouphanh, Chan; Tu, Wen Juan; Sutcliffe, Elissa L.; McCuaig, Robert; Sutton, Christopher R.; Zafar, Anjum; Munier, C. Mee Ling; Zaunders, John J.; Xu, Yin; Theodoratos, Angelo; Tan, Abel; Lim, Pek Siew; Knaute, Tobias; Masch, Antonia; Zerweck, Johannes; Brezar, Vedran; Milburn, Peter J.; Dunn, Jenny; Casarotto, Marco G.; Turner, Stephen J.; Seddiki, Nabila; Kelleher, Anthony D.

    2016-01-01

    ABSTRACT Memory T cells are characterized by their rapid transcriptional programs upon re-stimulation. This transcriptional memory response is facilitated by permissive chromatin, but exactly how the permissive epigenetic landscape in memory T cells integrates incoming stimulatory signals remains poorly understood. By genome-wide ChIP-sequencing ex vivo human CD4+ T cells, here, we show that the signaling enzyme, protein kinase C theta (PKC-θ) directly relays stimulatory signals to chromatin by binding to transcriptional-memory-responsive genes to induce transcriptional activation. Flanked by permissive histone modifications, these PKC-enriched regions are significantly enriched with NF-κB motifs in ex vivo bulk and vaccinia-responsive human memory CD4+ T cells. Within the nucleus, PKC-θ catalytic activity maintains the Ser536 phosphorylation on the p65 subunit of NF-κB (also known as RelA) and can directly influence chromatin accessibility at transcriptional memory genes by regulating H2B deposition through Ser32 phosphorylation. Furthermore, using a cytoplasm-restricted PKC-θ mutant, we highlight that chromatin-anchored PKC-θ integrates activating signals at the chromatin template to elicit transcriptional memory responses in human memory T cells. PMID:27149922

  16. Transcription and cancer.

    PubMed Central

    Cox, P. M.; Goding, C. R.

    1991-01-01

    The normal growth, development and function of an organism requires precise and co-ordinated control of gene expression. A major part of this control is exerted by regulating messenger RNA (mRNA) production and involves complex interactions between an array of transcriptionally active proteins and specific regulatory DNA sequences. The combination of such proteins and DNA sequences is specific for given gene or group of genes in a particular cell type and the proteins regulating the same gene may vary between cell types. In addition the expression or activity of these regulatory proteins may be modified depending on the state of differentiation of a cell or in response to an external stimulus. Thus, the differentiation of embryonic cells into diverse tissues is achieved and the mature structure and function of the organism is maintained. This review focusses on the role of perturbations of these transcriptional controls in neoplasia. Deregulation of transcription may result in the failure to express genes responsible for cellular differentiation, or alternatively, in the transcription of genes involved in cell division, through the inappropriate expression or activation of positively acting transcription factors and nuclear oncogenes. Whether the biochemical abnormalities that lead to the disordered growth and differentiation of a malignant tumour affect cell surface receptors, membrane or cytoplasmic signalling proteins or nuclear transcription factors, the end result is the inappropriate expression of some genes and failure to express others. Current research is starting to elucidate which of the elements of this complicated system are important in neoplasia. PMID:1645561

  17. The Enzyme-Like Domain of Arabidopsis Nuclear β-Amylases Is Critical for DNA Sequence Recognition and Transcriptional Activation[C][W][OPEN

    PubMed Central

    Soyk, Sebastian; Šimková, Klára; Zürcher, Evelyne; Luginbühl, Leonie; Brand, Luise H.; Vaughan, Cara K.; Wanke, Dierk; Zeeman, Samuel C.

    2014-01-01

    Plant BZR1-BAM transcription factors contain a β-amylase (BAM)–like domain, characteristic of proteins involved in starch breakdown. The enzyme-derived domains appear to be noncatalytic, but they determine the function of the two Arabidopsis thaliana BZR1-BAM isoforms (BAM7 and BAM8) during transcriptional initiation. Removal or swapping of the BAM domains demonstrates that the BAM7 BAM domain restricts DNA binding and transcriptional activation, while the BAM8 BAM domain allows both activities. Furthermore, we demonstrate that BAM7 and BAM8 interact on the protein level and cooperate during transcriptional regulation. Site-directed mutagenesis of residues in the BAM domain of BAM8 shows that its function as a transcriptional activator is independent of catalysis but requires an intact substrate binding site, suggesting it may bind a ligand. Microarray experiments with plants overexpressing truncated versions lacking the BAM domain indicate that the pseudo-enzymatic domain increases selectivity for the preferred cis-regulatory element BBRE (BZR1-BAM Responsive Element). Side specificity toward the G-box may allow crosstalk to other signaling networks. This work highlights the importance of the enzyme-derived domain of BZR1-BAMs, supporting their potential role as metabolic sensors. PMID:24748042

  18. AtC3H17, a Non-Tandem CCCH Zinc Finger Protein, Functions as a Nuclear Transcriptional Activator and Has Pleiotropic Effects on Vegetative Development, Flowering and Seed Development in Arabidopsis.

    PubMed

    Seok, Hye-Yeon; Woo, Dong-Hyuk; Park, Hee-Yeon; Lee, Sun-Young; Tran, Huong T; Lee, Eun-Hye; Vu Nguyen, Linh; Moon, Yong-Hwan

    2016-03-01

    Despite increasing reports that CCCH zinc finger proteins function in plant development and stress responses, the functions and molecular aspects of many CCCH zinc finger proteins remain uncharacterized. Here, we characterized the biological and molecular functions of AtC3H17, a unique Arabidopsis gene encoding a non-tandem CCCH zinc finger protein. AtC3H17 was ubiquitously expressed throughout the life cycle of Arabidopsis plants and their organs. The rate and ratio of seed germination of atc3h17 mutants were slightly slower and lower, respectively, than those of the wild type (WT), whereas AtC3H17-overexpressing transgenic plants (OXs) showed an enhanced germination rate. atc3h17 mutant seedlings were smaller and lighter than WT seedlings while AtC3H17 OX seedlings were larger and heavier. In regulation of flowering time, atc3h17 mutants showed delayed flowering, whereas AtC3H17 OXs showed early flowering compared with the WT. In addition, overexpression of AtC3H17 affected seed development, displaying abnormalities compared with the WT. AtC3H17 protein was localized to the nucleus and showed transcriptional activation activity in yeast and Arabidopsis protoplasts. The N-terminal region of AtC3H17, containing a conserved EELR-like motif, was necessary for transcriptional activation activity, and the two conserved glutamate residues in the EELR-like motif played an important role in transcriptional activation activity. Real-time PCR and transactivation analyses showed that AtC3H17 might be involved in seed development via transcriptional activation of OLEO1, OLEO2 and CRU3. Our results suggest that AtC3H17 has pleiotropic effects on vegetative development such as seed germination and seedling growth, flowering and seed development, and functions as a nuclear transcriptional activator in Arabidopsis.

  19. Different blue-light requirement for the accumulation of transcripts from nuclear genes for thylakoid proteins in Nicotiana tabacum and Lycopersicon esculentum.

    PubMed

    Palomares, R; Herrmann, R G; Oelmüller, R

    1991-11-01

    We have isolated recombinant lambda gt11 phages which carry cDNA clones for the major light-harvesting chlorophyll a/b-binding proteins of photosystem I (LHCPI) and II (LHCPII), subunit II of photosystem I, a chlorophyll a/b-binding protein of photosystem II (CP24), the Rieske iron-sulphur protein of the cytochrome b6/f complex, and the 33, 23 and 16 kDa proteins of the water-oxidizing complex of photosystem II from Nicotiana tabacum. The nucleotide sequences of cDNA clones encoding the precursors for LHCPI and the FeS protein are presented. If tobacco or tomato seedlings, or seedlings of a phytochrome-deficient aurea mutant of tomato which lacks more than 95% of the phytochrome of the isogenic wild type, are kept in blue light, the transcript level of each of these genes is higher than in seedlings grown in red light suggesting the involvement of a blue-UVA-light photoreceptor. In the case of LHCPI, a 1 min blue-light pulse applied to red-light-grown seedlings is sufficient to increase the transcript levels to those present in blue-light-grown seedlings, whereas almost no increase is observed for transcripts encoding the FeS and 33 kDa proteins. If dark-grown tomato seedlings receive a single far-red-light pulse, significant stimulation is detected for LHCPI transcripts, whereas transcripts encoding the FeS and 33 kDa proteins are not stimulated. It is concluded that the lower light requirement for the increase in the LHCPI transcript level is not specific for one of the light-dependent signal transduction chains.

  20. Transcription of the Tollip gene is elevated in intestinal epithelial cells through impaired O-GlcNAcylation-dependent nuclear translocation of the negative regulator Elf-1.

    PubMed

    Sugi, Yutaka; Takahashi, Kyoko; Nakano, Kou; Hosono, Akira; Kaminogawa, Shuichi

    2011-09-09

    Intestinal epithelial cells (IECs) must be tolerant of the large number of commensal bacteria inhabiting the intestinal tract to avoid excessive inflammatory reactions. Toll-interacting protein (Tollip), a negative regulator of Toll-like receptor signaling, is known to be expressed at high levels in IECs, and to thereby contribute to the hyporesponsiveness of IECs to commensals. In this study, we analyzed the underlying mechanisms for elevated transcription of the Tollip gene in IECs using a human IEC line, Caco-2, and a human monocyte line, THP-1, as a control. Elf-1 was identified as a transcription factor that negatively regulates Tollip gene expression. The transcription factor Elf-1 was localized in the nucleus by O-linked N-acetylglucosamine (O-GlcNAc) modification, whereas the unmodified form was detected only in the cytoplasm. Comparison of Caco-2 and THP-1 cells revealed that O-GlcNAc modification of Elf-1 was significantly lower in IECs than in monocytes. Collectively, the results indicate that insufficient O-GlcNAc modification prevents Elf-1-mediated transcriptional repression and thereby upregulates Tollip gene expression in IECs.

  1. Science review: Redox and oxygen-sensitive transcription factors in the regulation of oxidant-mediated lung injury: role for nuclear factor-κB

    PubMed Central

    Haddad, John J

    2002-01-01

    The primary role of pulmonary airways is to conduct air to the alveolar epithelium, where gas exchange can efficiently occur. Injuries to airways resulting from inhalation of airborne pollutants and parenteral exposure to ingested pollutants that cause oxidative stress have the potential to interfere with this process. A progressive rise of oxidative stress due to altered reduction–oxidation (redox) homeostasis appears to be one of the hallmarks of the processes that regulate gene transcription in lung physiology and pathophysiology. Reactive metabolites serve as signaling messengers for the evolution and perpetuation of the inflammatory process that is often associated with cell death and degeneration. Redox-sensitive transcription factors are often associated with the development and progression of many human disease states and inflammatory-related injury, particularly of the lung. The present review elaborates on the role of the redox-sensitive and oxygen-sensitive transcription factor NF-κB in mediating lung injury. Changes in the pattern of gene expression through regulatory transcription factors are crucial components of the machinery that determines cellular responses to oxidative and redox perturbations. Additionally, the discussion of the possible therapeutic approaches of antioxidants, thiol-related compounds and phosphodiesterase inhibitors as anti-inflammatory agents will thereby help understand the oxidant/redox-mediated lung injury mechanisms. PMID:12493069

  2. Targeting of Heat Shock Protein HSPA6 (HSP70B') to the Periphery of Nuclear Speckles is Disrupted by a Transcription Inhibitor Following Thermal Stress in Human Neuronal Cells.

    PubMed

    Becirovic, Larissa; Brown, Ian R

    2017-02-01

    Heat shock proteins (Hsps) are a set of highly conserved proteins involved in cellular repair and protective mechanisms. The intracellular localization of inducible members of the HSPA (HSP70) family can be used as an index to identify stress-sensitive sites in differentiated human neuronal cells. Following thermal stress, the little studied HSPA6 (HSP70B') was targeted to the periphery of nuclear speckles (perispeckles) that are sites of transcription factories. Triptolide, a fast-acting transcription inhibitor, knocked down levels of the large subunit of RNA polymerase II, RPB1, during the time-frame when HSPA6 associated with perispeckles. Administration of triptolide to heat shocked human neuronal SH-SY5Y cells, disrupted HSPA6 localization to perispeckles, suggesting the involvement of HSPA6 in transcriptional recovery after stress. The HSPA6 gene is present in the human genome but is not found in the genomes of the mouse and rat. Hence current animal models of neurodegenerative diseases lack a member of the HSPA family that exhibits the feature of stress-induced targeting to perispeckles.

  3. Transcription of Epstein-Barr virus-encoded nuclear antigen 1 promoter Qp is repressed by transforming growth factor-beta via Smad4 binding element in human BL cells.

    PubMed

    Liang, C L; Tsai, C N; Chung, P J; Chen, J L; Sun, C M; Chen, R H; Hong, J H; Chang, Y S

    2000-11-10

    In Epstein-Barr virus (EBV)-infected BL cells, the oncogenic EBV-encoded nuclear antigen 1 (EBNA 1) gene is directed from the latent promoter Qp. Yeast one-hybrid screen analysis using the -50 to -37 sequence of Qp as the bait was carried out to identify transcriptional factors that may control Qp activity. Results showed that Smad4 binds the -50 to -37 sequence of Qp, indicating that this promoter is potentially regulated by TGF-beta. The association of Smad4 with Qp was further confirmed by supershift of EMSA complexes using Smad4-specific antibody. The transfection of a Qp reporter construct in two EBV(+) BL cell lines, Rael and WW2, showed that Qp activity is repressed in response to the TGF-beta treatment. This repression involves the interaction of a Smad3/Smad4 complex and the transcriptional repressor TGIF, as determined by cotransfection assay and coimmunoprecipitation analysis. Results suggest that TGF-beta may transcriptionally repress Qp through the Smad4-binding site in human BL cells.

  4. Artemisinin triggers a G1 cell cycle arrest of human Ishikawa endometrial cancer cells and inhibits cyclin-dependent kinase-4 promoter activity and expression by disrupting nuclear factor-κB transcriptional signaling.

    PubMed

    Tran, Kalvin Q; Tin, Antony S; Firestone, Gary L

    2014-03-01

    Relatively little is known about the antiproliferative effects of artemisinin, a naturally occurring antimalarial compound from Artemisia annua, or sweet wormwood, in human endometrial cancer cells. Artemisinin induced a G1 cell cycle arrest in cultured human Ishikawa endometrial cancer cells and downregulated cyclin-dependent kinase-2 (CDK2) and CDK4 transcript and protein levels. Analysis of CDK4 promoter-luciferase reporter constructs showed that the artemisinin ablation of CDK4 gene expression was accounted for by the loss of CDK4 promoter activity. Chromatin immunoprecipitation demonstrated that artemisinin inhibited nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) subunit p65 and p50 interactions with the endogenous Ishikawa cell CDK4 promoter. Coimmunoprecipitation revealed that artemisinin disrupts endogenous p65 and p50 nuclear translocation through increased protein-protein interactions with IκB-α, an NF-κB inhibitor, and disrupts its interaction with the CDK4 promoter, leading to a loss of CDK4 gene expression. Artemisinin treatment stimulated the cellular levels of IκB-α protein without altering the level of IκB-α transcripts. Finally, expression of exogenous p65 resulted in the accumulation of this NF-κB subunit in the nucleus of artemisinin-treated and artemisinin-untreated cells, reversed the artemisinin downregulation of CDK4 protein expression and promoter activity, and prevented the artemisinin-induced G1 cell cycle arrest. Taken together, our results demonstrate that a key event in the artemisinin antiproliferative effects in endometrial cancer cells is the transcriptional downregulation of CDK4 expression by disruption of NF-κB interactions with the CDK4 promoter.

  5. Interaction of ApoA-IV with NR4A1 and NR1D1 Represses G6Pase and PEPCK Transcription: Nuclear Receptor-Mediated Downregulation of Hepatic Gluconeogenesis in Mice and a Human Hepatocyte Cell Line

    PubMed Central

    Li, Xiaoming; Xu, Min; Wang, Fei; Ji, Yong; DavidsoN, W. Sean; Li, Zongfang; Tso, Patrick

    2015-01-01

    We have previously shown that the nuclear receptor, NR1D1, is a cofactor in ApoA-IV-mediated downregulation of gluconeogenesis. Nuclear receptor, NR4A1, is involved in the transcriptional regulation of various genes involved in inflammation, apoptosis, and glucose metabolism. We investigated whether NR4A1 influences the effect of ApoA-IV on hepatic glucose metabolism. Our in situ proximity ligation assays and coimmunoprecipitation experiments indicated that ApoA-IV colocalized with NR4A1 in human liver (HepG2) and kidney (HEK-293) cell lines. The chromatin immunoprecipitation experiments and luciferase reporter assays indicated that the ApoA-IV and NR4A1 colocalized at the RORα response element of the human G6Pase promoter, reducing its transcriptional activity. Our RNA interference experiments showed that knocking down the expression of NR4A1 in primary mouse hepatocytes treated with ApoA-IV increased the expression of NR1D1, G6Pase, and PEPCK, and that knocking down NR1D1 expression increased the level of NR4A1. We also found that ApoA-IV induced the expression of endogenous NR4A1 in both cultured primary mouse hepatocytes and in the mouse liver, and decreased glucose production in primary mouse hepatocytes. Our findings showed that ApoA-IV colocalizes with NR4A1, which suppresses G6Pase and PEPCK gene expression at the transcriptional level, reducing hepatic glucose output and lowering blood glucose. The ApoA-IV-induced increase in NR4A1 expression in hepatocytes mediates further repression of gluconeogenesis. Our findings suggest that NR1D1 and NR4A1 serve similar or complementary functions in the ApoA-IV-mediated regulation of gluconeogenesis. PMID:26556724

  6. Diarctigenin, a lignan constituent from Arctium lappa, down-regulated zymosan-induced transcription of inflammatory genes through suppression of DNA binding ability of nuclear factor-kappaB in macrophages.

    PubMed

    Kim, Byung Hak; Hong, Seong Su; Kwon, Soon Woo; Lee, Hwa Young; Sung, Hyeran; Lee, In-Jeong; Hwang, Bang Yeon; Song, Sukgil; Lee, Chong-Kil; Chung, Daehyun; Ahn, Byeongwoo; Nam, Sang-Yoon; Han, Sang-Bae; Kim, Youngsoo

    2008-11-01

    Diarctigenin was previously isolated as an inhibitor of nitric oxide (NO) production in macrophages from the seeds of Arctium lappa used as an alternative medicine for the treatment of inflammatory disorders. However, little is known about the molecular basis of these effects. Here, we demonstrated that diarctigenin inhibited the production of NO, prostaglandin E(2), tumor necrosis factor-alpha, and interleukin (IL)-1beta and IL-6 with IC(50) values of 6 to 12 miciroM in zymosan- or lipopolysaccharide-(LPS) activated macrophages. Diarctigenin attenuated zymosan-induced mRNA synthesis of inducible NO synthase (iNOS) and also inhibited promoter activities of iNOS and cytokine genes in the cells. Because nuclear factor (NF)-kappaB plays a pivotal role in inflammatory gene transcription, we next investigated the effect of diarctigenin on NF-kappaB activation. Diarctigenin inhibited the transcriptional activity and DNA binding ability of NF-kappaB in zymosan-activated macrophages but did not affect the degradation and phosphorylation of inhibitory kappaB (IkappaB) proteins. Moreover, diarctigenin suppressed expression vector NF-kappaB p65-elicited NF-kappaB activation and also iNOS promoter activity, indicating that the compound could directly target an NF-kappa-activating signal cascade downstream of IkappaB degradation and inhibit NF-kappaB-regulated iNOS expression. Diarctigenin also inhibited the in vitro DNA binding ability of NF-kappaB but did not affect the nuclear import of NF-kappaB p65 in the cells. Taken together, diarctigenin down-regulated zymosan- or LPS-induced inflammatory gene transcription in macrophages, which was due to direct inhibition of the DNA binding ability of NF-kappaB. Finally, this study provides a pharmacological potential of diarctigenin in the NF-kappaB-associated inflammatory disorders.

  7. Toxicogenomic Dissection of the Perfluorooctanoic Acid Transcript Profile in Mouse Liver: Evidence for the Involvement of Nuclear Receptors PPARα and CAR

    EPA Science Inventory

    A number of perfluorinated alkyl acids including perfluorooctanoic acid (PFOA) elicit effects similar to peroxisome proliferator chemicals (PPC) in mouse and rat liver. There is strong evidence that PPC cause many of their effects linked to liver cancer through the nuclear recep...

  8. PRDM16 enhances nuclear receptor-dependent transcription of the brown fat-specific Ucp1 gene through interactions with Mediator subunit MED1

    PubMed Central

    Iida, Satoshi; Chen, Wei; Nakadai, Tomoyoshi; Ohkuma, Yoshiaki

    2015-01-01

    PR domain-containing 16 (PRDM16) induces expression of brown fat-specific genes in brown and beige adipocytes, although the underlying transcription-related mechanisms remain largely unknown. Here, in vitro studies show that PRDM16, through its zinc finger domains, directly interacts with the MED1 subunit of the Mediator complex, is recruited to the enhancer of the brown fat-specific uncoupling protein 1 (Ucp1) gene through this interaction, and enhances thyroid hormone receptor (TR)-driven transcription in a biochemically defined system in a Mediator-dependent manner, thus providing a direct link to the general transcription machinery. Complementary cell-based studies show that upon forskolin treatment, PRDM16 induces Ucp1 expression in undifferentiated murine embryonic fibroblasts, that this induction depends on MED1 and TR, and, consistent with a direct effect, that PRDM16 is recruited to the Ucp1 enhancer. Related studies have defined MED1 and PRDM16 interaction domains important for Ucp1 versus Ppargc1a induction by PRDM16. These results reveal novel mechanisms for PRDM16 function through the Mediator complex. PMID:25644605

  9. SPT5, an essential gene important for normal transcription in Saccharomyces cerevisiae, encodes an acidic nuclear protein with a carboxy-terminal repeat.

    PubMed Central

    Swanson, M S; Malone, E A; Winston, F

    1991-01-01

    Mutations in the SPT5 gene of Saccharomyces cerevisiae were isolated previously as suppressors of delta insertion mutations at HIS4 and LYS2. In this study we have shown that spt5 mutations suppress the his4-912 delta and lys2-128 delta alleles by altering transcription. We cloned the SPT5 gene and found that either an increase or a decrease in the copy number of the wild-type SPT5 gene caused an Spt- phenotype. Construction and analysis of an spt5 null mutation demonstrated that SPT5 is essential for growth, suggesting that SPT5 may be required for normal transcription of a large number of genes. The SPT5 DNA sequence was determined; it predicted a 116-kDa protein with an extremely acidic amino terminus and a novel six-amino-acid repeat at the carboxy terminus (consensus = S-T/A-W-G-G-A/Q). By indirect immunofluorescence microscopy we showed that a bifunctional SPT5-beta-galactosidase protein was located in the yeast nucleus. This molecular analysis of the SPT5 gene revealed a number of interesting similarities to the previously characterized SPT6 gene of S. cerevisiae. These results suggest that SPT5 and SPT6 act in a related fashion to influence essential transcriptional processes in S. cerevisiae. Images PMID:1840633

  10. Co-operation of the transcription factor hepatocyte nuclear factor-4 with Sp1 or Sp3 leads to transcriptional activation of the human haem oxygenase-1 gene promoter in a hepatoma cell line.

    PubMed

    Takahashi, Shigeru; Matsuura, Naomi; Kurokawa, Takako; Takahashi, Yuji; Miura, Takashi

    2002-11-01

    We reported previously that the 5'-flanking region (nucleotides -1976 to -1655) of the human haem oxygenase-1 ( hHO-1 ) gene enhances hHO-1 promoter activity in human hepatoma HepG2 cells, but not in HeLa cells [Takahashi, Takahashi, Ito, Nagano, Shibahara and Miura (1999) Biochim. Biophys. Acta 1447, 231-235]. To define more precisely the regulatory elements involved, in the present study we have functionally dissected this region and localized the enhancer to a 50 bp fragment (-1793 to -1744). Site-direct mutagenesis analysis revealed that two regions were responsible for this enhancer activity, i.e. a hepatocyte nuclear factor-4 (HNF-4) homologous region and a GC box motif homologous region. Mutation in either region alone moderately decreased enhancer activity. However, mutations in both regions reduced promoter activity to the basal level. Electrophoretic mobility-shift assays demonstrated that the P5-2 fragment (-1793 to -1744) interacted with at least two nuclear factors, i.e. HNF-4 and Sp1/Sp3. Co-transfection experiments using Drosophila SL2 cells revealed that HNF-4 and Sp1/Sp3 synergistically stimulated the enhancer activity of the P5-2 fragment. These results indicate that co-operation of HNF-4 with Sp1 or Sp3 leads to the activation of hHO-1 gene expression in hepatoma cells.

  11. The RelA/cRel nuclear factor-κB (NF-κB) dimer, crucial for inflammation resolution, mediates the transcription of the key enzyme in melatonin synthesis in RAW 264.7 macrophages.

    PubMed

    Muxel, Sandra Marcia; Laranjeira-Silva, Maria Fernanda; Carvalho-Sousa, Claudia Emanuelle; Floeter-Winter, Lucile Maria; Markus, Regina P

    2016-05-01

    Lipopolysaccharide (LPS) modulates the transcription of the gene that codifies the enzyme arylalkylamine-N-acetyltransferase (AA-NAT) through nuclear translocation of the transcription factor nuclear factor-κ-light-chain-enhancer of activated B cells (NF-κB). AA-NAT converts serotonin to N-acetylserotonin, the ultimate precursor of melatonin. Activation of kappa B elements (aa-nat-κB), localized in the promoter (nat-κB1 and nat-κB2), leads to Aa-nat transcription in RAW 264.7 macrophages. Competitive electrophoretic mobility shift assay (EMSA) with oligonucleotide probes corresponding to each of the two elements, as well as a NF-κB consensus corresponding probe, revealed different specificities for each κB element. In addition, activator protein-1 (AP-1) as well as signal transducers and activator of transcription-1 and 3 (STAT-1; STAT-3) competed with NF-κB for binding to nat-κB1, while only STAT-3 competed with NF-κB for binding to nat-κB2. According to co-immunoprecipitation (ChiP) assays, these two sites are able to distinguish NF-κB subunits. The sequence nat-κB1 bound dimers containing p52, RelA, and cRel, while nat-κB2 bound preferentially p50, p52, and RelA, and did not bind cRel. The expression of RelA and cRel is essential for the induction of Aa-nat expression and melatonin synthesis. Considering that the expression of cRel is induced by the earlier expressed p50/RelA, the differential effects of NF-κB dimers may be intimately associated with the temporal regulation of inflammatory responses, with the resolution phase being associated with paracrine and autocrine melatonin effects. Such data suggest that the proven effects of exogenous melatonin in the resolution phase of inflammation are paralleled by the effects of locally synthesized melatonin in immune cells.

  12. Interactions of U2 Gene Loci and Their Nuclear Transcripts with Cajal (Coiled) Bodies: Evidence for PreU2 within Cajal Bodies

    PubMed Central

    Smith, Kelly P.; Lawrence, Jeanne Bentley

    2000-01-01

    The Cajal (coiled) body (CB) is a structure enriched in proteins involved in mRNA, rRNA, and snRNA metabolism. CBs have been shown to interact with specific histone and snRNA gene loci. To examine the potential role of CBs in U2 snRNA metabolism, we used a variety of genomic and oligonucleotide probes to visualize in situ newly synthesized U2 snRNA relative to U2 loci and CBs. Results demonstrate that long spacer sequences between U2 coding repeats are transcribed, supporting other recent evidence that U2 transcription proceeds past the 3′ box. The presence of bright foci of this U2 locus RNA differed between alleles within the same nucleus; however, this did not correlate with the loci's association with a CB. Experiments with specific oligonucleotide probes revealed signal for preU2 RNA within CBs. PreU2 was also detected in the locus-associated RNA foci, whereas sequences 3′ of preU2 were found only in these foci, not in CBs. This suggests that a longer primary transcript is processed before entry into CBs. Although this work shows that direct contact of a U2 locus with a CB is not simply correlated with RNA at that locus, it provides the first evidence of new preU2 transcripts within CBs. We also show that, in contrast to CBs, SMN gems do not associate with U2 gene loci and do not contain preU2. Because other evidence indicates that preU2 is processed in the cytoplasm before assembly into snRNPs, results point to an involvement of CBs in modification or transport of preU2 RNA. PMID:10982395

  13. Ethanol extract of Zhongtian hawthorn lowers serum cholesterol in mice by inhibiting transcription of 3-hydroxy-3-methylglutaryl-CoA reductase via nuclear factor-kappa B signal pathway.

    PubMed

    Hu, Hai-Jie; Luo, Xue-Gang; Dong, Qing-Qing; Mu, Ai; Shi, Guo-Long; Wang, Qiu-Tong; Chen, Xiao-Ying; Zhou, Hao; Zhang, Tong-Cun; Pan, Li-Wen

    2016-03-01

    Hawthorn is a berry-like fruit from the species of Crataegus. In China, it has another more famous name, Shan-Zha, which has been used to improve digestion as a traditional Chinese medicine or food for thousands of years. Moreover, during the last decades, hawthorn has received more attention because of its potential to treat cardiovascular diseases. However, currently, only fruits of C. pinnatifida and C. pinnatifida var. major are included as Shan-Zha in the Chinese Pharmacopoeia. In this study, our results showed that the ethanol extract of Zhongtian hawthorn, a novel grafted cultivar of C. cuneata (wild Shan-Zha), could markedly reduce body weight and levels of serum total cholesterol, triglyceride, low-density lipoprotein cholesterol, and liver cholesterol of hyperlipidemia mice. It could suppress the stimulation effect of high-fat diet on the transcription of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and p65, and counteract the downregulation of CYP7A1 and LDLR. In addition, the results of luciferase reporter assay and Western blot showed that the transcriptional activity of HMGCR promoter was inhibited by Zhongtian hawthorn ethanol extract in a dose-dependent manner, while overexpression of p65 could reverse this transcriptional repression effect. These results suggested that Zhongtian hawthorn could provide health benefits by counteracting the high-fat diet-induced hypercholesteolemic and hyperlipidemic effects in vivo, and the mechanism underlying this event was mainly dependent on the suppressive effect of Zhongtian hawthorn ethanol extract on the transcription of HMGCR via nuclear factor-kappa B (NF-κB) signal pathway. Therefore, this novel cultivar of hawthorn cultivar which has much bigger fruits, early bearing, high yield, cold resistance, and drought resistance, might be considered as a good alternative to Shan-Zha and has great value in the food and medicine industry. In addition, to our best knowledge, this is also the first report that the

  14. JACALIN-LECTIN LIKE1 Regulates the Nuclear Accumulation of GLYCINE-RICH RNA-BINDING PROTEIN7, Influencing the RNA Processing of FLOWERING LOCUS C Antisense Transcripts and Flowering Time in Arabidopsis1[OPEN

    PubMed Central

    Xiao, Jun; Li, Chunhua; Xu, Shujuan; Xing, Lijing; Xu, Yunyuan; Chong, Kang

    2015-01-01

    Lectins selectively recognize sugars or glycans for defense in living cells, but less is known about their roles in the development process and the functional network with other factors. Here, we show that Arabidopsis (Arabidopsis thaliana) JACALIN-LECTIN LIKE1 (AtJAC1) functions in flowering time control. Loss of function of AtJAC1 leads to precocious flowering, whereas overexpression of AtJAC1 causes delayed flowering. AtJAC1 influences flowering through regulation of the key flowering repressor gene FLOWERING LOCUS C (FLC). Genetic analysis revealed that AtJAC1’s function is mostly dependent on GLYCINE-RICH RNA-BINDING PROTEIN7 (GRP7), an upstream regulator of FLC. Biochemical and cell biological data indicated that AtJAC1 interacted physically with GRP7 specifically in the cytoplasm. AtJAC1 influences the nucleocytoplasmic distribution of GRP7, with predominant nuclear localization of GRP7 when AtJAC1 function is lost but retention of GRP7 in the cytoplasm when AtJAC1 is overexpressed. A temporal inducible assay suggested that AtJAC1’s regulation of flowering could be compromised by the nuclear accumulation of GRP7. In addition, GRP7 binds to the antisense precursor messenger RNA of FLC through a conserved RNA motif. Loss of GRP7 function leads to the elevation of total FLC antisense transcripts and reduced proximal-distal polyadenylation ratio, as well as histone methylation changes in the FLC gene body region and increased total functional sense FLC transcript. Attenuating the direct binding of GRP7 with competing artificial RNAs leads to changes of FLC antisense precursor messenger RNA processing and flowering transition. Taken together, our study indicates that AtJAC1 coordinates with GRP7 in shaping plant development through the regulation of RNA processing in Arabidopsis. PMID:26392261

  15. TAF4, a subunit of transcription factor II D, directs promoter occupancy of nuclear receptor HNF4A during post-natal hepatocyte differentiation.

    PubMed

    Alpern, Daniil; Langer, Diana; Ballester, Benoit; Le Gras, Stephanie; Romier, Christophe; Mengus, Gabrielle; Davidson, Irwin

    2014-09-10

    The functions of the TAF subunits of mammalian TFIID in physiological processes remain poorly characterised. In this study, we describe a novel function of TAFs in directing genomic occupancy of a transcriptional activator. Using liver-specific inactivation in mice, we show that the TAF4 subunit of TFIID is required for post-natal hepatocyte maturation. TAF4 promotes pre-initiation complex (PIC) formation at post-natal expressed liver function genes and down-regulates a subset of embryonic expressed genes by increased RNA polymerase II pausing. The TAF4-TAF12 heterodimer interacts directly with HNF4A and in vivo TAF4 is necessary to maintain HNF4A-directed embryonic gene expression at post-natal stages and promotes HNF4A occupancy of functional cis-regulatory elements adjacent to the transcription start sites of post-natal expressed genes. Stable HNF4A occupancy of these regulatory elements requires TAF4-dependent PIC formation highlighting that these are mutually dependent events. Local promoter-proximal HNF4A-TFIID interactions therefore act as instructive signals for post-natal hepatocyte differentiation.

  16. Arabidopsis histone deacetylase HDA6 is required for maintenance of transcriptional gene silencing and determines nuclear organization of rDNA repeats.

    PubMed

    Probst, Aline V; Fagard, Mathilde; Proux, Florence; Mourrain, Philippe; Boutet, Stéphanie; Earley, Keith; Lawrence, Richard J; Pikaard, Craig S; Murfett, Jane; Furner, Ian; Vaucheret, Hervé; Mittelsten Scheid, Ortrun

    2004-04-01

    Histone acetylation and deacetylation are connected with transcriptional activation and silencing in many eukaryotic organisms. Gene families for enzymes that accomplish these modifications show a surprising multiplicity in sequence and expression levels, suggesting a high specificity for different targets. We show that mutations in Arabidopsis (Arabidopsis thaliana) HDA6, a putative class I histone deacetylase gene, result in loss of transcriptional silencing from several repetitive transgenic and endogenous templates. Surprisingly, total levels of histone H4 acetylation are only slightly affected, whereas significant hyperacetylation is restricted to the nucleolus organizer regions that contain the rDNA repeats. This switch coincides with an increase of histone 3 methylation at Lys residue 4, a modified DNA methylation pattern, and a concomitant decondensation of the chromatin. These results indicate that HDA6 might play a role in regulating activity of rRNA genes, and this control might be functionally linked to silencing of other repetitive templates and to its previously assigned role in RNA-directed DNA methylation.

  17. Blue-light mediated accumulation of nuclear-encoded transcripts coding for proteins of the thylakoid membrane is absent in the phytochrome-deficient aurea mutant of tomato.

    PubMed

    Oelmüller, R; Kendrick, R E; Briggs, W R

    1989-08-01

    Polyclonal antibodies against pea phytochrome detect 2 protein bands (about 116 and 120 kDa) on blots of crude protein extracts and protein of microsomal preparations of dark-grown tomato seedlings. Both protein bands are undetectable in Western blots of the aurea mutant extracts. Neither protein band is detectable after isogenic wild-type seedlings are illuminated with 3 h of red light, either in the crude extract or in the membrane fraction of the irradiated seedlings; this result is consistent with the hypothesis that both bands are phytochrome. When dark-grown wild-type seedlings are illuminated with 3 h of red light or blue light against a red light background, the transcript levels for chlorophyll a/b-binding proteins of photosystem I and II, plastocyanin, and the subunit II of photosystem I increase. In all cases, the same fluence rate of blue light is much more effective than red light alone, a result that indicates the involvement of a blue/UV-A light photoreceptor in addition to the involvement of the far-red-absorbing form of phytochrome, Pfr. The aurea mutant responds neither to red light nor to blue light. Thus, no Pfr-independent induction of the four transcripts by a blue/UV-A light photoreceptor can be measured in the aurea mutant.

  18. The nuclear retention of transcription factor FOXO3a correlates with a DNA damage response and increased glutamine synthetase expression by astrocytes suggesting a neuroprotective role in the ageing brain

    PubMed Central

    Fluteau, Adeline; Ince, Paul G.; Minett, Thais; Matthews, Fiona E.; Brayne, Carol; Garwood, Claire J.; Ratcliffe, Laura E.; Morgan, Sarah; Heath, Paul R.; Shaw, Pamela J.; Wharton, Stephen B.; Simpson, Julie E.

    2015-01-01

    The accumulation of reactive oxygen species leading to oxidative damage and cell death plays an important role in a number of neurodegenerative disorders. FOXO3a, the main isoform of FOXO transcription factors, mediates the cellular response to oxidative stress by regulating the expression of genes involved in DNA repair and glutamine metabolism, including glutamine synthetase (GS). Immunohistochemical investigation of the population-based neuropathology cohort of the Medical Research Council’s Cognitive Function and Ageing Study (MRC CFAS) demonstrates that nuclear retention of FOXO3a significantly correlates with a DNA damage response and with GS expression by astrocytes. Furthermore, we show that GS expression correlates with increasing Alzheimer-type pathology in this ageing cohort. Our findings suggest that in response to oxidative stress, the nuclear retention of FOXO3a in astrocytes upregulates expression of GS as a neuroprotective mechanism. However, the activity of GS may be compromised by increasing levels of oxidative stress in the ageing brain resulting in dysfunctional enzyme activity, neuronal excitotoxic damage and cognitive impairment. PMID:26455863

  19. The nuclear retention of transcription factor FOXO3a correlates with a DNA damage response and increased glutamine synthetase expression by astrocytes suggesting a neuroprotective role in the ageing brain.

    PubMed

    Fluteau, Adeline; Ince, Paul G; Minett, Thais; Matthews, Fiona E; Brayne, Carol; Garwood, Claire J; Ratcliffe, Laura E; Morgan, Sarah; Heath, Paul R; Shaw, Pamela J; Wharton, Stephen B; Simpson, Julie E

    2015-11-16

    The accumulation of reactive oxygen species leading to oxidative damage and cell death plays an important role in a number of neurodegenerative disorders. FOXO3a, the main isoform of FOXO transcription factors, mediates the cellular response to oxidative stress by regulating the expression of genes involved in DNA repair and glutamine metabolism, including glutamine synthetase (GS). Immunohistochemical investigation of the population-based neuropathology cohort of the Medical Research Council's Cognitive Function and Ageing Study (MRC CFAS) demonstrates that nuclear retention of FOXO3a significantly correlates with a DNA damage response and with GS expression by astrocytes. Furthermore, we show that GS expression correlates with increasing Alzheimer-type pathology in this ageing cohort. Our findings suggest that in response to oxidative stress, the nuclear retention of FOXO3a in astrocytes upregulates expression of GS as a neuroprotective mechanism. However, the activity of GS may be compromised by increasing levels of oxidative stress in the ageing brain resulting in dysfunctional enzyme activity, neuronal excitotoxic damage and cognitive impairment.

  20. Arabidopsis inositol polyphosphate 6-/3-kinase is a nuclear protein that complements a yeast mutant lacking a functional ArgR-Mcm1 transcription complex.

    PubMed

    Xia, Hui-Jun; Brearley, Charles; Elge, Stephan; Kaplan, Boaz; Fromm, Hillel; Mueller-Roeber, Bernd

    2003-02-01

    Inositol 1,4,5-trisphosphate 3-kinase, and more generally inositol polyphosphate kinases (Ipk), play important roles in signal transduction in animal cells; however, their functions in plant cells remain to be elucidated. Here, we report the molecular cloning of a cDNA (AtIpk2beta) from a higher plant, Arabidopsis. Arabidopsis AtIpk2beta is a 33-kD protein that exhibits weak homology ( approximately 25% identical amino acids) with Ipk proteins from animals and yeast and lacks a calmodulin binding site, as revealed by sequence analysis and calmodulin binding assays. However, recombinant AtIpk2beta phosphorylates inositol 1,4,5-trisphosphate to inositol 1,4,5,6-tetrakisphosphate and also converts it to inositol 1,3,4,5,6-pentakisphosphate [Ins(1,3,4,5,6)P(5)]. AtIpk2beta also phosphorylates inositol 1,3,4,5-tetrakisphosphate to Ins(1,3,4,5,6)P(5). Thus, the enzyme is a D3/D6 dual-specificity inositol phosphate kinase. AtIpk2beta complements a yeast ARG82/IPK2 mutant lacking a functional ArgR-Mcm1 transcription complex. This complex is involved in regulating Arg metabolism-related gene expression and requires inositol polyphosphate kinase activity to function. AtIpk2beta was found to be located predominantly in the nucleus of plant cells, as demonstrated by immunolocalization and fusion to green fluorescent protein. RNA gel blot analysis and promoter-beta-glucuronidase reporter gene studies demonstrated AtIpk2beta gene expression in various organs tested. These data suggest a role for AtIpk2beta as a transcriptional control mediator in plants.

  1. Heat shock protein 90-mediated inactivation of nuclear factor-κB switches autophagy to apoptosis through becn1 transcriptional inhibition in selenite-induced NB4 cells.

    PubMed

    Jiang, Qian; Wang, Yuhan; Li, Tianjiao; Shi, Kejian; Li, Zhushi; Ma, Yushi; Li, Feng; Luo, Hui; Yang, Yang; Xu, Caimin

    2011-04-15

    Autophagy can protect cells while also contributing to cell damage, but the precise interplay between apoptosis and autophagy and the contribution of autophagy to cell death are still not clear. Previous studies have shown that supranutritional doses of sodium selenite promote apoptosis in human leukemia NB4 cells. Here, we report that selenite treatment triggers opposite patterns of autophagy in the NB4, HL60, and Jurkat leukemia cell lines during apoptosis and provide evidence that the suppressive effect of selenite on autophagy in NB4 cells is due to the decreased expression of the chaperone protein Hsp90 (heat shock protein 90), suggesting a novel regulatory function of Hsp90 in apoptosis and autophagy. Excessive or insufficient expression indicates that Hsp90 protects NB4 cells from selenite-induced apoptosis, and selenite-induced decreases in the expression of Hsp90, especially in NB4 cells, inhibit the activities of the IκB kinase/nuclear factor-κB (IKK/NF-κB) signaling pathway, leading to less nuclear translocation and inactivation of NF-κB and the subsequent weak binding of the becn1 promoter, which facilitates the transition from autophagy to apoptosis. Taken together, our observations provide novel insights into the mechanisms underlying the balance between apoptosis and autophagy, and we also identified Hsp90-NF-κB-Beclin1 as a potential biological pathway for signaling the switch from autophagy to apoptosis in selenite-treated NB4 cells.

  2. Targeting the Nuclear Cathepsin L CCAAT Displacement Protein/Cut Homeobox Transcription Factor-Epithelial Mesenchymal Transition Pathway in Prostate and Breast Cancer Cells with the Z-FY-CHO Inhibitor.

    PubMed

    Burton, Liza J; Dougan, Jodi; Jones, Jasmine; Smith, Bethany N; Randle, Diandra; Henderson, Veronica; Odero-Marah, Valerie A

    2017-03-01

    The epithelial mesenchymal transition (EMT) promotes tumor migration and invasion by downregulating epithelial markers such as E-cadherin and upregulating mesenchymal markers such as vimentin. Cathepsin L (Cat L) is a cysteine protease that can proteolytically activate CCAAT displacement protein/cut homeobox transcription factor (CUX1). We hypothesized that nuclear Cat L may promote EMT via CUX1 and that this could be antagonized with the Cat L-specific inhibitor Z-FY-CHO. Mesenchymal prostate (ARCaP-M and ARCaP-E overexpressing Snail) and breast (MDA-MB-468, MDA-MB-231, and MCF-7 overexpressing Snail) cancer cells expressed lower E-cadherin activity, higher Snail, vimentin, and Cat L activity, and a p110/p90 active CUX1 form, compared to epithelial prostate (ARCaP-E and ARCaP-Neo) and breast (MCF-7 and MCF-7 Neo) cancer cells. There was increased binding of CUX1 to Snail and the E-cadherin promoter in mesenchymal cells compared to epithelial prostate and breast cells. Treatment of mesenchymal cells with the Cat L inhibitor Z-FY-CHO led to nuclear-to-cytoplasmic relocalization of Cat L, decreased binding of CUX1 to Snail and the E-cadherin promoter, reversed EMT, and decreased cell migration/invasion. Overall, our novel data suggest that a positive feedback loop between Snail-nuclear Cat L-CUX1 drives EMT, which can be antagonized by Z-FY-CHO. Therefore, Z-FY-CHO may be an important therapeutic tool to antagonize EMT and cancer progression.

  3. The nuclear corepressors NCoR and SMRT are key regulators of both ligand- and 8-bromo-cyclic AMP-dependent transcriptional activity of the human progesterone receptor.

    PubMed

    Wagner, B L; Norris, J D; Knotts, T A; Weigel, N L; McDonnell, D P

    1998-03-01

    Previously, we defined a novel class of ligands for the human progesterone receptor (PR) which function as mixed agonists. These compounds induce a conformational change upon binding the receptor that is different from those induced by agonists and antagonists. This establishes a correlation between the structure of a ligand-receptor complex and its transcriptional activity. In an attempt to define the cellular components which distinguish between different ligand-induced PR conformations, we have determined, by using a mammalian two-hybrid assay, that the nuclear receptor corepressor (NCoR) and the silencing mediator for retinoid and thyroid hormone receptor (SMRT) differentially associate with PR depending upon the class of ligand bound to the receptor. Specifically, we observed that the corepressors preferentially associate with antagonist-occupied PR and that overexpression of these corepressors suppresses the partial agonist activity of antagonist-occupied PR. Binding studies performed in vitro, however, reveal that recombinant SMRT can interact with PR in a manner which is not influenced by the nature of the bound ligand. Thus, the inability of SMRT or NCoR to interact with agonist-activated PR when assayed in vivo may relate more to the increased affinity of PR for coactivators, with a subsequent displacement of corepressors, than to an inherent low affinity for the corepressor proteins. Previous work from other groups has shown that 8-bromo-cyclic AMP (8-bromo-cAMP) can convert the PR antagonist RU486 into an agonist and, additionally, can potentiate the transcriptional activity of agonist-bound PR. In this study, we show that exogenous expression of NCoR or SMRT suppresses all 8-bromo-cAMP-mediated potentiation of PR transcriptional activity. Further analysis revealed that 8-bromo-cAMP addition decreases the association of NCoR and SMRT with PR. Thus, we propose that 8-bromo-cAMP-mediated potentiation of PR transcriptional activity is due, at least in part

  4. The Nuclear Corepressors NCoR and SMRT Are Key Regulators of Both Ligand- and 8-Bromo-Cyclic AMP-Dependent Transcriptional Activity of the Human Progesterone Receptor

    PubMed Central

    Wagner, Brandee L.; Norris, John D.; Knotts, Trina A.; Weigel, Nancy L.; McDonnell, Donald P.

    1998-01-01

    Previously, we defined a novel class of ligands for the human progesterone receptor (PR) which function as mixed agonists. These compounds induce a conformational change upon binding the receptor that is different from those induced by agonists and antagonists. This establishes a correlation between the structure of a ligand-receptor complex and its transcriptional activity. In an attempt to define the cellular components which distinguish between different ligand-induced PR conformations, we have determined, by using a mammalian two-hybrid assay, that the nuclear receptor corepressor (NCoR) and the silencing mediator for retinoid and thyroid hormone receptor (SMRT) differentially associate with PR depending upon the class of ligand bound to the receptor. Specifically, we observed that the corepressors preferentially associate with antagonist-occupied PR and that overexpression of these corepressors suppresses the partial agonist activity of antagonist-occupied PR. Binding studies performed in vitro, however, reveal that recombinant SMRT can interact with PR in a manner which is not influenced by the nature of the bound ligand. Thus, the inability of SMRT or NCoR to interact with agonist-activated PR when assayed in vivo may relate more to the increased affinity of PR for coactivators, with a subsequent displacement of corepressors, than to an inherent low affinity for the corepressor proteins. Previous work from other groups has shown that 8-bromo-cyclic AMP (8-bromo-cAMP) can convert the PR antagonist RU486 into an agonist and, additionally, can potentiate the transcriptional activity of agonist-bound PR. In this study, we show that exogenous expression of NCoR or SMRT suppresses all 8-bromo-cAMP-mediated potentiation of PR transcriptional activity. Further analysis revealed that 8-bromo-cAMP addition decreases the association of NCoR and SMRT with PR. Thus, we propose that 8-bromo-cAMP-mediated potentiation of PR transcriptional activity is due, at least in part

  5. Vaccination inhibits TLR2 transcription via suppression of GR nuclear translocation and binding to TLR2 promoter in porcine lung infected with Mycoplasma hyopneumoniae.

    PubMed

    Sun, Zhiyuan; Liu, Maojun; Zou, Huafeng; Li, Xian; Shao, Guoqing; Zhao, Ruqian

    2013-12-27

    Toll-like receptors (TLRs) and glucocorticoid receptor (GR) act respectively as effectors of innate immune and stress responses. The crosstalk between them is critical for the maintenance of homeostasis during the immune response. Vaccination is known to boost adaptive immunity, yet it remains elusive whether vaccination may affect GR/TLR interactions following infection. Duroc×Meishan crossbred piglets were allocated to three groups. The control group (CC) received neither vaccination nor infection; the non-vaccinated infection group (NI) was artificially infected intratracheally with Mycoplasma hyopneumoniae (M. hyopneumoniae); while the vaccinated, infected group (VI) was vaccinated intramuscularly with inactivated M. hyopneumoniae one month before infection. The clinical signs and macroscopic lung lesions were significantly reduced by vaccination. However, vaccination did not affect the concentration of M. hyopneumoniae DNA in the lung. Serum cortisol was significantly decreased in both NI and VI pigs (P<0.01), but only VI pigs demonstrated significantly diminished nuclear GR content. TLRs 1-10 were all expressed in lung, among which TLR2 was the most abundant and was significantly up-regulated (P<0.05) in NI pigs, but not in VI pigs. Accordingly, GR binding to the GR response element on TLR2 promoter was significantly increased (P<0.05) in NI pigs, but not in VI pigs. These results suggest that the inhibition of GR nuclear translocation and binding to the TLR2 promoter, which results in diminished TLR2 expression, is associated with the protective effect of vaccination on M. hyopneumoniae-induced lung lesions in the pig.

  6. Increased Energy Expenditure, Ucp1 Expression, and Resistance to Diet-induced Obesity in Mice Lacking Nuclear Factor-Erythroid-2-related Transcription Factor-2 (Nrf2).

    PubMed

    Schneider, Kevin; Valdez, Joshua; Nguyen, Janice; Vawter, Marquis; Galke, Brandi; Kurtz, Theodore W; Chan, Jefferson Y

    2016-04-01

    The NRF2 (also known as NFE2L2) transcription factor is a critical regulator of genes involved in defense against oxidative stress. Previous studies suggest thatNrf2plays a role in adipogenesisin vitro, and deletion of theNrf2gene protects against diet-induced obesity in mice. Here, we demonstrate that resistance to diet-induced obesity inNrf2(-/-)mice is associated with a 20-30% increase in energy expenditure. Analysis of bioenergetics revealed thatNrf2(-/-)white adipose tissues exhibit greater oxygen consumption. White adipose tissue showed a >2-fold increase inUcp1gene expression. Oxygen consumption is also increased nearly 2.5-fold inNrf2-deficient fibroblasts. Oxidative stress induced by glucose oxidase resulted in increasedUcp1expression. Conversely, antioxidant chemicals (such asN-acetylcysteine and Mn(III)tetrakis(4-benzoic acid)porphyrin chloride) and SB203580 (a known suppressor ofUcp1expression) decreasedUcp1and oxygen consumption inNrf2-deficient fibroblasts. These findings suggest that increasing oxidative stress by limitingNrf2function in white adipocytes may be a novel means to modulate energy balance as a treatment of obesity and related clinical disorders.

  7. Studies on differential nuclear translocation mechanism and assembly of the three subunits of the Arabidopsis thaliana transcription factor NF-Y.

    PubMed

    Hackenberg, Dieter; Wu, Yanfang; Voigt, Andrea; Adams, Robert; Schramm, Peter; Grimm, Bernhard

    2012-07-01

    The eukaryotic transcription factor NF-Y consists of three subunits (A, B, and C), which are encoded in Arabidopsis thaliana in multigene families consisting of 10, 13, and 13 genes, respectively. In principle, all potential combinations of the subunits are possible for the assembly of the heterotrimeric complex. We aimed at assessing the probability of each subunit to participate in the assembly of NF-Y. The evaluation of physical interactions among all members of the NF-Y subunit families indicate a strong requirement for NF-YB/NF-YC heterodimerization before the entire complex can be accomplished. By means of a modified yeast two-hybrid system assembly of all three subunits to a heterotrimeric complex was demonstrated. Using GFP fusion constructs, NF-YA and NF-YC localization in the nucleus was demonstrated, while NF-YB is solely imported into the nucleus as a NF-YC-associated heterodimer NF-YC. This piggyback transport of the two Arabidopsis subunits differs from the import of the NF-Y heterotrimer of heterotrophic organisms. Based on a peptide structure model of the histone-fold-motifs, disulfide bonding among intramolecular conserved cysteine residues of NF-YB, which is responsible for the redox-regulated assembly of NF-YB and NF-YC in human and Aspergillus nidulans, can be excluded for Arabidopsis NF-YB.

  8. Nuclear localization of vascular endothelial growth factor-D and regulation of c-Myc-dependent transcripts in human lung fibroblasts.

    PubMed

    El-Chemaly, Souheil; Pacheco-Rodriguez, Gustavo; Malide, Daniela; Meza-Carmen, Victor; Kato, Jiro; Cui, Ye; Padilla, Philip I; Samidurai, Arun; Gochuico, Bernadette R; Moss, Joel

    2014-07-01

    Lymphangiogenesis and angiogenesis are processes that are, in part, regulated by vascular endothelial growth factor (VEGF)-D. The formation of lymphatic structures has been implicated in multiple lung diseases, including pulmonary fibrosis. VEGF-D is a secreted protein produced by fibroblasts and macrophages, which induces lymphangiogenesis by signaling via VEGF receptor-3, and angiogenesis through VEGF receptor-2. VEGF-D contains a central VEGF homology domain, which is the biologically active domain, with flanking N- and C-terminal propeptides. Full-length VEGF-D (∼ 50 kD) is proteolytically processed in the extracellular space, to generate VEGF homology domain that contains the VEGF-D receptor-binding sites. Here, we report that, independent of its cell surface receptors, full-length VEGF-D accumulated in nuclei of fibroblasts, and that this process appears to increase with cell density. In nuclei, full-length VEGF-D associated with RNA polymerase II and c-Myc. In cells depleted of VEGF-D, the transcriptionally regulated genes appear to be modulated by c-Myc. These findings have potential clinical implications, as VEGF-D was found in fibroblast nuclei in idiopathic pulmonary fibrosis, a disease characterized by fibroblast proliferation. These findings are consistent with actions of full-length VEGF-D in cellular homeostasis in health and disease, independent of its receptors.

  9. Inhibitory effects of black pepper (Piper nigrum) extracts and compounds on human tumor cell proliferation, cyclooxygenase enzymes, lipid peroxidation and nuclear transcription factor-kappa-B.

    PubMed

    Liu, Yunbao; Yadev, Vivek R; Aggarwal, Bharat B; Nair, Muraleedharan G

    2010-08-01

    Black pepper (Piper nigrum) and hot pepper (Capsicum spp.) are widely used in traditional medicines. Although hot Capsicum spp. extracts and its active principles, capsaicinoids, have been linked with anticancer and anti-inflammatory activities, whether black pepper and its active principle exhibit similar activities is not known. In this study, we have evaluated the antioxidant, anti-inflammatory and anticancer activities of extracts and compounds from black pepper by using proinflammatory transcription factor NF-kappaB, COX-1 and -2 enzymes, human tumor cell proliferation and lipid peroxidation (LPO). The capsaicinoids, the alkylamides, isolated from the hot pepper Scotch Bonnet were also used to compare the bioactivities of alkylamides and piperine from black pepper. All compounds derived from black pepper suppressed TNF-induced NF-kappaB activation, but alkyl amides, compound 4 from black pepper and 5 from hot pepper, were most effective. The human cancer cell proliferation inhibitory activities of piperine and alklyl amides in Capsicum and black pepper were dose dependant. The inhibitory concentrations 50% (IC50) of the alklylamides were in the range 13-200 microg/mL. The extracts of black pepper at 200 microg/mL and its compounds at 25 microg/mL inhibited LPO by 45-85%, COX enzymes by 31-80% and cancer cells proliferation by 3.5-86.8%. Overall, these results suggest that black pepper and its constituents like hot pepper, exhibit anti-inflammatory, antioxidant and anticancer activities.

  10. p14ARF post-transcriptional regulation of nuclear cyclin D1 in MCF-7 breast cancer cells: discrimination between a good and bad prognosis?

    PubMed

    McGowan, Eileen M; Tran, Nham; Alling, Nikki; Yagoub, Daniel; Sedger, Lisa M; Martiniello-Wilks, Rosetta

    2012-01-01

    As part of a cell's inherent protection against carcinogenesis, p14ARF is upregulated in response to hyperproliferative signalling to induce cell cycle arrest. This property makes p14ARF a leading candidate for cancer therapy. This study explores the consequences of reactivating p14ARF in breast cancer and the potential of targeting p14ARF in breast cancer treatment. Our results show that activation of the p14ARF-p53-p21-Rb pathway in the estrogen sensitive MCF-7 breast cancer cells induces many hallmarks of senescence including a large flat cell morphology, multinucleation, senescence-associated-β-gal staining, and rapid G1 and G2/M phase cell cycle arrest. P14ARF also induces the expression of the proto-oncogene cyclin D1, which is most often associated with a transition from G1-S phase and is highly expressed in breast cancers with poor clinical prognosis. In this study, siRNA knockdown of cyclin D1, p21 and p53 show p21 plays a pivotal role in the maintenance of high cyclin D1 expression, cell cycle and growth arrest post-p14ARF induction. High p53 and p14ARF expression and low p21/cyclin D1 did not cause cell-cycle arrest. Knockdown of cyclin D1 stops proliferation but does not reverse senescence-associated cell growth. Furthermore, cyclin D1 accumulation in the nucleus post-p14ARF activation correlated with a rapid loss of nucleolar Ki-67 protein and inhibition of DNA synthesis. Latent effects of the p14ARF-induced cellular processes resulting from high nuclear cyclin D1 accumulation included a redistribution of Ki-67 into the nucleoli, aberrant nuclear growth (multinucleation), and cell proliferation. Lastly, downregulation of cyclin D1 through inhibition of ER abrogated latent recurrence. The mediation of these latent effects by continuous expression of p14ARF further suggests a novel mechanism whereby dysregulation of cyclin D1 could have a double-edged effect. Our results suggest that p14ARF induced-senescence is related to late-onset breast cancer in

  11. Expression and secretion of chemotactic cytokines IL-8 and MCP-1 by human endothelial cells after Rickettsia rickettsii infection: regulation by nuclear transcription factor NF-kappaB.

    PubMed

    Clifton, Dawn R; Rydkina, Elena; Huyck, Heidie; Pryhuber, Gloria; Freeman, Robert S; Silverman, David J; Sahni, Sanjeev K

    2005-08-01

    Infection of endothelial cells (EC) with Rickettsia rickettsii results in Rocky Mountain spotted fever, an acute illness characterized by systemic inflammation. Interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) are important chemokines for activating neutrophils and monocytes, respectively, and recruiting these circulating immune cells to the sites of inflammation. In this study, we have measured the expression and secretion of these chemokines during R. rickettsii infection of cultured human EC. In comparison to uninfected controls, increased mRNA expression of IL-8 and MCP-1 in R. rickettsii-infected EC was evident as early as 3 h and was sustained up to 21 h. Subsequent analysis of culture supernatants revealed significantly enhanced secretion of both chemokines at 3, 8, and 18 h post-infection (5-28-fold increase in IL-8 and 4-16-fold increase in MCP-1). The presence of peptide-aldehyde compound MG132 to inhibit proteasome-mediated degradation of the inhibitory protein IkappaBalpha and synthetic peptide SN-50 to inhibit the nuclear translocation of nuclear factor-kappa B (NF-kappaB) resulted in significant inhibition of the chemokine response. Also, T24 cells expressing a super-repressor mutant of IkappaBalpha (to render NF-kappaB inactivatable) secreted significantly lower quantities of IL-8 than mock-transfected cells. A neutralizing antibody against IL-1alpha or an IL-1 specific receptor antagonist had no effect on the early phase of R. rickettsii-induced NF-kappaB activation and IL-8/ MCP-1 secretion at 3 h. Both of these treatments, however, diminished late-phase NF-kappaB activation by about 33% and only partially suppressed the infection-induced chemokine release at 21 h. Thus, while chemokine response early during the infection likely depends on the direct activation of NF-kappaB, subtle autocrine effects of newly synthesized IL-1alpha may contribute, in part, to the control of NF-kappaB activation and chemokine production at later

  12. Bisphenol A increases aP2 expression in 3T3L1 by enhancing the transcriptional activity of nuclear receptors at the promoter

    PubMed Central

    Atlas, Ella; Pope, Louise; Wade, Mike G; Kawata, Alice; Boudreau, Adele; Boucher, Jonathan G

    2014-01-01

    Environmental pollutants, such as bisphenol A (BPA), have the potential to affect the differentiation processes and the biology of the adipose tissue. The 3T3-L1 model is one of the murine cell models used extensively for the investigation of the molecular events that govern the differentiation of adipocytes from a committed preadipocyte to a mature, lipid laden adipocyte. Most of the studies investigating the effects of BPA on preadipocyte differentiation have investigated the effects of this chemical in the presence of an optimal differentiation cocktail containing high concentrations of the synthetic glucocorticoid dexamethasone, conditions that result in 90% to 100% of differentiated adipocytes. Our studies employed the 3T3-L1 cell model in the absence of exogenous glucocorticoids. We show that BPA is able to increase the differentiation of the 3T3-L1 cells under these conditions. Furthermore, the effect of BPA was observed in the absence of the synthetic glucocorticoid (dexamethasone), a hormone known to be required for the differentiation of the 3T3-L1 cells. In addition, BPA upregulated the mRNA expression and protein levels of the terminal marker of adipogenesis the fatty acid binding protein (aP2) in these cells. Interestingly, the known modulators of adipogenesis such as the peroxisome proliferator-activated receptor (PPAR) γ or CCAAT enhancer binding protein (C/EBP) α were not elevated at the mRNA or protein level in response to BPA. Furthermore, BPA upregulated the expression levels of the marker of adipogenesis aP2, through an effect on the transcriptional activity of C/EBPδ and the glucocorticoid receptor (GR) at its promoter. PMID:25068083

  13. Nuclear Factor of Activated T Cells-dependent Down-regulation of the Transcription Factor Glioma-associated Protein 1 (GLI1) Underlies the Growth Inhibitory Properties of Arachidonic Acid*

    PubMed Central

    Comba, Andrea; Almada, Luciana L.; Tolosa, Ezequiel J.; Iguchi, Eriko; Marks, David L.; Vara Messler, Marianela; Silva, Renata; Fernandez-Barrena, Maite G.; Enriquez-Hesles, Elisa; Vrabel, Anne L.; Botta, Bruno; Di Marcotulio, Lucia; Ellenrieder, Volker; Eynard, Aldo R.; Pasqualini, Maria E.; Fernandez-Zapico, Martin E.

    2016-01-01

    Numerous reports have demonstrated a tumor inhibitory effect of polyunsaturated fatty acids (PUFAs). However, the molecular mechanisms modulating this phenomenon are in part poorly understood. Here, we provide evidence of a novel antitumoral mechanism of the PUFA arachidonic acid (AA). In vivo and in vitro experiments showed that AA treatment decreased tumor growth and metastasis and increased apoptosis. Molecular analysis of this effect showed significantly reduced expression of a subset of antiapoptotic proteins, including BCL2, BFL1/A1, and 4-1BB, in AA-treated cells. We demonstrated that down-regulation of the transcription factor glioma-associated protein 1 (GLI1) in AA-treated cells is the underlying mechanism controlling BCL2, BFL1/A1, and 4-1BB expression. Using luciferase reporters, chromatin immunoprecipitation, and expression studies, we found that GLI1 binds to the promoter of these antiapoptotic molecules and regulates their expression and promoter activity. We provide evidence that AA-induced apoptosis and down-regulation of antiapoptotic genes can be inhibited by overexpressing GLI1 in AA-sensitive cells. Conversely, inhibition of GLI1 mimics AA treatments, leading to decreased tumor growth, cell viability, and expression of antiapoptotic molecules. Further characterization showed that AA represses GLI1 expression by stimulating nuclear translocation of NFATc1, which then binds the GLI1 promoter and represses its transcription. AA was shown to increase reactive oxygen species. Treatment with antioxidants impaired the AA-induced apoptosis and down-regulation of GLI1 and NFATc1 activation, indicating that NFATc1 activation and GLI1 repression require the generation of reactive oxygen species. Collectively, these results define a novel mechanism underlying AA antitumoral functions that may serve as a foundation for future PUFA-based therapeutic approaches. PMID:26601952

  14. Transcriptional up-regulation of nitric oxide synthase II by nuclear factor-kappaB at rostral ventrolateral medulla in a rat mevinphos intoxication model of brain stem death.

    PubMed

    Chan, Julie Y H; Wu, Carol H Y; Tsai, Ching-Yi; Cheng, Hsiao-Lei; Dai, Kuang-Yu; Chan, Samuel H H; Chang, Alice Y W

    2007-06-15

    As the origin of a 'life-and-death' signal that reflects central cardiovascular regulatory failure during brain stem death, the rostral ventrolateral medulla (RVLM) is a suitable neural substrate for mechanistic delineation of this vital phenomenon. Using a clinically relevant animal model that employed the organophosphate pesticide mevinphos (Mev) as the experimental insult, we evaluated the hypothesis that transcriptional up-regulation of nitric oxide synthase I or II (NOS I or II) gene expression by nuclear factor-kappaB (NF-kappaB) on activation of muscarinic receptors in the RVLM underlies brain stem death. In Sprague-Dawley rats maintained under propofol anaesthesia, co-microinjection of muscarinic M2R (methoctramine) or M4R (tropicamide), but not M1R (pirenzepine) or M3R (4-diphenylacetoxy-N-dimethylpiperidinium) antagonist significantly reduced the enhanced NOS I-protein kinase G signalling ('pro-life' phase) or augmented NOS II-peroxynitrite cascade ('pro-death' phase) in ventrolateral medulla, blunted the biphasic increase and decrease in baroreceptor reflex-mediated sympathetic vasomotor tone that reflect the transition from life to death, and diminished the elevated DNA binding activity or nucleus-bound translocation of NF-kappaB in RVLM neurons induced by microinjection of Mev into the bilateral RVLM. However, NF-kappaB inhibitors (diethyldithiocarbamate or pyrrolidine dithiocarbamate) or double-stranded kappaB decoy DNA preferentially antagonized the augmented NOS II-peroxynitrite cascade and the associated cardiovascular depression exhibited during the 'pro-death' phase. We conclude that transcriptional up-regulation of NOS II gene expression by activation of NF-kappaB on selective stimulation of muscarinic M2 or M4 subtype receptors in the RVLM underlies the elicited cardiovascular depression during the 'pro-death' phase in our Mev intoxication model of brain stem death.

  15. Transcription of mitochondrial DNA.

    PubMed

    Tabak, H F; Grivell, L A; Borst, P

    1983-01-01

    While mitochondrial DNA (mtDNA) is the simplest DNA in nature, coding for rRNAs and tRNAs, results of DNA sequence, and transcript analysis have demonstrated that both the synthesis and processing of mitochondrial RNAs involve remarkably intricate events. At one extreme, genes in animal mtDNAs are tightly packed, both DNA strands are completely transcribed (symmetric transcription), and the appearance of specific mRNAs is entirely dependent on processing at sites signalled by the sequences of the tRNAs, which abut virtually every gene. At the other extreme, gene organization in yeast (Saccharomyces) is anything but compact, with long stretches of AT-rich DNA interspaced between coding sequences and no obvious logic to the order of genes. Transcription is asymmetric and several RNAs are initiated de novo. Nevertheless, extensive RNA processing occurs due largely to the presence of split genes. RNA splicing is complex, is controlled by both mitochondrial and nuclear genes, and in some cases is accompanied by the formation of RNAs that behave as covalently closed circles. The present article reviews current knowledge of mitochondrial transcription and RNA processing in relation to possible mechanisms for the regulation of mitochondrial gene expression.

  16. Interaction of hepatocyte nuclear factors in transcriptional regulation of tissue specific hormonal expression of human multidrug resistance-associated protein 2 (abcc2)

    SciTech Connect

    Qadri, Ishtiaq Hu, L.-J.; Iwahashi, Mieko; Al-Zuabi, Subhi; Quattrochi, Linda C.; Simon, Francis R.

    2009-02-01

    Multidrug resistance-associated protein 2 (MRP2) (ABCC2) is an ATP-binding cassette membrane protein located primarily on apical surface of hepatocytes that mediates transport of conjugated xenobiotics and endogenous compounds into bile. MRP2 is highly expressed in hepatocytes, and at lower levels in small intestines, stomach and kidney. Previous reports have characterized mammalian MRP2 promoters, but none have established the molecular mechanism(s) involved in liver enriched expression. This study aims to investigate the mechanism of hepatic MRP2 regulation. A 2130 bp of MRP2 promoter was cloned from PAC-1 clone P108G1-7, to identify putative liver specific/hormone responsive functional DNA binding sites. Using deletion analysis, site specific mutagenesis and co-transfection studies, liver specific expression was determined. MRP2 promoter-LUC constructs were highly expressed in liver cell lines compared to non-liver cells. The region extending from - 3 to+ 458 bp of MRP2 promoter starting from AUG contained the potential binding sites for CAAATT box enhancer binding protein (C/EBP), hepatocytes nuclear factor 1, 3 and 4 (HNF1, HNF3, and HNF4. Only HNF1 and HNF4 co-transfection with MRP2 luciferase increased expression. Site specific mutational analysis of HNF1 binding site indicated an important role for HNF1{alpha}. HNF4{alpha} induction of MRP2 was independent of HNF1 binding site. C/EBP, HNF3, and HNF6 inhibited HNF1{alpha} while HNF4{alpha} induced MRP2 luciferase expression and glucocorticoids stimulated MRP2 expression. This study emphasizes the complex regulation of MRP2 with HNF1{alpha} and HNF4{alpha} playing a central role. The coordinated regulation of xenobiotic transporters and oxidative conjugation may determine the adaptive responses to cellular detoxification processes.

  17. Conditional Expression of Parkinson disease-related Mutant α-synuclein in the Midbrain Dopaminergic Neurons causes Progressive Neurodegeneration and Degradation of Transcription Factor Nuclear Receptor Related 1

    PubMed Central

    Lin, Xian; Parisiadou, Loukia; Sgobio, Carmelo; Liu, Guoxiang; Yu, Jia; Sun, Lixin; Shim, Hoon; Gu, Xing-Long; Luo, Jing; Long, Cai-Xia; Ding, Jinhui; Mateo, Yolanda; Sullivan, Patricia H.; Wu, Ling-Gang; Goldstein, David S.; Lovinger, David; Cai, Huaibin

    2012-01-01

    α-synuclein(α-syn) plays a prominent role in the degeneration of midbrain dopaminergic (mDA) neurons in Parkinson disease (PD). However, only a few studies on α-syn have been carried out in the mDA neurons in vivo, which may be attributed to a lack of α-syn transgenic mice that develop PD-like severe degeneration of mDA neurons. To gain mechanistic insights into the α-syn-induced mDA neurodegeneration, we generated a new line of tetracycline-regulated inducible transgenic mice that overexpressed the PD-related α-syn A53T missense mutation in the mDA neurons. Here we show that the mutant mice developed profound motor disabilities and robust mDA neurodegeneration, resembling some key motor and pathological phenotypes of PD. We further systematically examined the subcellular abnormalities appeared in the mDA neurons of mutant mice, and observed a profound decrease of dopamine release, the fragmentation of Golgi apparatus, and impairments of autophagy/lysosome degradation pathways in these neurons. To further understand the specific molecular events leading to the α-syn-dependent degeneration of mDA neurons, we found that over-expression of α-syn promoted a proteasome-dependent degradation of nuclear receptor related 1 protein (Nurr1); while inhibition of Nurr1 degradation ameliorated the α-syn-induced loss of mDA neurons. Given that Nurr1 plays an essential role in maintaining the normal function and survival of mDA neurons, our studies suggest that the α-syn-mediated suppression of Nurr1 protein expression may contribute to the preferential vulnerability of mDA neurons in the pathogenesis of PD. PMID:22764233

  18. Isobutyrylshikonin inhibits lipopolysaccharide-induced nitric oxide and prostaglandin E2 production in BV2 microglial cells by suppressing the PI3K/Akt-mediated nuclear transcription factor-κB pathway.

    PubMed

    Jayasooriya, Rajapaksha Gedara Prasad Tharanga; Lee, Kyoung-Tae; Kang, Chang-Hee; Dilshara, Matharage Gayani; Lee, Hak-Ju; Choi, Yung Hyun; Choi, Il-Whan; Kim, Gi-Young

    2014-12-01

    Microglia are important macrophages to defend against pathogens in the central nervous system (CNS); however, persistent or acute inflammation of microglia lead to CNS disorders via neuronal cell death. Therefore, we theorized that a good strategy for the treatment of CNS disorders would be to target inflammatory mediators from microglia in disease. Consequently, we investigated whether isobutyrylshikonin (IBS) attenuates the production of proinflammatory mediators, such as nitric oxide (NO) and prostaglandin E2, in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Treatment with IBS inhibited the secretion of NO and prostaglandin E2 (as well as the expression of their key regulatory genes), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2). Isobutyrylshikonin also suppressed LPS-induced DNA-binding activity of nuclear transcription factor-κB (NF-κB), by inhibiting the nuclear translocation of p50 and p65 in addition to blocking the phosphorylation and degradation of IκBα. Pretreatment with pyrrolidine dithiocarbamate, a specific NF-κB inhibitor, showed the down-regulation of LPS-induced iNOS and COX-2 messenger RNA by suppressing NF-κB activity. This indirectly suggests that IBS-mediated NF-κB inhibition is the main signaling pathway involved in the inhibition of iNOS and COX-2 expression. In addition, IBS attenuated LPS-induced phosphorylation of PI3K and Akt, which are upstream molecules of NF-κB, in LPS-stimulated BV2 microglial cells. The functional aspects of the PI3K/Akt signaling pathway were analyzed with LY294002, which is a specific PI3K/Akt inhibitor that attenuated LPS-induced iNOS and COX-2 expression by suppressing NF-κB activity. These data suggest that an IBS-mediated anti-inflammatory effect may be involved in suppressing the PI3K/Akt-mediated NF-κB signaling pathway.

  19. The transcription factor, Nuclear factor, erythroid 2 (Nfe2), is a regulator of the oxidative stress response during Danio rerio development

    PubMed Central

    Williams, Larissa M.; Lago, Briony A.; McArthur, Andrew G.; Raphenya, Amogelang R.; Pray, Nicholas; Saleem, Nabil; Salas, Sophia; Paulson, Katherine; Mangar, Roshni S.; Liu, Yang; Vo, Andy H.; Shavit, Jordan A.

    2017-01-01

    Development is a complex and well-defined process characterized by rapid cell proliferation and apoptosis. At this stage in life, a developmentally young organism is more sensitive to toxicants as compared to an adult. In response to pro-oxidant exposure, members of the Cap’n’Collar (CNC) basic leucine zipper (b-ZIP) transcription factor family (including Nfe2 and Nfe2-related factors, Nrfs) activate the expression of genes whose protein products contribute to reduced toxicity. Here, we studied the role of the CNC protein, Nfe2, in the developmental response to pro-oxidant exposure in the zebrafish (Danio rerio). Following acute waterborne exposures to diquat or tert-buytlhydroperoxide (tBOOH) at one of three developmental stages, wildtype (WT) and nfe2 knockout (KO) embryos and larvae were morphologically scored and their transcriptomes sequenced. Early in development, KO animals suffered from hypochromia that was made more severe through exposure to pro-oxidants; this phenotype in the KO may be linked to decreased expression of alas2, a gene involved in heme synthesis. WT and KO eleutheroembryos and larvae were phenotypically equally affected by exposure to pro-oxidants, where tBOOH caused more pronounced phenotypes as compared to diquat. Comparing diquat and tBOOH exposed embryos relative to the WT untreated control, a greater number of genes were up-regulated in the tBOOH condition as compared to diquat (tBOOH: 304 vs diquat: 148), including those commonly found to be differentially regulated in the vertebrate oxidative stress response (OSR) (e.g. hsp70.2, txn1, and gsr). When comparing WT and KO across all treatments and times, there were 1170 genes that were differentially expressed, of which 33 are known targets of the Nrf proteins Nrf1 and Nrf2. More specifically, in animals exposed to pro-oxidants a total of 968 genes were differentially expressed between WT and KO across developmental time, representing pathways involved in coagulation, embryonic

  20. Requirement of nuclear localization and transcriptional activity of p53 for its targeting to the yolk syncytial layer (YSL) nuclei in zebrafish embryo and its use for apoptosis assay

    SciTech Connect

    Chen, G.-D.; Chou, C.-M.; Hwang, S.-P.L.; Wang, F.-F.; Chen, Y.-C.; Hung, C.-C.; Chen, Jeou-Yuan . E-mail: bmchen@ibms.sinica.edu.tw; Huang, C.-J. . E-mail: cjibc@gate.sinica.edu.tw

    2006-05-26

    We expressed zebrafish p53 protein fused to GFP by a neuron-specific HuC promoter in zebrafish embryos. Instead of displaying neuronal expression patterns, p53-GFP was targeted to zebrafish YSL nuclei. This YSL targeting is p53 sequence-specific because GFP fusion proteins of p63 and p73 displayed neuronal-specific patterns. To dissect the underlying mechanisms, various constructs encoding a series of p53 mutant proteins under the control of different promoters were generated. Our results showed that expression of p53, in early zebrafish embryo, is preferentially targeted to the nuclei of YSL, which is mediated by importin. Similarly, this targeting is abrogated when p53 nuclear localization signal is disrupted. In addition, the transcriptional activity of p53 is required for this targeting. We further showed that fusion of pro-apoptotic BAD protein to p53-GFP led to apoptosis of YSL cells, and subsequent imperfect microtubule formation and abnormal blastomere movements.

  1. Vitamin A-dependent transcriptional activation of the nuclear factor of activated T cells c1 (NFATc1) is critical for the development and survival of B1 cells.

    PubMed

    Maruya, Mikako; Suzuki, Keiichiro; Fujimoto, Hanae; Miyajima, Michio; Kanagawa, Osami; Wakayama, Teruhiko; Fagarasan, Sidonia

    2011-01-11

    B1 cells represent a distinct subset of B cells that produce most of the natural serum IgM and much of the gut IgA and function as an important component of early immune responses to pathogens. The development of B1 cells depends on the nuclear factor of activated T cells c1 (NFATc1), a transcription factor abundantly expressed by B1 cells but not by conventional B2 cells. However, the factors that regulate the expression of NFATc1 in B1 cells remain unknown. Here we show that a vitamin A-deficient diet results in reduction of NFATc1 expression in B1 cells and almost complete loss of the B1 cell compartment. As a consequence, vitamin A-deficient mice have reduced serum IgM and are unable to mount T cell-independent antibody responses against bacterial antigens. We demonstrate that injection of all-trans retinoic acid induces the expression of NFATc1, particularly from the constitutive P2 promoter, and leads to the increase of the B1 cells. Thus, the retinoic acid-dependent pathway is critical for regulating NFATc1 expression and for maintenance of the natural memory B cell compartment.

  2. Expression of alpha V integrin is modulated by Epstein-Barr virus nuclear antigen 3C and the metastasis suppressor Nm23-H1 through interaction with the GATA-1 and Sp1 transcription factors

    SciTech Connect

    Choudhuri, Tathagata; Verma, Subhash C.; Lan, Ke; Robertson, Erle S. . E-mail: erle@mail.med.upenn.edu

    2006-07-20

    Epstein-Barr virus (EBV) is a lymphotrophic herpesvirus infecting most of the world's population. It is associated with a number of human lymphoid and epithelial tumors and lymphoproliferative diseases in immunocompromised patients. A subset of latent EBV antigens is required for immortalization of primary B-lymphocytes. The metastatic suppressor Nm23-H1 which is downregulated in human invasive breast carcinoma reduces the migration and metastatic activity of breast carcinoma cells when expressed from a heterologous promoter. Interestingly, the EBV nuclear antigen 3C (EBNA3C) reverses these activities of Nm23-H1. The alpha V integrins recognize a variety of ligands for signaling and are involved in cell migration and proliferation and also serve as major receptors for extracellular-matrix-mediated cell adhesion and migration. The goal of this study was to determine if Nm23-H1 and EBNA3C can modulate alpha V integrin expression and downstream activities. The results of our studies indicate that Nm23-H1 downregulates alpha V intregrin expression in a dose responsive manner. In contrast, EBNA3C can upregulate alpha V integrin expression. Furthermore, the study showed that the association of the Sp1 and GATA transcription factors with Nm23-H1 is required for modulation of the alpha V integrin activity. Thus, these results suggest a direct correlation between the alpha V integrin expression and the interaction of Nm23-H1 with EBNA3C.

  3. Genome-Wide Mapping of Uncapped and Cleaved Transcripts Reveals a Role for the Nuclear mRNA Cap-Binding Complex in Cotranslational RNA Decay in Arabidopsis[OPEN

    PubMed Central

    Willmann, Matthew R.

    2016-01-01

    RNA turnover is necessary for controlling proper mRNA levels posttranscriptionally. In general, RNA degradation is via exoribonucleases that degrade RNA either from the 5′ end to the 3′ end, such as XRN4, or in the opposite direction by the multisubunit exosome complex. Here, we use genome-wide mapping of uncapped and cleaved transcripts to reveal the global landscape of cotranslational mRNA decay in the Arabidopsis thaliana transcriptome. We found that this process leaves a clear three nucleotide periodicity in open reading frames. This pattern of cotranslational degradation is especially evident near the ends of open reading frames, where we observe accumulation of cleavage events focused 16 to 17 nucleotides upstream of the stop codon because of ribosomal pausing during translation termination. Following treatment of Arabidopsis plants with the translation inhibitor cycloheximide, cleavage events accumulate 13 to 14 nucleotides upstream of the start codon where initiating ribosomes have been stalled with these sequences in their P site. Further analysis in xrn4 mutant plants indicates that cotranslational RNA decay is XRN4 dependent. Additionally, studies in plants lacking CAP BINDING PROTEIN80/ABA HYPERSENSITIVE1, the largest subunit of the nuclear mRNA cap binding complex, reveal a role for this protein in cotranslational decay. In total, our results demonstrate the global prevalence and features of cotranslational RNA decay in a plant transcriptome. PMID:27758893

  4. Nucleolar localization of myc transcripts.

    PubMed Central

    Bond, V C; Wold, B

    1993-01-01

    In situ hybridization has revealed a striking subnuclear distribution of c-myc RNA transcripts. A major fraction of the sense-strand nuclear c-myc transcripts was localized to the nucleoli. myc intron 1-containing RNAs were noticeably absent from nucleoli, accumulating instead in the nucleoplasm. The localization of myc RNA to nucleoli was shown to be common to a number of diverse cell types, including primary Sertoli cells and several cell lines. Furthermore, nucleolar localization was not restricted to c-myc and N-myc and myoD transcripts also displayed this phenomenon. In contrast, gamma-actin or lactate dehydrogenase transcripts did not display nucleolar localization. These observations suggest a new role for the nucleolus in transport and/or turnover of potential mRNAs. Images PMID:7684491

  5. Analysis of nuclear reconstitution, nuclear envelope assembly, and nuclear pore assembly using Xenopus in vitro assays.

    PubMed

    Bernis, Cyril; Forbes, Douglass J

    2014-01-01

    The large and complex eukaryotic nucleus is the arbiter of DNA replication, RNA transcription, splicing, and ribosome assembly. With the advent of in vitro nuclear reconstitution extracts derived from Xenopus eggs in the 1980s, it became possible to assemble multiple nuclei in vitro around added DNA or chromatin substrates. Such reconstituted nuclei contain a nuclear lamina, double nuclear membranes, nuclear pores, and are competent for DNA replication and nuclear import. In vitro nuclear reconstitution has allowed the assembly of "wild-type" and "biochemically mutant" nuclei in which the impact of individual components can be assessed. Here, we describe protocols for preparation of the nuclear reconstitution extract, nuclear reconstitution in vitro, assessment of nuclear membrane integrity, and a more specialized assay for nuclear pore assembly into preformed pore-free nuclear intermediates.

  6. Analysis of Nuclear Reconstitution, Nuclear Envelope Assembly, and Nuclear Pore Assembly Using Xenopus In Vitro Assays

    PubMed Central

    Bernis, Cyril; Forbes, Douglass J.

    2015-01-01

    The large and complex eukaryotic nucleus is the arbiter of DNA replication, RNA transcription, splicing, and ribosome assembly. With the advent of in vitro nuclear reconstitution extracts derived from Xenopus eggs in the 1980s, it became possible to assemble multiple nuclei in vitro around added DNA or chromatin substrates. Such reconstituted nuclei contain a nuclear lamina, double nuclear membranes, nuclear pores, and are competent for DNA replication and nuclear import. In vitro nuclear reconstitution has allowed the assembly of “wild-type” and “biochemically mutant” nuclei in which the impact of individual components can be assessed. Here, we describe protocols for preparation of the nuclear reconstitution extract, nuclear reconstitution in vitro, assessment of nuclear membrane integrity, and a more specialized assay for nuclear pore assembly into preformed pore-free nuclear intermediates. PMID:24857730

  7. The human clotting factor VIII cDNA contains an autonomously replicating sequence consensus- and matrix attachment region-like sequence that binds a nuclear factor, represses heterologous gene expression, and mediates the transcriptional effects of sodium butyrate.

    PubMed Central

    Fallaux, F J; Hoeben, R C; Cramer, S J; van den Wollenberg, D J; Briët, E; van Ormondt, H; van Der Eb, A J

    1996-01-01

    Expression of the human blood-clotting factor VIII (FVIII) cDNA is hampered by the presence of sequences located in the coding region that repress transcription. We have previously identified a 305-bp fragment within the FVIII cDNA that is involved in the repression (R.C. Hoeben, F.J. Fallaux, S.J. Cramer, D.J.M. van den Wollenberg, H. van Ormondt, E. Briet, and A.J. van der Eb, Blood 85:2447-2454, 1995). Here, we show that this 305-bp region of FVIII cDNA contains sequences that resemble the yeast (Saccharomyces cerevisiae) autonomously replicating sequence consensus. Two of these DNA elements coincide with AT-rich sequences that are often found in matrix attachment regions or scaffold-attached regions. One of these elements, consisting of nucleotides 1569 to 1600 of the FVIII cDNA (nucleotide numbering is according to the system of Wood et al. (W.I. Wood, D.J. Capon, C.C. Simonsen, D.L. Eaton, J. Gitschier, D. Keyt, P.H. Seeburg, D.H. Smith, P. Hollingshead, K.L. Wion, et al., Nature [London] 312:330-337,1984), binds a nuclear factor in vitro but loses this capacity after four of its base pairs have been changed. A synthetic heptamer of this segment can repress the expression of a chloramphenicol acetyltransferase (CAT) reporter gene and also loses this capacity upon mutation. Furthermore, we demonstrate that repression by FVIII sequences can be relieved by sodium butyrate. We demonstrate that the synthetic heptamer (FVIII nucleotides 1569 to 1600), when placed upstream of the Moloney murine leukemia virus long terminal repeat promoter that drives the CAT reporter, can render the CAT reporter inducible by butyrate. This effect was absent when the same element was mutated. The stimulatory effect of butyrate could not be attributed to butyrate-responsive elements in the studied long terminal repeat promoters. Our data provide a functional characterization of the sequences that repress expression of the FVIII cDNA. These data also suggest a link between

  8. Comparison of proliferating cell nuclear antigen, thyroid transcription factor-1, Ki-67, p63, p53 and high-molecular weight cytokeratin expressions in papillary thyroid carcinoma, follicular carcinoma, and follicular adenoma.

    PubMed

    Tan, Ayca; Etit, Demet; Bayol, Umit; Altinel, Deniz; Tan, Sedat

    2011-04-01

    The searching of the reliable and repeatable immunohistochemical markers in the differential diagnosis of the thyroid's differentiated follicular epithelial neoplasms has been continuing. Recently, the studies have majored on immunohistochemical markers such as high-molecular weight cytokeratin (HMW-CK), galectin-3, cytokeratin 19, and p27. We aimed to evaluate the differences of the expressions of the proliferating cell nuclear antigen (PCNA), thyroid transcription factor-1 (TTF-1), Ki-67, p63, p53, and HMW-CK among the papillary thyroid carcinomas (PTCs), follicular carcinomas (FCs), and follicular adenomas (FAs). Thirty-nine patients with the diagnoses of the PTC, FC, and FA in the archives of the Izmir Tepecik Training and Research Hospital Pathology Laboratory registries in between 2004 and 2009 were included in the study. Immunohistochemical stains for PCNA, TTF-1, Ki-67, p63, p53, and HMW-CK were applied. The results were analyzed statistically by using Statistical Package for the Social Sciences (SPSS) for Windows 16.0 program (SPSS Inc., IBM, Somers, New York, USA). In all 3 groups, all tumors showed PCNA and TTF-1 positivity. Ki-67 proliferation index varied in a wide range in all groups. Although it was not statistically significant, 19 of 39 tumors (7 PTCs, 2 FCs, and 10 FAs) were stained with p63. The results of the immunoreactivity seen in PTCs with p53 (41.2%) and HMW-CK (52.9%) were statistically significant. The tumors in the other 2 groups (FC and FA) showed no reactivity with HMW-CK. Although the differential diagnosis of the thyroid follicular neoplasms are based on the histologic and cytomorphological criteria, p53 and HMW-CK positivity might be undertaken in favor of the diagnosis of the PTC.

  9. A diarylheptanoid from lesser galangal (Alpinia officinarum) inhibits proinflammatory mediators via inhibition of mitogen-activated protein kinase, p44/42, and transcription factor nuclear factor-kappa B.

    PubMed

    Yadav, Prem N; Liu, Zhihua; Rafi, Mohamed M

    2003-06-01

    The diarylheptanoid 7-(4'-hydroxy-3'-methoxyphenyl)-1-phenylhept-4-en-3-one (HMP) is a naturally occurring phytochemical found in lesser galangal (Alpinia officinarum). In the present study, we have demonstrated the anti-inflammatory properties of this compound on mouse macrophage cell line (RAW 264.7) and human peripheral blood mononuclear cells (PBMCs) in vitro. Treatment of RAW 264.7 cells with HMP (6.25-25 microM) significantly inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production. This compound also inhibited the release of LPS-induced proinflammatory cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) from human PB-MCs in vitro. In addition, Western blotting and reverse transcription-polymerase chain reaction analysis demonstrated that HMP decreased LPS-induced inducible nitric-oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein and mRNA expression in RAW 264.7 cells. Furthermore, HMP treatment also reduced nuclear factor-kappa B (NF-kappa B) DNA binding induced by LPS in RAW 264.7 cells. To elucidate the molecular mechanism for inhibition of proinflammatory mediators by HMP (25 microM), we have studied the effect of HMP on LPS-induced p38 and p44/42 mitogen-activated protein kinase (MAPK). We observed that the phosphorylation of p44/42 MAPK in LPS-stimulated RAW 264.7 cells was markedly inhibited by HMP, whereas activation of p38 MAPK was not affected. These results suggested that HMP from lesser galangal suppressed the LPS-induced production of NO, IL-1 beta, and TNF-alpha and expression of iNOS and COX-2 gene expression by inhibiting NF-kappa B activation and phosphorylation of p44/42 MAPK.

  10. Activation of the transcription factor nuclear factor-kappa B in uterine luminal epithelial cells by interleukin 1 Beta 2: a novel interleukin 1 expressed by the elongating pig conceptus.

    PubMed

    Mathew, Daniel J; Newsom, Emily M; Guyton, Jennifer M; Tuggle, Christopher K; Geisert, Rodney D; Lucy, Matthew C

    2015-04-01

    Conceptus mortality is greatest in mammals during the peri-implantation period, a time when conceptuses appose and attach to the uterine surface epithelium while releasing proinflammatory molecules. Interleukin 1 beta (IL1B), a master proinflammatory cytokine, is released by the primate, rodent, and pig blastocyst during the peri-implantation period and is believed to be essential for establishment of pregnancy. The gene encoding IL1B has duplicated in the pig, resulting in a novel gene. Preliminary observations indicate that the novel IL1B is specifically expressed by pig conceptuses during the peri-implantation period. To verify this, IL1B was cloned from mRNA isolated from Day 12 pig conceptuses and compared with IL1B cloned from mRNA isolated from pig peripheral blood leukocytes (PBLs). The pig conceptuses, but not the PBLs, expressed a novel IL1B, referred to here as interleukin 1 beta 2 (IL1B2). Porcine endometrium was treated with recombinant porcine interleukin 1 beta 1 (IL1B1), the prototypical cytokine, and IL1B2 proteins. Immunohistochemistry and real-time RT-PCR were used to measure activation of nuclear factor-kappa B (NFKB) and NFKB-regulated transcripts, respectively, within the endometrium. Both IL1B1 and IL1B2 activated NFKB in the uterine luminal epithelium within 4 h. The NFKB activation and related gene expression, however, were lower in endometrium treated with IL1B2, suggesting that the conceptus-derived cytokine may have reduced activity within the uterus. In conclusion, the peri-implantation pig conceptus expresses a novel IL1B that can activate NFKB within the uterine surface epithelium, likely creating a proinflammatory microenvironment during establishment of pregnancy in the pig.

  11. Basiliolides, a class of tetracyclic C19 dilactones from Thapsia garganica, release Ca(2+) from the endoplasmic reticulum and regulate the activity of the transcription factors nuclear factor of activated T cells, nuclear factor-kappaB, and activator protein 1 in T lymphocytes.

    PubMed

    Navarrete, Carmen; Sancho, Rocío; Caballero, Francisco J; Pollastro, Federica; Fiebich, Bernd L; Sterner, Olov; Appendino, Giovanni; Muñoz, Eduardo

    2006-10-01

    Calcium concentration within the endoplasmic reticulum (ER) plays an essential role in cell physiology. We have investigated the effects of basiliolides, a novel class of C19 dilactones isolated from Thapsia garganica, on Ca(2+) mobilization in T cells. Basiliolide A1 induced a rapid mobilization of intracellular Ca(2+) in the leukemia T-cell line Jurkat. First, a rapid calcium peak was observed and inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester. This initial calcium mobilization was followed by a sustained elevation, mediated by the entry of extracellular calcium through store-operated calcium release-activated Ca(2+) (CRAC) channels and sensitive to inhibition by EGTA, and by the CRAC channel inhibitor N-{4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP-2). Basiliolide A1 mobilized Ca(2+) from ER stores, but in contrast to thapsigargin, it did not induce apoptosis. Basiliolide A1 induced nuclear factor of activated T cells 1 dephosphorylation and activation that was inhibited by BTP-2 and cyclosporine A. In addition, we found that basiliolide A1 alone did not mediate IkappaBalpha degradation or RelA phosphorylation (ser536), but it synergized with phorbol 12-myristate 13-acetate to induce a complete degradation of the nuclear factor-kappaB inhibitory protein and to activate the c-Jun NH(2)-terminal kinase. Moreover, basiliolide A1 regulated both interleukin-2 and tumor necrosis factor-alpha gene expression at the transcriptional level. In basiliolide B, oxidation of one of the two geminal methyls to a carboxymethyl group retained most of the activity of basiliolide A1. In contrast, basiliolide C, where the 15-carbon is oxidized to an acetoxymethine, was much less active. These findings qualify these compounds as new probes to investigate intracellular calcium homeostasis.

  12. Transcriptional control of plant defence responses.

    PubMed

    Buscaill, Pierre; Rivas, Susana

    2014-08-01

    Mounting of efficient plant defence responses depends on the ability to trigger a rapid defence reaction after recognition of the invading microbe. Activation of plant resistance is achieved by modulation of the activity of multiple transcriptional regulators, both DNA-binding transcription factors and their regulatory proteins, that are able to reprogram transcription in the plant cell towards the activation of defence signalling. Here we provide an overview of recent developments on the transcriptional control of plant defence responses and discuss defence-related hormone signalling, the role of WRKY transcription factors during the regulation of plant responses to pathogens, nuclear functions of plant immune receptor proteins, as well as varied ways by which microbial effectors subvert plant transcriptional reprogramming to promote disease.

  13. Heme and heme biosynthesis intermediates induce heme oxygenase-1 and cytochrome P450 2A5, enzymes with putative sequential roles in heme and bilirubin metabolism: different requirement for transcription factor nuclear factor erythroid- derived 2-like 2.

    PubMed

    Lämsä, Virpi; Levonen, Anna-Liisa; Sormunen, Raija; Yamamoto, Masayuki; Hakkola, Jukka

    2012-11-01

    Cytochrome P450 2A5 (CYP2A5) oxidizes bilirubin to biliverdin and represents a putative candidate for maintaining bilirubin at safe but adequate antioxidant levels. Curiously, CYP2A5 is induced by both excessive heme and chemicals that inhibit heme synthesis. We hypothesized that heme homeostasis is a key modifier of Cyp2a5 expression via transcription factor nuclear factor erythroid-derived 2-like 2 (Nrf2) and characterized the coordination of CYP2A5 and heme oxygenase-1 (HMOX1) responses using wild-type and Nrf2(-/-) primary mouse hepatocytes. HMOX1 was rapidly elevated by exogenous hemin, thereby limiting the transactivation of Cyp2a5 until high heme (> 5µM) exposure. Nrf2 was mandatory for CYP2A5 but not for HMOX1 induction by heme. CYP2A5 was intensively and HMOX1 moderately elevated in heme synthesis blockades by succinylacetone and N-methyl protoporphyrin IX, and Nrf2 partially mediated the induction of CYP2A5. Immunoelectron microscopy revealed that CYP2A5 is targeted Nrf2 dependently both to the endoplasmic reticulum (ER) and mitochondria. However, excessive heme increased CYP2A5 predominantly in the ER. Phenobarbital, dibutyryl-cAMP, and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) overexpression stimulate heme biosynthesis and induce CYP2A5. Acute but not chronic CYP2A5 induction by phenobarbital required Nrf2, whereas CYP2A5 induction by dibutyryl-cAMP and PGC-1α was potentiated by Nrf2 knockout. Collectively, heme homeostasis is established as a crucial regulator of hepatic Cyp2a5 expression mediated via Nrf2 activation, whereas Nrf2 is redundant for Hmox1 induction by heme. Similar subcellular targeting and coordination of CYP2A5 and HMOX1 responses suggest favorable conditions for enhanced CYP2A5-mediated bilirubin maintenance in altered heme homeostasis that predisposes to oxidative stress.

  14. Effect of in vitro administered 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on DNA-binding activities of nuclear transcription factors in NIH-3T3 mouse fibroblasts.

    PubMed

    Hwang, Seong Gu; Sasagawa, Hiromi; Matsumura, Fumio

    2007-01-01

    TCDD is a very toxic environmental contaminant which is known to cause a variety of toxic symptoms in many species. Because of a myriad of biochemical changes TCDD is known to induce in many test animals, it has been difficult to pinpoint the causative event common to all those symptoms in different species. One of the research avenues we have been following is identification of the pattern of TCDD-induced changes in DNA binding characteristics of nuclear transcription factors (NTFs), each of which has the property to trigger a set of coordinated changes in many gene expressions. Since in our previous work we studied animals affected by TCDD in vivo using gel mobility shift assay approached with32P labeled oligonucleotide probes, we examined in the current study the possibility whether we could establish an equivalent in vitro system in NIH-3T3 mouse fibroblast cells so as to be able to learn the similarities and the dissimilarities of TCDD-induced responses of NTFs between in vitro and in vivo. The results indicated that, for a large part, this in vitro test system could reasonably reproduce the pattern of changes occurring in vivo at the early stages of TCDD's action in terms of induced changes in binding of thes NTSs to DNA. The key features were TCDD induced upregulation of NTFs binding to the response elements for AP-1, dioxin (DRE) and T3 (thyroid hormone) and down-regulation of those to response elements (REs) for c-Myc, Sp-1 and retinoic acid receptor (RARE). However, the time course required the changes in DNA binding activity was much shorter in vitro. To study the basic cause for such changes in NTF binding, we studied the effects of exogenously added EGF, forskolin, TPA (12-0-tetradecanoylphorbol acetate) and TNFalpha on the expression of TCDD's action on some of these NTFs. The results showed that these agents indeed greatly influence the outcome. The most influential agents were TNFalpha, forskolin and EGF. These results indicate that this in vitro

  15. Nuclear RNA Isolation and Sequencing.

    PubMed

    Dhaliwal, Navroop K; Mitchell, Jennifer A

    2016-01-01

    Most transcriptome studies involve sequencing and quantification of steady-state mRNA by isolating and sequencing poly (A) RNA. Although this type of sequencing data is informative to determine steady-state mRNA levels it does not provide information on transcriptional output and thus may not always reflect changes in transcriptional regulation of gene expression. Furthermore, sequencing poly (A) RNA may miss transcribed regions of the genome not usually modified by polyadenylation which includes many long noncoding RNAs. Here, we describe nuclear-RNA sequencing (nucRNA-seq) which investigates the transcriptional landscape through sequencing and quantification of nuclear RNAs which are both unspliced and spliced transcripts for protein-coding genes and nuclear-retained long noncoding RNAs.

  16. Transcription by RNA polymerases I and III

    PubMed Central

    Paule, Marvin R.; White, Robert J.

    2000-01-01

    The task of transcribing nuclear genes is shared between three RNA polymerases in eukaryotes: RNA polymerase (pol) I synthesises the large rRNA, pol II synthesises mRNA and pol III synthesises tRNA and 5S rRNA. Although pol II has received most attention, pol I and pol III are together responsible for the bulk of transcriptional activity. This survey will summarise what is known about the process of transcription by pol I and pol III, how it happens and the proteins involved. Attention will be drawn to the similarities between the three nuclear RNA polymerase systems and also to their differences. PMID:10684922

  17. Sry is a transcriptional activator.

    PubMed

    Dubin, R A; Ostrer, H

    1994-09-01

    The SRY gene functions as a genetic switch in gonadal ridge initiating testis determination. The mouse Sry and human SRY open reading frames (ORFs) share a conserved DNA-binding domain (the HMG-box) yet exhibit no additional homology outside this region. As judged by the accumulation of lacZ-SRY hybrid proteins in the nucleus, both the human and mouse SRY ORFs contain a nuclear localization signal. The mouse Sry HMG-box domain selectively binds the sequence NACAAT in vitro when challenged with a random pool of oligonucleotides and binds AACAAT with the highest affinity. When put under the control of a heterologous promotor, the mouse Sry gene activated transcription of a reporter gene containing multiple copies of the AACAAT binding site. Activation was likewise observed for a GAL4-responsive reporter gene, when the mouse Sry gene was linked to the DNA-binding domain of GAL4. Using this system, the activation function was mapped to a glutamine/histidine-rich domain. In addition, LexA-mouse Sry fusion genes activated a LexA-responsive reporter gene in yeast. In contrast, a GAL4-human SRY fusion gene did not cause transcriptional activation. These studies suggest that both the human and the mouse SRY ORFs encode nuclear, DNA-binding proteins and that the mouse Sry ORF can function as a transcriptional activator with separable DNA-binding and activator domains.

  18. Nuclear rights - nuclear wrongs

    SciTech Connect

    Paul, E.F.; Miller, F.D.; Paul, J.; Ahrens, J.

    1986-01-01

    This book contains 11 selections. The titles are: Three Ways to Kill Innocent Bystanders: Some Conundrums Concerning the Morality of War; The International Defense of Liberty; Two Concepts of Deterrence; Nuclear Deterrence and Arms Control; Ethical Issues for the 1980s; The Moral Status of Nuclear Deterrent Threats; Optimal Deterrence; Morality and Paradoxical Deterrence; Immoral Risks: A Deontological Critique of Nuclear Deterrence; No War Without Dictatorship, No Peace Without Democracy: Foreign Policy as Domestic Politics; Marxism-Leninism and its Strategic Implications for the United States; Tocqueveille War.

  19. Targeting Transcription Factors in Cancer

    PubMed Central

    Bhagwat, Anand S.; Vakoc, Christopher R.

    2015-01-01

    Transcription factors (TFs) are commonly deregulated in the pathogenesis of human cancer and are a major class of cancer cell dependencies. Consequently, targeting of TFs can be highly effective in treating particular malignancies, as highlighted by the clinical efficacy of agents that target nuclear hormone receptors. In this review we discuss recent advances in our understanding of TFs as drug targets in oncology, with an emphasis on the emerging chemical approaches to modulate TF function. The remarkable diversity and potency of TFs as drivers of cell transformation justifies a continued pursuit of TFs as therapeutic targets for drug discovery. PMID:26645049

  20. Epicatechin induces NF-kappaB, activator protein-1 (AP-1) and nuclear transcription factor erythroid 2p45-related factor-2 (Nrf2) via phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) and extracellular regulated kinase (ERK) signalling in HepG2 cells.

    PubMed

    Granado-Serrano, Ana Belén; Martín, María Angeles; Haegeman, Guy; Goya, Luis; Bravo, Laura; Ramos, Sonia

    2010-01-01

    The dietary flavonoid epicatechin has been reported to exhibit a wide range of biological activities. The objective of the present study was to investigate the time-dependent regulation by epicatechin on the activity of the main transcription factors (NF-kappaB, activator protein-1 (AP-1) and nuclear transcription factor erythroid 2p45-related factor (Nrf2)) related to antioxidant defence and survival and proliferation pathways in HepG2 cells. Treatment of cells with 10 microm-epicatechin induced the NF-kappaB pathway in a time-dependent manner characterised by increased levels of IkappaB kinase (IKK) and phosphorylated inhibitor of kappaB subunit-alpha (p-IkappaBalpha) and proteolytic degradation of IkappaB, which was consistent with an up-regulation of the NF-kappaB-binding activity. Time-dependent activation of the AP-1 pathway, in concert with enhanced c-Jun nuclear levels and induction of Nrf2 translocation and phosphorylation were also demonstrated. Additionally, epicatechin-induced NF-kappaB and Nrf2 were connected to reactive oxygen species intracellular levels and to the activation of cell survival and proliferation pathways, being phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) and extracellular regulated kinase (ERK) associated to Nrf2 modulation and ERK to NF-kappaB induction. These data suggest that the epicatechin-induced survival effect occurs by the induction of redox-sensitive transcription factors through a tight regulation of survival and proliferation pathways.

  1. Nuclear Medicine.

    ERIC Educational Resources Information Center

    Badawi, Ramsey D.

    2001-01-01

    Describes the use of nuclear medicine techniques in diagnosis and therapy. Describes instrumentation in diagnostic nuclear medicine and predicts future trends in nuclear medicine imaging technology. (Author/MM)

  2. Transcriptional enhancers: Transcription, function and flexibility.

    PubMed

    Melamed, Philippa; Yosefzon, Yahav; Rudnizky, Sergei; Pnueli, Lilach

    2016-01-01

    Active transcriptional enhancers are often transcribed to eRNAs, whose changing levels mirror those of the target gene mRNA. We discuss some of the reported functions of these eRNAs and their likely diversity to allow utilization of distinct cis regulatory regions to enhance transcription in diverse developmental and cellular contexts.

  3. Transcription in archaea

    NASA Technical Reports Server (NTRS)

    Kyrpides, N. C.; Ouzounis, C. A.; Woese, C. R. (Principal Investigator)

    1999-01-01

    Using the sequences of all the known transcription-associated proteins from Bacteria and Eucarya (a total of 4,147), we have identified their homologous counterparts in the four complete archaeal genomes. Through extensive sequence comparisons, we establish the presence of 280 predicted transcription factors or transcription-associated proteins in the four archaeal genomes, of which 168 have homologs only in Bacteria, 51 have homologs only in Eucarya, and the remaining 61 have homologs in both phylogenetic domains. Although bacterial and eukaryotic transcription have very few factors in common, each exclusively shares a significantly greater number with the Archaea, especially the Bacteria. This last fact contrasts with the obvious close relationship between the archaeal and eukaryotic transcription mechanisms per se, and in particular, basic transcription initiation. We interpret these results to mean that the archaeal transcription system has retained more ancestral characteristics than have the transcription mechanisms in either of the other two domains.

  4. SUMOylation of ROR{alpha} potentiates transcriptional activation function

    SciTech Connect

    Hwang, Eun Ju; Lee, Ji Min; Jeong, Jiyeong; Park, Joo Hyeon; Yang, Young; Lim, Jong-Seok; Kim, Jung Hwa; Baek, Sung Hee; Kim, Keun Il

    2009-01-16

    SUMOylation regulates a variety of cellular processes, including control of transcriptional activities of nuclear receptors. Here, we present SUMOylation of orphan nuclear receptor, ROR{alpha} by both SUMO-1 and SUMO-2. SUMOylation of ROR{alpha} occurred on the 240th lysine residue at the hinge region of human protein. PIAS family members, PIASx{alpha}, PIAS3, and PIASy, increased SUMOylation of ROR{alpha}, whereas SENP2 specifically removed SUMO from ROR{alpha}. SUMOylation-defective mutant form of ROR{alpha} exhibited decreased transcriptional activity on ROR{alpha}-responsive promoters indicating that SUMOylation may positively regulate transcriptional function of ROR{alpha}.

  5. The Role of Nuclear Bodies in Gene Expression and Disease

    PubMed Central

    Morimoto, Marie; Boerkoel, Cornelius F.

    2013-01-01

    This review summarizes the current understanding of the role of nuclear bodies in regulating gene expression. The compartmentalization of cellular processes, such as ribosome biogenesis, RNA processing, cellular response to stress, transcription, modification and assembly of spliceosomal snRNPs, histone gene synthesis and nuclear RNA retention, has significant implications for gene regulation. These functional nuclear domains include the nucleolus, nuclear speckle, nuclear stress body, transcription factory, Cajal body, Gemini of Cajal body, histone locus body and paraspeckle. We herein review the roles of nuclear bodies in regulating gene expression and their relation to human health and disease. PMID:24040563

  6. Nuclear lamins and neurobiology.

    PubMed

    Young, Stephen G; Jung, Hea-Jin; Lee, John M; Fong, Loren G

    2014-08-01

    Much of the work on nuclear lamins during the past 15 years has focused on mutations in LMNA (the gene for prelamin A and lamin C) that cause particular muscular dystrophy, cardiomyopathy, partial lipodystrophy, and progeroid syndromes. These disorders, often called "laminopathies," mainly affect mesenchymal tissues (e.g., striated muscle, bone, and fibrous tissue). Recently, however, a series of papers have identified important roles for nuclear lamins in the central nervous system. Studies of knockout mice uncovered a key role for B-type lamins (lamins B1 and B2) in neuronal migration in the developing brain. Also, duplications of LMNB1 (the gene for lamin B1) have been shown to cause autosome-dominant leukodystrophy. Finally, recent studies have uncovered a peculiar pattern of nuclear lamin expression in the brain. Lamin C transcripts are present at high levels in the brain, but prelamin A expression levels are very low-due to regulation of prelamin A transcripts by microRNA 9. This form of prelamin A regulation likely explains why "prelamin A diseases" such as Hutchinson-Gilford progeria syndrome spare the central nervous system. In this review, we summarize recent progress in elucidating links between nuclear lamins and neurobiology.

  7. Transcriptional Control of the TNF Gene

    PubMed Central

    Falvo, James V.; Tsytsykova, Alla V.; Goldfeld, Anne E.

    2016-01-01

    The cytokine TNF is a critical mediator of immune and inflammatory responses. The TNF gene is an immediate early gene, rapidly transcribed in a variety of cell types following exposure to a broad range of pathogens and signals of inflammation and stress. Regulation of TNF gene expression at the transcriptional level is cell type- and stimulus-specific, involving the recruitment of distinct sets of transcription factors to a compact and modular promoter region. In this review, we describe our current understanding of the mechanisms through which TNF transcription is specifically activated by a variety of extracellular stimuli in multiple cell types, including T cells, B cells, macrophages, mast cells, dendritic cells, and fibroblasts. We discuss the role of nuclear factor of activated T cells and other transcription factors and coactivators in enhanceosome formation, as well as the contradictory evidence for a role for nuclear factor κB as a classical activator of the TNF gene. We describe the impact of evolutionarily conserved cis-regulatory DNA motifs in the TNF locus upon TNF gene transcription, in contrast to the neutral effect of single nucleotide polymorphisms. We also assess the regulatory role of chromatin organization, epigenetic modifications, and long-range chromosomal interactions at the TNF locus. PMID:20173386

  8. Transcriptional coregulator RIP140: an essential regulator of physiology.

    PubMed

    Nautiyal, Jaya

    2017-04-01

    Transcriptional coregulators drive gene regulatory decisions in the transcriptional space. Although transcription factors including all nuclear receptors provide a docking platform for coregulators to bind, these proteins bring enzymatic capabilities to the gene regulatory sites. RIP140 is a transcriptional coregulator essential for several physiological processes, and aberrations in its function may lead to diseased states. Unlike several other coregulators that are known either for their coactivating or corepressing roles, in gene regulation, RIP140 is capable of acting both as a coactivator and a corepressor. The role of RIP140 in female reproductive axis and recent findings of its role in carcinogenesis and adipose biology have been summarised.

  9. Nuclear hormone receptors in podocytes

    PubMed Central

    2012-01-01

    Nuclear receptors are a family of ligand-activated, DNA sequence-specific transcription factors that regulate various aspects of animal development, cell proliferation, differentiation, and homeostasis. The physiological roles of nuclear receptors and their ligands have been intensively studied in cancer and metabolic syndrome. However, their role in kidney diseases is still evolving, despite their ligands being used clinically to treat renal diseases for decades. This review will discuss the progress of our understanding of the role of nuclear receptors and their ligands in kidney physiology with emphasis on their roles in treating glomerular disorders and podocyte injury repair responses. PMID:22995171

  10. Global analysis of transcriptionally engaged yeast RNA polymerase III reveals extended tRNA transcripts

    PubMed Central

    Turowski, Tomasz W.; Leśniewska, Ewa; Delan-Forino, Clementine; Sayou, Camille; Boguta, Magdalena; Tollervey, David

    2016-01-01

    RNA polymerase III (RNAPIII) synthesizes a range of highly abundant small stable RNAs, principally pre-tRNAs. Here we report the genome-wide analysis of nascent transcripts attached to RNAPIII under permissive and restrictive growth conditions. This revealed strikingly uneven polymerase distributions across transcription units, generally with a predominant 5′ peak. This peak was higher for more heavily transcribed genes, suggesting that initiation site clearance is rate-limiting during RNAPIII transcription. Down-regulation of RNAPIII transcription under stress conditions was found to be uneven; a subset of tRNA genes showed low response to nutrient shift or loss of the major transcription regulator Maf1, suggesting potential “housekeeping” roles. Many tRNA genes were found to generate long, 3′-extended forms due to read-through of the canonical poly(U) terminators. The degree of read-through was anti-correlated with the density of U-residues in the nascent tRNA, and multiple, functional terminators can be located far downstream. The steady-state levels of 3′-extended pre-tRNA transcripts are low, apparently due to targeting by the nuclear surveillance machinery, especially the RNA binding protein Nab2, cofactors for the nuclear exosome, and the 5′-exonuclease Rat1. PMID:27206856

  11. Transcription Regulation in Archaea

    PubMed Central

    Gehring, Alexandra M.; Walker, Julie E.

    2016-01-01

    The known diversity of metabolic strategies and physiological adaptations of archaeal species to extreme environments is extraordinary. Accurate and responsive mechanisms to ensure that gene expression patterns match the needs of the cell necessitate regulatory strategies that control the activities and output of the archaeal transcription apparatus. Archaea are reliant on a single RNA polymerase for all transcription, and many of the known regulatory mechanisms employed for archaeal transcription mimic strategies also employed for eukaryotic and bacterial species. Novel mechanisms of transcription regulation have become apparent by increasingly sophisticated in vivo and in vitro investigations of archaeal species. This review emphasizes recent progress in understanding archaeal transcription regulatory mechanisms and highlights insights gained from studies of the influence of archaeal chromatin on transcription. PMID:27137495

  12. Nuclear Scans

    MedlinePlus

    Nuclear scans use radioactive substances to see structures and functions inside your body. They use a special ... images. Most scans take 20 to 45 minutes. Nuclear scans can help doctors diagnose many conditions, including ...

  13. Nuclear Winter.

    ERIC Educational Resources Information Center

    Ehrlich, Anne

    1984-01-01

    "Nuclear Winter" was recently coined to describe the climatic and biological effects of a nuclear war. These effects are discussed based on models, simulations, scenarios, and projections. Effects on human populations are also considered. (JN)

  14. Nuclear Chemistry.

    ERIC Educational Resources Information Center

    Chemical and Engineering News, 1979

    1979-01-01

    Provides a brief review of the latest developments in nuclear chemistry. Nuclear research today is directed toward increased activity in radiopharmaceuticals and formation of new isotopes by high-energy, heavy-ion collisions. (Author/BB)

  15. A nuclear protein tyrosine phosphatase TC-PTP is a potential negative regulator of the PRL-mediated signaling pathway: dephosphorylation and deactivation of signal transducer and activator of transcription 5a and 5b by TC-PTP in nucleus.

    PubMed

    Aoki, Naohito; Matsuda, Tsukasa

    2002-01-01

    In the present study we examined involvement of nuclear protein tyrosine phosphatase TC-PTP in PRL-mediated signaling. TC-PTP could dephosphorylate signal transducer and activator of transcription 5a (STAT5a) and STAT5b, but the apparent dephosphorylation activity of TC-PTP was weaker than that of cytosolic PTP1B 30 min after PRL stimulation in transfected COS-7 cells, whereas both STAT5a and STAT5b were dephosphorylated to the same extent by recombinant TC-PTP and PTP1B in vitro. Tyrosine-phosphorylated STAT5 was coimmunoprecipitated with substrate trapping mutants of TC-PTP, suggesting that STAT5 is a specific substrate of TC-PTP. These observations were further extended in mammary epithelial COMMA-1D cells stably expressing TC-PTP. A time-course study revealed that dephosphorylation of STAT5 by TC-PTP was delayed compared with that by cytosolic PTP1B due to nuclear localization of TC-PTP throughout PRL stimulation in mammary epithelial cells. Endogenous beta-casein gene expression and beta-casein gene promoter activation in COS-7 cells were largely suppressed by TC-PTP wild type as well as catalytically inactive mutants, suggesting that stable complexes formed between STAT5 and TC-PTP in the nucleus. Taken together, we conclude that TC-PTP is catalytically competent with respect to dephosphorylation and deactivation of PRL-activated STAT5 in the nucleus.

  16. Nascent RNA sequencing reveals distinct features in plant transcription

    PubMed Central

    Hetzel, Jonathan; Duttke, Sascha H.; Benner, Christopher; Chory, Joanne

    2016-01-01

    Transcriptional regulation of gene expression is a major mechanism used by plants to confer phenotypic plasticity, and yet compared with other eukaryotes or bacteria, little is known about the design principles. We generated an extensive catalog of nascent and steady-state transcripts in Arabidopsis thaliana seedlings using global nuclear run-on sequencing (GRO-seq), 5′GRO-seq, and RNA-seq and reanalyzed published maize data to capture characteristics of plant transcription. De novo annotation of nascent transcripts accurately mapped start sites and unstable transcripts. Examining the promoters of coding and noncoding transcripts identified comparable chromatin signatures, a conserved “TGT” core promoter motif and unreported transcription factor-binding sites. Mapping of engaged RNA polymerases showed a lack of enhancer RNAs, promoter-proximal pausing, and divergent transcription in Arabidopsis seedlings and maize, which are commonly present in yeast and humans. In contrast, Arabidopsis and maize genes accumulate RNA polymerases in proximity of the polyadenylation site, a trend that coincided with longer genes and CpG hypomethylation. Lack of promoter-proximal pausing and a higher correlation of nascent and steady-state transcripts indicate Arabidopsis may regulate transcription predominantly at the level of initiation. Our findings provide insight into plant transcription and eukaryotic gene expression as a whole. PMID:27729530

  17. Omeprazole Induces NAD(P)H Quinone Oxidoreductase 1 via Aryl Hydrocarbon Receptor-Independent Mechanisms: Role of the Transcription Factor Nuclear Factor Erythroid 2–Related Factor 2

    PubMed Central

    Zhang, Shaojie; Patel, Ananddeep; Moorthy, Bhagavatula; Shivanna, Binoy

    2015-01-01

    Activation of the aryl hydrocarbon receptor (AhR) transcriptionally induces phase I (cytochrome P450 (CYP) 1A1) and phase II (NAD(P)H quinone oxidoreductase 1 (NQO1) detoxifying enzymes. The effects of the classical and nonclassical AhR ligands on phase I and II enzymes are well studied in human hepatocytes. Additionally, we observed that the proton pump inhibitor, omeprazole (OM), transcriptionally induces CYP1A1 in the human adenocarcinoma cell line, H441 cells via AhR. Whether OM activates AhR and induces the phase II enzyme, NAD(P)H quinone oxidoreductase 1 (NQO1), in fetal primary human pulmonary microvascular endothelial cells (HPMEC) is unknown. Therefore, we tested the hypothesis that OM will induce NQO1 in HPMEC via the AhR. The concentrations of OM used in our experiments did not result in cytotoxicity. OM activated AhR as evident by increased CYP1A1 mRNA expression. However, contrary to our hypothesis, OM increased NQO1 mRNA and protein via an AhR-independent mechanism as AhR knockdown failed to abrogate OM-mediated increase in NQO1 expression. Interestingly, OM activated Nrf2 as evident by increased phosphoNrf2 (S40) expression in OM-treated compared to vehicle-treated cells. Furthermore, Nrf2 knockdown abrogated OM-mediated increase in NQO1 expression. In conclusion, we provide evidence that OM induces NQO1 via AhR-independent, but Nrf2-dependent mechanisms. PMID:26441083

  18. Transcriptional Control of Synaptic Plasticity by Transcription Factor NF-κB.

    PubMed

    Engelmann, Christian; Haenold, Ronny

    2016-01-01

    Activation of nuclear factor kappa B (NF-κB) transcription factors is required for the induction of synaptic plasticity and memory formation. All components of this signaling pathway are localized at synapses, and transcriptionally active NF-κB dimers move to the nucleus to translate synaptic signals into altered gene expression. Neuron-specific inhibition results in altered connectivity of excitatory and inhibitory synapses and functionally in selective learning deficits. Recent research on transgenic mice with impaired or hyperactivated NF-κB gave important insights into plasticity-related target gene expression that is regulated by NF-κB. In this minireview, we update the available data on the role of this transcription factor for learning and memory formation and comment on cross-sectional activation of NF-κB in the aged and diseased brain that may directly or indirectly affect κB-dependent transcription of synaptic genes.

  19. Nuclear weapons, nuclear effects, nuclear war

    SciTech Connect

    Bing, G.F.

    1991-08-20

    This paper provides a brief and mostly non-technical description of the militarily important features of nuclear weapons, of the physical phenomena associated with individual explosions, and of the expected or possible results of the use of many weapons in a nuclear war. Most emphasis is on the effects of so-called ``strategic exchanges.``

  20. Nuclear Fuels.

    ERIC Educational Resources Information Center

    Nash, J. Thomas

    1983-01-01

    Trends in and factors related to the nuclear industry and nuclear fuel production are discussed. Topics addressed include nuclear reactors, survival of the U.S. uranium industry, production costs, budget cuts by the Department of Energy and U.S. Geological survey for resource studies, mining, and research/development activities. (JN)

  1. Characterization of transcriptional regulatory domains of ankyrin repeat cofactor-1

    SciTech Connect

    Zhang, Aihua; Li, Chia-Wei; Chen, J. Don . E-mail: chenjd@umdnj.edu

    2007-07-13

    The ankyrin repeats cofactor-1 (ANCO-1) was recently identified as a p160 coactivator-interacting protein that may inhibit transcriptional activity of nuclear receptors. Here, we have characterized the transcriptional regulatory domains of ANCO-1. Two intrinsic repression domains (RD) were identified: an N-terminal RD1 at residues 318-611 and a C-terminal RD2 at 2369-2663. ANCO-1 also contains an activation domain (AD) capable of stimulating transcription in both mammalian and yeast cells. The minimal AD was delimited to a 70-amino acid region at residues 2076-2145. Overall, full-length ANCO-1 exhibited transcriptional repressor activity, suggesting that RD domains may suppress the AD activity. We further demonstrated that ANCO-1 silencing by siRNA enhanced progesterone receptor-mediated transcription. Together, these results indicate that the transcriptional potential of ANCO-1 may be modulated by a combination of repression and activation signals.

  2. Coupling pre-mRNA processing to transcription on the RNA factory assembly line

    PubMed Central

    Lee, Kuo-Ming; Tarn, Woan-Yuh

    2013-01-01

    It has been well-documented that nuclear processing of primary transcripts of RNA polymerase II occurs co-transcriptionally and is functionally coupled to transcription. Moreover, increasing evidence indicates that transcription influences pre-mRNA splicing and even several post-splicing RNA processing events. In this review, we discuss the issues of how RNA polymerase II modulates co-transcriptional RNA processing events via its carboxyl terminal domain, and the protein domains involved in coupling of transcription and RNA processing events. In addition, we describe how transcription influences the expression or stability of mRNAs through the formation of distinct mRNP complexes. Finally, we delineate emerging findings that chromatin modifications function in the regulation of RNA processing steps, especially splicing, in addition to transcription. Overall, we provide a comprehensive view that transcription could integrate different control systems, from epigenetic to post-transcriptional control, for efficient gene expression. PMID:23392244

  3. Innate immunity and inflammation: a transcriptional paradigm.

    PubMed

    Hawiger, J

    2001-01-01

    The innate immune response and the process of inflammation are interwoven. Excessive and continuing cytokine production in response to bacterial lipopolysacharides (LPS) or superantigens is a hallmark of the systemic inflammatory response (IR), which can be life-threatening. Dissemination of these bacterial products induces waves of proinflammatory cytokines that cause vascular injury and multiple organ dysfunction. Both LPS and superantigens induce signaling to the nucleus in mononuclear phagocytes and T cells, respectively. These signaling pathways are mediated by NF-kappaB and other stress-responsive transcription factors (SRTFs), which play a critical role in reprogramming gene expression. The nuclear import of NF-kappaB allows transcriptional activation of over 100 genes that encode mediators of inflammatory and immune responses. We have developed a novel method to block nuclear import of NF-kappaB through cell-permeable peptide transduction in monocytes, macrophages, T lymphocytes, and endothelial cells. Strikingly, a cell-permeable peptide that antagonizes nuclear import of NF-kappaB and other SRTFs, suppressed the systemic production of proinflammatory cytokines (TNFalpha and interferon gamma) in mice challenged with a lethal dose of LPS, and increased their survival by at least 90%. Thus, systemic inflammatory responses are critically dependent on the transcriptional activation ofcytokine genes that are controlled by NF-kappaB and other SRTFs.

  4. Nuclear functions of prefoldin

    PubMed Central

    Millán-Zambrano, Gonzalo; Chávez, Sebastián

    2014-01-01

    Prefoldin is a cochaperone, present in all eukaryotes, that cooperates with the chaperonin CCT. It is known mainly for its functional relevance in the cytoplasmic folding of actin and tubulin monomers during cytoskeleton assembly. However, both canonical and prefoldin-like subunits of this heterohexameric complex have also been found in the nucleus, and are functionally connected with nuclear processes in yeast and metazoa. Plant prefoldin has also been detected in the nucleus and physically associated with a gene regulator. In this review, we summarize the information available on the involvement of prefoldin in nuclear phenomena, place special emphasis on gene transcription, and discuss the possibility of a global coordination between gene regulation and cytoplasmic dynamics mediated by prefoldin. PMID:25008233

  5. Genome wide analysis of human genes transcriptionally and post-transcriptionally regulated by the HTLV-I protein p30

    PubMed Central

    Taylor, John M; Ghorbel, Sofiane; Nicot, Christophe

    2009-01-01

    Background Human T-cell leukemia virus type 1 (HTLV-I) is a human retrovirus that is etiologically linked to adult T-cell leukemia (ATL), an aggressive and fatal lymphoproliferative disease. The viral transactivator, Tax, is thought to play an important role during the initial stages of CD4+ T-cell immortalization by HTLV-1. Tax has been shown to activate transcription through CREB/ATF and NF-KB, and to alter numerous signaling pathways. These pleiotropic effects of Tax modify the expression of a wide array of cellular genes. Another viral protein encoded by HTLV-I, p30, has been shown to affect virus replication at the transcriptional and posttranscriptional levels. Little is currently known regarding the effect of p30 on the expression and nuclear export of cellular host mRNA transcripts. Identification of these RNA may reveal new targets and increase our understanding of HTLV-I pathogenesis. In this study, using primary peripheral blood mononuclear cells, we report a genome wide analysis of human genes transcriptionally and post-transcriptionally regulated by the HTLV-I protein p30. Results Using microarray analysis, we analyzed total and cytoplasmic cellular mRNA transcript levels isolated from PBMCs to assess the effect of p30 on cellular RNA transcript expression and their nuclear export. We report p30-dependent transcription resulting in the 2.5 fold up-regulation of 15 genes and the down-regulation of 65 human genes. We further tested nuclear export of cellular mRNA and found that p30 expression also resulted in a 2.5 fold post-transcriptional down-regulation of 90 genes and the up-regulation of 33 genes. Conclusion Overall, our study describes that expression of the HTLV-I protein p30 both positively and negatively alters the expression of cellular transcripts. Our study identifies for the first time the cellular genes for which nuclear export is affected by p30. These results suggest that p30 may possess a more global function with respect to m

  6. Inhibitory effects of the standardized extract (DA-9601) of Artemisia asiatica Nakai on phorbol ester-induced ornithine decarboxylase activity, papilloma formation, cyclooxygenase-2 expression, inducible nitric oxide synthase expression and nuclear transcription factor kappa B activation in mouse skin.

    PubMed

    Seo, Hyo-Joung; Park, Kwang-Kyun; Han, Seong Su; Chung, Won-Yoon; Son, Mi-Won; Kim, Won-Bae; Surh, Young-Joon

    2002-08-01

    Artemisia asiatica Nakai has been used in traditional Asian medicine for the treatment of inflammatory and other disorders. Previous studies have revealed that the formulated ethanol extract (DA-9601) of A. asiatica has pronounced antioxidative and antiinflammatory activities and exhibits cytoprotective effects against experimentally induced gastrointestinal, hepatic and pancreatic damage. In the present study, we assessed the inhibitory effect of DA-9601 on tumor promotion, which is closely linked to inflammatory tissue damage. As an initial approach to evaluating the possible antitumor-promoting potential of DA-9601, its effects on TPA-induced ear edema were examined in female ICR mice. Pretreatment of the inner surface of the mouse ear with DA-9601 30 min prior to topical application of TPA inhibited ear edema at 5 hr. TPA-stimulated expression of epidermal COX-2 and iNOS was also mitigated by topical application of the same extract. Moreover, DA-9601 abrogated the TPA-mediated activation of NF-kappa B/Rel and AP-1 in mouse epidermis. Suppression of epidermal NF-kappa B by DA-9601 appeared to be mediated in part through inhibition of I kappa B alpha degradation, thereby blocking the nuclear translocation of p65, the functional subunit of NF-kappa B. DA-9601 also significantly suppressed TPA-induced ODC activity and papilloma formation in mouse skin. Taken together, these findings suggest that DA-9601 derived from A. asiatica possesses potential chemopreventive activities.

  7. Analysis of nuclear reprogramming following nuclear transfer to Xenopus oocyte.

    PubMed

    Jullien, Jerome

    2015-01-01

    Germinal vesicle of stage V-VI Xenopus Laevis oocytes (at the prophase I stage of meiosis) can be used to transplant mammalian nuclei. In this type of interspecies nuclear transfer no cell division occurs and no new cell types are generated. However, the transplanted nuclei undergo extensive transcriptional reprogramming. Here, it is first explained how to carry out transplantation of multiple mammalian cell nuclei to Xenopus oocytes. It is then described how to perform RT-qPCR, Western Blot, Chromatin Immunoprecipitation, and live imaging analysis to monitor transcriptional reprogramming of the nuclei transplanted to oocytes.

  8. ASTP Onboard Voice Transcription

    NASA Technical Reports Server (NTRS)

    1975-01-01

    The transcription is presented of the Apollo-Soyuz Test Project voice communications as recorded on the command module data storage equipment. Data from this recorder are telemetered (dumped) to Space Tracking and Data Network sites for retransmission to the Johnson Space Center. The transcript is divided into three columns -- time, speaker, and text. The Greenwich mean time column consists of three two-digit numbers representing hours, minutes, and seconds (e.g., 22 34 14) for the Julian dates shown at the top of the page on which a new day begins. The speaker column indicates the source of a transmission; the text column contains the verbatim transcript of the communications.

  9. Evolution of a transcriptional regulator from a transmembrane nucleoporin.

    PubMed

    Franks, Tobias M; Benner, Chris; Narvaiza, Iñigo; Marchetto, Maria C N; Young, Janet M; Malik, Harmit S; Gage, Fred H; Hetzer, Martin W

    2016-05-15

    Nuclear pore complexes (NPCs) emerged as nuclear transport channels in eukaryotic cells ∼1.5 billion years ago. While the primary role of NPCs is to regulate nucleo-cytoplasmic transport, recent research suggests that certain NPC proteins have additionally acquired the role of affecting gene expression at the nuclear periphery and in the nucleoplasm in metazoans. Here we identify a widely expressed variant of the transmembrane nucleoporin (Nup) Pom121 (named sPom121, for "soluble Pom121") that arose by genomic rearrangement before the divergence of hominoids. sPom121 lacks the nuclear membrane-anchoring domain and thus does not localize to the NPC. Instead, sPom121 colocalizes and interacts with nucleoplasmic Nup98, a previously identified transcriptional regulator, at gene promoters to control transcription of its target genes in human cells. Interestingly, sPom121 transcripts appear independently in several mammalian species, suggesting convergent innovation of Nup-mediated transcription regulation during mammalian evolution. Our findings implicate alternate transcription initiation as a mechanism to increase the functional diversity of NPC components.

  10. Evolution of a transcriptional regulator from a transmembrane nucleoporin

    PubMed Central

    Franks, Tobias M.; Benner, Chris; Narvaiza, Iñigo; Marchetto, Maria C.N.; Young, Janet M.; Malik, Harmit S.; Gage, Fred H.; Hetzer, Martin W.

    2016-01-01

    Nuclear pore complexes (NPCs) emerged as nuclear transport channels in eukaryotic cells ∼1.5 billion years ago. While the primary role of NPCs is to regulate nucleo–cytoplasmic transport, recent research suggests that certain NPC proteins have additionally acquired the role of affecting gene expression at the nuclear periphery and in the nucleoplasm in metazoans. Here we identify a widely expressed variant of the transmembrane nucleoporin (Nup) Pom121 (named sPom121, for “soluble Pom121”) that arose by genomic rearrangement before the divergence of hominoids. sPom121 lacks the nuclear membrane-anchoring domain and thus does not localize to the NPC. Instead, sPom121 colocalizes and interacts with nucleoplasmic Nup98, a previously identified transcriptional regulator, at gene promoters to control transcription of its target genes in human cells. Interestingly, sPom121 transcripts appear independently in several mammalian species, suggesting convergent innovation of Nup-mediated transcription regulation during mammalian evolution. Our findings implicate alternate transcription initiation as a mechanism to increase the functional diversity of NPC components. PMID:27198230

  11. Nuclear astrophysics

    SciTech Connect

    Haxton, W.C.

    1992-01-01

    The problem of core-collapse supernovae is used to illustrate the many connections between nuclear astrophysics and the problems nuclear physicists study in terrestrial laboratories. Efforts to better understand the collapse and mantle ejection are also motivated by a variety of interdisciplinary issues in nuclear, particle, and astrophysics, including galactic chemical evolution, neutrino masses and mixing, and stellar cooling by the emission of new particles. The current status of theory and observations is summarized.

  12. Nuclear astrophysics

    SciTech Connect

    Haxton, W.C.

    1992-12-31

    The problem of core-collapse supernovae is used to illustrate the many connections between nuclear astrophysics and the problems nuclear physicists study in terrestrial laboratories. Efforts to better understand the collapse and mantle ejection are also motivated by a variety of interdisciplinary issues in nuclear, particle, and astrophysics, including galactic chemical evolution, neutrino masses and mixing, and stellar cooling by the emission of new particles. The current status of theory and observations is summarized.

  13. Nuclear Fission

    NASA Astrophysics Data System (ADS)

    Denschlag, J. O.

    This chapter first gives a survey on the history of the discovery of nuclear fission. It briefly presents the liquid-drop and shell models and their application to the fission process. The most important quantities accessible to experimental determination such as mass yields, nuclear charge distribution, prompt neutron emission, kinetic energy distribution, ternary fragment yields, angular distributions, and properties of fission isomers are presented as well as the instrumentation and techniques used for their measurement. The contribution concentrates on the fundamental aspects of nuclear fission. The practical aspects of nuclear fission are discussed in http://dx.doi.org/10.1007/978-1-4419-0720-2_57 of Vol. 6.

  14. RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond.

    PubMed

    Castel, Stephane E; Martienssen, Robert A

    2013-02-01

    A growing number of functions are emerging for RNA interference (RNAi) in the nucleus, in addition to well-characterized roles in post-transcriptional gene silencing in the cytoplasm. Epigenetic modifications directed by small RNAs have been shown to cause transcriptional repression in plants, fungi and animals. Additionally, increasing evidence indicates that RNAi regulates transcription through interaction with transcriptional machinery. Nuclear small RNAs include small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) and are implicated in nuclear processes such as transposon regulation, heterochromatin formation, developmental gene regulation and genome stability.

  15. DNA supercoiling during transcription

    PubMed Central

    Ma, Jie; Wang, Michelle D.

    2017-01-01

    The twin-supercoiled-domain model describes how transcription can drive DNA supercoiling, and how DNA supercoiling, in turn plays an important role in regulating gene transcription. In vivo and in vitro experiments have disclosed many details of the complex interactions in this relationship, and recently new insights have been gained with the help of genome-wide DNA supercoiling mapping techniques and single molecule methods. This review summarizes the general mechanisms of the interplay between DNA supercoiling and transcription, considers the biological implications, and focuses on recent important discoveries and technical advances in this field. We highlight the significant impact of DNA supercoiling in transcription, but also more broadly in all processes operating on DNA.

  16. Nuclear structure of Trypanosoma cruzi.

    PubMed

    Schenkman, Sergio; Pascoalino, Bruno dos Santos; Nardelli, Sheila C

    2011-01-01

    The presence of nucleus in living organisms characterizes the Eukaryote domain. The nucleus compartmentalizes the genetic material surrounded by a double membrane called nuclear envelope. The nucleus has been observed since the advent of the light microscope, and sub-compartments such as nucleoli, diverse nuclear bodies and condensed chromosomes have been later recognized, being part of highly organized and dynamic structure. The significance and function of such organization has increased with the understanding of transcription, replication, DNA repair, recombination processes. It is now recognized as consequence of adding complexity and regulation in more complex eukaryotic cells. Here we provide a description of the actual stage of knowledge of the nuclear structure of Trypanosoma cruzi. As an early divergent eukaryote, it presents unique and/or reduced events of DNA replication, transcription and repair as well as RNA processing and transport to the cytosol. Nevertheless, it shows peculiar structure changes accordingly to the cell cycle and stage of differentiation. T. cruzi proliferates only as epimastigote and amastigote stages, and when these forms differentiate in trypomastigote forms, their cell cycle is arrested. This arrested stage is capable of invading mammalian cells and of surviving harsh conditions, such as the gut of the insect vector and mammalian macrophages. Transcription and replication decrease during transformation in trypomastigotes implicating large alterations in the nuclear structure. Recent evidences also suggest that T. cruzi nucleus respond to oxidative and nutritional stresses. Due to the phylogenetic proximity with other well-known trypanosomes, such as Trypanosoma brucei and Leishmania major, they are expected to have similar nuclear organization, although differences are noticed due to distinct life cycles, cellular organizations and the specific adaptations for surviving in different host environments. Therefore, the general

  17. Nuclear safety

    NASA Technical Reports Server (NTRS)

    Buden, D.

    1991-01-01

    Topics dealing with nuclear safety are addressed which include the following: general safety requirements; safety design requirements; terrestrial safety; SP-100 Flight System key safety requirements; potential mission accidents and hazards; key safety features; ground operations; launch operations; flight operations; disposal; safety concerns; licensing; the nuclear engine for rocket vehicle application (NERVA) design philosophy; the NERVA flight safety program; and the NERVA safety plan.

  18. Transcription upregulation via force-induced direct stretching of chromatin

    NASA Astrophysics Data System (ADS)

    Tajik, Arash; Zhang, Yuejin; Wei, Fuxiang; Sun, Jian; Jia, Qiong; Zhou, Wenwen; Singh, Rishi; Khanna, Nimish; Belmont, Andrew S.; Wang, Ning

    2016-12-01

    Mechanical forces play critical roles in the function of living cells. However, the underlying mechanisms of how forces influence nuclear events remain elusive. Here, we show that chromatin deformation as well as force-induced transcription of a green fluorescent protein (GFP)-tagged bacterial-chromosome dihydrofolate reductase (DHFR) transgene can be visualized in a living cell by using three-dimensional magnetic twisting cytometry to apply local stresses on the cell surface via an Arg-Gly-Asp-coated magnetic bead. Chromatin stretching depended on loading direction. DHFR transcription upregulation was sensitive to load direction and proportional to the magnitude of chromatin stretching. Disrupting filamentous actin or inhibiting actomyosin contraction abrogated or attenuated force-induced DHFR transcription, whereas activating endogenous contraction upregulated force-induced DHFR transcription. Our findings suggest that local stresses applied to integrins propagate from the tensed actin cytoskeleton to the LINC complex and then through lamina-chromatin interactions to directly stretch chromatin and upregulate transcription.

  19. Nuclear privatization

    SciTech Connect

    Jeffs, E.

    1995-11-01

    The United Kingdom government announced in May 1995 plans to privatize the country`s two nuclear generating companies, Nuclear Electric and Scottish Nuclear. Under the plan, the two companies will become operating divisions of a unified holding company, to be called British Electric, with headquarters in Scotland. Britain`s nuclear plants were left out of the initial privatization in 1989 because the government believed the financial community would be unwilling to accept the open-ended liability of decommissioning the original nine stations based on the Magnox gas-cooled reactor. Six years later, the government has found a way around this by retaining these power stations in state ownership, leaving the new nuclear company with the eight Advanced Gas-cooled Reactor (AGR) stations and the recently completed Sizewell B PWR stations. The operating Magnox stations are to be transferred to BNFL, which operates two Magnox stations of their own at Calder Hall and Chapelcross.

  20. Transcriptional regulation of the uncoupling protein-1 gene.

    PubMed

    Villarroya, Francesc; Peyrou, Marion; Giralt, Marta

    2017-03-01

    Regulated transcription of the uncoupling protein-1 (UCP1) gene, and subsequent UCP1 protein synthesis, is a hallmark of the acquisition of the differentiated, thermogenically competent status of brown and beige/brite adipocytes, as well as of the responsiveness of brown and beige/brite adipocytes to adaptive regulation of thermogenic activity. The 5' non-coding region of the UCP1 gene contains regulatory elements that confer tissue specificity, differentiation dependence, and neuro-hormonal regulation to UCP1 gene transcription. Two main regions-a distal enhancer and a proximal promoter region-mediate transcriptional regulation through interactions with a plethora of transcription factors, including nuclear hormone receptors and cAMP-responsive transcription factors. Co-regulators, such as PGC-1α, play a pivotal role in the concerted regulation of UCP1 gene transcription. Multiple interactions of transcription factors and co-regulators at the promoter region of the UCP1 gene result in local chromatin remodeling, leading to activation and increased accessibility of RNA polymerase II and subsequent gene transcription. Moreover, a commonly occurring A-to-G polymorphism in close proximity to the UCP1 gene enhancer influences the extent of UCP1 gene transcription. Notably, it has been reported that specific aspects of obesity and associated metabolic diseases are associated with human population variability at this site. On another front, the unique properties of the UCP1 promoter region have been exploited to develop brown adipose tissue-specific gene delivery tools for experimental purposes.

  1. In vivo delivery of transcription factors with multifunctional oligonucleotides

    NASA Astrophysics Data System (ADS)

    Lee, Kunwoo; Rafi, Mohammad; Wang, Xiaojian; Aran, Kiana; Feng, Xuli; Lo Sterzo, Carlo; Tang, Richard; Lingampalli, Nithya; Kim, Hyun Jin; Murthy, Niren

    2015-07-01

    Therapeutics based on transcription factors have the potential to revolutionize medicine but have had limited clinical success as a consequence of delivery problems. The delivery of transcription factors is challenging because it requires the development of a delivery vehicle that can complex transcription factors, target cells and stimulate endosomal disruption, with minimal toxicity. Here, we present a multifunctional oligonucleotide, termed DARTs (DNA assembled recombinant transcription factors), which can deliver transcription factors with high efficiency in vivo. DARTs are composed of an oligonucleotide that contains a transcription-factor-binding sequence and hydrophobic membrane-disruptive chains that are masked by acid-cleavable galactose residues. DARTs have a unique molecular architecture, which allows them to bind transcription factors, trigger endocytosis in hepatocytes, and stimulate endosomal disruption. The DARTs have enhanced uptake in hepatocytes as a result of their galactose residues and can disrupt endosomes efficiently with minimal toxicity, because unmasking of their hydrophobic domains selectively occurs in the acidic environment of the endosome. We show that DARTs can deliver the transcription factor nuclear erythroid 2-related factor 2 (Nrf2) to the liver, catalyse the transcription of Nrf2 downstream genes, and rescue mice from acetaminophen-induced liver injury.

  2. Homocysteine induces inflammatory transcriptional signaling in monocytes

    PubMed Central

    Meng, Shu; Ciment, Stephen; Jan, Michael; Tran, Tran; Pham, Hung; Cueto, Ramón; Yang, Xiao-Feng; Wang, Hong

    2013-01-01

    Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease. This study is to investigate transcriptional mechanism underlying homocysteine (Hcy)-induced and monocytes (MC)-derived inflammatory response. We identified 11 Hcy-induced genes, 17 anti-inflammatory cytokine interleukin 10-induced, 8 pro-inflammatory cytokine interferon γ (IFNγ)-induced and 8 pro-inflammatory cytokine tumor necrosis factor α (TNFα)-induced genes through literature search. Binding frequency of 36 transcription factors (TFs) implicated in inflammation and MC differentiation were analyzed within core promoter regions of identified genes, and classified into 3 classes based on the significant binding frequency to the promoter of Hcy-induced genes. Class 1 TFs exert high significant binding frequency in Hcy-induced genes. Class 2 and 3 TFs have low and no significant binding frequency, respectively. Class 1 TF binding occurrence in Hcy-induced genes is similar to that in IFNγ-induced genes, but not that in TNFα-induced. We conclude that Hcy is a pro-inflammatory amino acid and induces inflammatory transcriptional signal pathways mediated by class 1 TF. We term class 1 TF, which includes heat shock factor, MC enhancer factor-2, nuclear factor of activated T-cells, nuclear factor kappa light chain enhancer of activated B cells and Krueppel-like factor 4, as putative Hcy-responsive TFs. PMID:23276953

  3. Nuclear stress test

    MedlinePlus

    ... Persantine stress test; Thallium stress test; Stress test - nuclear; Adenosine stress test; Regadenoson stress test; CAD - nuclear stress; Coronary artery disease - nuclear stress; Angina - nuclear ...

  4. Nuclear protein extraction from frozen porcine myocardium.

    PubMed

    Kuster, Diederik W D; Merkus, Daphne; Jorna, Huub J J; Dekkers, Dick H W; Duncker, Dirk J; Verhoeven, Adrie J M

    2011-06-01

    Protocols for the extraction of nuclear proteins have been developed for cultured cells and fresh tissue, but sometimes only frozen tissue is available. We have optimized the homogenization procedure and subsequent fractionation protocol for the preparation of nuclear protein extracts from frozen porcine left ventricular (LV) tissue. This method gave a highly reproducible protein yield (6.5±0.7% of total protein; mean±SE, n=9) and a 6-fold enrichment of the nuclear marker protein B23. The nuclear protein extracts were essentially devoid of cytosolic, myofilament, and histone proteins. Compared to nuclear extracts from fresh LV tissue, some loss of nuclear proteins to the cytosolic fraction was observed. Using this method, we studied the distribution of tyrosine phosphorylated signal transducer and activator of transcription 3 (PY-STAT3) in LV tissue of animals treated with the β-agonist dobutamine. Upon treatment, PY-STAT3 increased 30.2±8.5-fold in total homogenates, but only 6.9±2.1-fold (n=4, P=0.03) in nuclear protein extracts. Of all PY-STAT3 formed, only a minor fraction appeared in the nuclear fraction. This simple and reproducible protocol yielded nuclear protein extracts that were highly enriched in nuclear proteins with almost complete removal of cytosolic and myofilament proteins. This nuclear protein extraction protocol is therefore well-suited for nuclear proteome analysis of frozen heart tissue collected in biobanks.

  5. Nuclear reprogramming.

    PubMed

    Halley-Stott, Richard P; Pasque, Vincent; Gurdon, J B

    2013-06-01

    There is currently particular interest in the field of nuclear reprogramming, a process by which the identity of specialised cells may be changed, typically to an embryonic-like state. Reprogramming procedures provide insight into many mechanisms of fundamental cell biology and have several promising applications, most notably in healthcare through the development of human disease models and patient-specific tissue-replacement therapies. Here, we introduce the field of nuclear reprogramming and briefly discuss six of the procedures by which reprogramming may be experimentally performed: nuclear transfer to eggs or oocytes, cell fusion, extract treatment, direct reprogramming to pluripotency and transdifferentiation.

  6. Improving Mode of Action Analysis Using Transcript Profiling in Nullizygous Mouse Models

    EPA Science Inventory

    A number of nuclear receptors (NR) mediate transcriptional, hepatocyte growth and carcinogenic effects in the rodent liver after chemical exposure. These receptors include the constitutive activated/androstane receptor (CAR), pregnane X receptor (PXR), and peroxisome proliferator...

  7. Regulation of Transcript Elongation

    PubMed Central

    Belogurov, Georgiy A.; Artsimovitch, Irina

    2015-01-01

    Bacteria lack subcellular compartments and harbor a single RNA polymerase that synthesizes both structural and protein-coding RNAs, which are cotranscriptionally processed by distinct pathways. Nascent rRNAs fold into elaborate secondary structures and associate with ribosomal proteins, whereas nascent mRNAs are translated by ribosomes. During elongation, nucleic acid signals and regulatory proteins modulate concurrent RNA-processing events, instruct RNA polymerase where to pause and terminate transcription, or act as roadblocks to the moving enzyme. Communications among complexes that carry out transcription, translation, repair, and other cellular processes ensure timely execution of the gene expression program and survival under conditions of stress. This network is maintained by auxiliary proteins that act as bridges between RNA polymerase, ribosome, and repair enzymes, blurring boundaries between separate information-processing steps and making assignments of unique regulatory functions meaningless. Understanding the regulation of transcript elongation thus requires genome-wide approaches, which confirm known and reveal new regulatory connections. PMID:26132790

  8. Transcriptional control of cardiac fibroblast plasticity.

    PubMed

    Lighthouse, Janet K; Small, Eric M

    2016-02-01

    Cardiac fibroblasts help maintain the normal architecture of the healthy heart and are responsible for scar formation and the healing response to pathological insults. Various genetic, biomechanical, or humoral factors stimulate fibroblasts to become contractile smooth muscle-like cells called myofibroblasts that secrete large amounts of extracellular matrix. Unfortunately, unchecked myofibroblast activation in heart disease leads to pathological fibrosis, which is a major risk factor for the development of cardiac arrhythmias and heart failure. A better understanding of the molecular mechanisms that control fibroblast plasticity and myofibroblast activation is essential to develop novel strategies to specifically target pathological cardiac fibrosis without disrupting the adaptive healing response. This review highlights the major transcriptional mediators of fibroblast origin and function in development and disease. The contribution of the fetal epicardial gene program will be discussed in the context of fibroblast origin in development and following injury, primarily focusing on Tcf21 and C/EBP. We will also highlight the major transcriptional regulatory axes that control fibroblast plasticity in the adult heart, including transforming growth factor β (TGFβ)/Smad signaling, the Rho/myocardin-related transcription factor (MRTF)/serum response factor (SRF) axis, and Calcineurin/transient receptor potential channel (TRP)/nuclear factor of activated T-Cell (NFAT) signaling. Finally, we will discuss recent strategies to divert the fibroblast transcriptional program in an effort to promote cardiomyocyte regeneration. This article is a part of a Special Issue entitled "Fibrosis and Myocardial Remodeling".

  9. HIV-1 transcription and latency: an update

    PubMed Central

    2013-01-01

    Combination antiretroviral therapy, despite being potent and life-prolonging, is not curative and does not eradicate HIV-1 infection since interruption of treatment inevitably results in a rapid rebound of viremia. Reactivation of latently infected cells harboring transcriptionally silent but replication-competent proviruses is a potential source of persistent residual viremia in cART-treated patients. Although multiple reservoirs may exist, the persistence of resting CD4+ T cells carrying a latent infection represents a major barrier to eradication. In this review, we will discuss the latest reports on the molecular mechanisms that may regulate HIV-1 latency at the transcriptional level, including transcriptional interference, the role of cellular factors, chromatin organization and epigenetic modifications, the viral Tat trans-activator and its cellular cofactors. Since latency mechanisms may also operate at the post-transcriptional level, we will consider inhibition of nuclear RNA export and inhibition of translation by microRNAs as potential barriers to HIV-1 gene expression. Finally, we will review the therapeutic approaches and clinical studies aimed at achieving either a sterilizing cure or a functional cure of HIV-1 infection, with a special emphasis on the most recent pharmacological strategies to reactivate the latent viruses and decrease the pool of viral reservoirs. PMID:23803414

  10. Polyphenol Compound as a Transcription Factor Inhibitor

    PubMed Central

    Park, Seyeon

    2015-01-01

    A target-based approach has been used to develop novel drugs in many therapeutic fields. In the final stage of intracellular signaling, transcription factor–DNA interactions are central to most biological processes and therefore represent a large and important class of targets for human therapeutics. Thus, we focused on the idea that the disruption of protein dimers and cognate DNA complexes could impair the transcriptional activation and cell transformation regulated by these proteins. Historically, natural products have been regarded as providing the primary leading compounds capable of modulating protein–protein or protein-DNA interactions. Although their mechanism of action is not fully defined, polyphenols including flavonoids were found to act mostly as site-directed small molecule inhibitors on signaling. There are many reports in the literature of screening initiatives suggesting improved drugs that can modulate the transcription factor interactions responsible for disease. In this review, we focus on polyphenol compound inhibitors against dimeric forms of transcription factor components of intracellular signaling pathways (for instance, c-jun/c-fos (Activator Protein-1; AP-1), c-myc/max, Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and β-catenin/T cell factor (Tcf)). PMID:26529010

  11. O-GlcNAc inhibits interaction between Sp1 and Elf-1 transcription factors

    SciTech Connect

    Lim, Kihong; Chang, Hyo-Ihl

    2009-03-13

    The novel protein modification, O-linked N-acetylglucosamine (O-GlcNAc), plays an important role in various aspects of cell regulation. Although most of nuclear transcription regulatory factors are modified by O-GlcNAc, O-GlcNAc effects on transcription remain largely undefined yet. In this study, we show that O-GlcNAc inhibits a physical interaction between Sp1 and Elf-1 transcription factors, and negatively regulates transcription of placenta and embryonic expression oncofetal protein gene (Pem). These findings suggest that O-GlcNAc inhibits Sp1-mediated gene transcription possibly by interrupting Sp1 interaction with its cooperative factor.

  12. Co-dependence between trypanosome nuclear lamina components in nuclear stability and control of gene expression

    PubMed Central

    Maishman, Luke; Obado, Samson O.; Alsford, Sam; Bart, Jean-Mathieu; Chen, Wei-Ming; Ratushny, Alexander V.; Navarro, Miguel; Horn, David; Aitchison, John D.; Chait, Brian T.; Rout, Michael P.; Field, Mark C.

    2016-01-01

    The nuclear lamina is a filamentous structure subtending the nuclear envelope and required for chromatin organization, transcriptional regulation and maintaining nuclear structure. The trypanosomatid coiled-coil NUP-1 protein is a lamina component functionally analogous to lamins, the major lamina proteins of metazoa. There is little evidence for shared ancestry, suggesting the presence of a distinct lamina system in trypanosomes. To find additional trypanosomatid lamina components we identified NUP-1 interacting proteins by affinity capture and mass-spectrometry. Multiple components of the nuclear pore complex (NPC) and a second coiled-coil protein, which we termed NUP-2, were found. NUP-2 has a punctate distribution at the nuclear periphery throughout the cell cycle and is in close proximity to NUP-1, the NPCs and telomeric chromosomal regions. RNAi-mediated silencing of NUP-2 leads to severe proliferation defects, gross alterations to nuclear structure, chromosomal organization and nuclear envelope architecture. Further, transcription is altered at telomere-proximal variant surface glycoprotein (VSG) expression sites (ESs), suggesting a role in controlling ES expression, although NUP-2 silencing does not increase VSG switching. Transcriptome analysis suggests specific alterations to Pol I-dependent transcription. NUP-1 is mislocalized in NUP-2 knockdown cells and vice versa, implying that NUP-1 and NUP-2 form a co-dependent network and identifying NUP-2 as a second trypanosomatid nuclear lamina component. PMID:27625397

  13. (Nuclear theory). [Research in nuclear physics

    SciTech Connect

    Haxton, W.

    1990-01-01

    This report discusses research in nuclear physics. Topics covered in this paper are: symmetry principles; nuclear astrophysics; nuclear structure; quark-gluon plasma; quantum chromodynamics; symmetry breaking; nuclear deformation; and cold fusion. (LSP)

  14. The transcription factor encyclopedia.

    PubMed

    Yusuf, Dimas; Butland, Stefanie L; Swanson, Magdalena I; Bolotin, Eugene; Ticoll, Amy; Cheung, Warren A; Zhang, Xiao Yu Cindy; Dickman, Christopher T D; Fulton, Debra L; Lim, Jonathan S; Schnabl, Jake M; Ramos, Oscar H P; Vasseur-Cognet, Mireille; de Leeuw, Charles N; Simpson, Elizabeth M; Ryffel, Gerhart U; Lam, Eric W-F; Kist, Ralf; Wilson, Miranda S C; Marco-Ferreres, Raquel; Brosens, Jan J; Beccari, Leonardo L; Bovolenta, Paola; Benayoun, Bérénice A; Monteiro, Lara J; Schwenen, Helma D C; Grontved, Lars; Wederell, Elizabeth; Mandrup, Susanne; Veitia, Reiner A; Chakravarthy, Harini; Hoodless, Pamela A; Mancarelli, M Michela; Torbett, Bruce E; Banham, Alison H; Reddy, Sekhar P; Cullum, Rebecca L; Liedtke, Michaela; Tschan, Mario P; Vaz, Michelle; Rizzino, Angie; Zannini, Mariastella; Frietze, Seth; Farnham, Peggy J; Eijkelenboom, Astrid; Brown, Philip J; Laperrière, David; Leprince, Dominique; de Cristofaro, Tiziana; Prince, Kelly L; Putker, Marrit; del Peso, Luis; Camenisch, Gieri; Wenger, Roland H; Mikula, Michal; Rozendaal, Marieke; Mader, Sylvie; Ostrowski, Jerzy; Rhodes, Simon J; Van Rechem, Capucine; Boulay, Gaylor; Olechnowicz, Sam W Z; Breslin, Mary B; Lan, Michael S; Nanan, Kyster K; Wegner, Michael; Hou, Juan; Mullen, Rachel D; Colvin, Stephanie C; Noy, Peter John; Webb, Carol F; Witek, Matthew E; Ferrell, Scott; Daniel, Juliet M; Park, Jason; Waldman, Scott A; Peet, Daniel J; Taggart, Michael; Jayaraman, Padma-Sheela; Karrich, Julien J; Blom, Bianca; Vesuna, Farhad; O'Geen, Henriette; Sun, Yunfu; Gronostajski, Richard M; Woodcroft, Mark W; Hough, Margaret R; Chen, Edwin; Europe-Finner, G Nicholas; Karolczak-Bayatti, Magdalena; Bailey, Jarrod; Hankinson, Oliver; Raman, Venu; LeBrun, David P; Biswal, Shyam; Harvey, Christopher J; DeBruyne, Jason P; Hogenesch, John B; Hevner, Robert F; Héligon, Christophe; Luo, Xin M; Blank, Marissa Cathleen; Millen, Kathleen Joyce; Sharlin, David S; Forrest, Douglas; Dahlman-Wright, Karin; Zhao, Chunyan; Mishima, Yuriko; Sinha, Satrajit; Chakrabarti, Rumela; Portales-Casamar, Elodie; Sladek, Frances M; Bradley, Philip H; Wasserman, Wyeth W

    2012-01-01

    Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe.

  15. The Transcription Factor Encyclopedia

    PubMed Central

    2012-01-01

    Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe. PMID:22458515

  16. Nuclear battlefields

    SciTech Connect

    Arkin, W.M.; Fieldhouse, R.W.

    1985-01-01

    This book provides complete data on the nuclear operations and research facilities in the U.S.A., the U.S.S.R., France, China and the U.K. It describes detailed estimates on the U.S.S.R.'s nuclear stockpile for over 500 locations. It shows how non-nuclear countries cooperate with the world-wide war machine. And it maps the U.S. nuclear facilities from Little America, WY, and Charleston, SC, to the battleships patroling the world's oceans and subs stalking under the sea. The data were gathered from unclassified sources through the Freedom of Information Act, from data supplied to military installations, and from weapons source books. It provides guidance for policymakers, government and corporate officials.

  17. NUCLEAR REACTOR

    DOEpatents

    Sherman, J.; Sharbaugh, J.E.; Fauth, W.L. Jr.; Palladino, N.J.; DeHuff, P.G.

    1962-10-23

    A nuclear reactor incorporating seed and blanket assemblies is designed. Means are provided for obtaining samples of the coolant from the blanket assemblies and for varying the flow of coolant through the blanket assemblies. (AEC)

  18. Nuclear Medicine

    MedlinePlus

    ... here Home » Science Education » Science Topics » Nuclear Medicine SCIENCE EDUCATION SCIENCE EDUCATION Science Topics Resource Links for ... administered by inhalation, by oral ingestion, or by direct injection into an organ. The mode of tracer ...

  19. Nuclear accidents

    SciTech Connect

    Mobley, J.A.

    1982-05-01

    A nuclear accident with radioactive contamination can happen anywhere in the world. Because expert nuclear emergency teams may take several hours to arrive at the scene, local authorities must have a plan of action for the hours immediately following an accident. The site should be left untouched except to remove casualties. Treatment of victims includes decontamination and meticulous wound debridement. Acute radiation syndrome may be an overwhelming sequela.

  20. Nuclear cardiac

    SciTech Connect

    Slutsky, R.; Ashburn, W.L.

    1982-01-01

    The relationship between nuclear medicine and cardiology has continued to produce a surfeit of interesting, illuminating, and important reports involving the analysis of cardiac function, perfusion, and metabolism. To simplify the presentation, this review is broken down into three major subheadings: analysis of myocardial perfusion; imaging of the recent myocardial infarction; and the evaluation of myocardial function. There appears to be an increasingly important relationship between cardiology, particularly cardiac physiology, and nuclear imaging techniques. (KRM)

  1. Nuclear Data

    SciTech Connect

    White, Morgan C.

    2014-01-23

    PowerPoint presentation targeted for educational use. Nuclear data comes from a variety of sources and in many flavors. Understanding where the data you use comes from and what flavor it is can be essential to understand and interpret your results. This talk will discuss the nuclear data pipeline with particular emphasis on providing links to additional resources that can be used to explore the issues you will encounter.

  2. Nuclear Nonproliferation

    SciTech Connect

    Atkins-Duffin, C E

    2008-12-10

    With an explosion equivalent of about 20kT of TNT, the Trinity test was the first demonstration of a nuclear weapon. Conducted on July 16, 1945 in Alamogordo, NM this site is now a Registered National Historic Landmark. The concept and applicability of nuclear power was demonstrated on December 20, 1951 with the Experimental Breeder Reactor Number One (EBR-1) lit four light bulbs. This reactor is now a Registered National Historic Landmark, located near Arco, ID. From that moment forward it had been clearly demonstrated that nuclear energy has both peaceful and military applications and that the civilian and military fuel cycles can overlap. For the more than fifty years since the Atoms for Peace program, a key objective of nuclear policy has been to enable the wider peaceful use of nuclear energy while preventing the spread of nuclear weapons. Volumes have been written on the impact of these two actions on the world by advocates and critics; pundits and practioners; politicians and technologists. The nations of the world have woven together a delicate balance of treaties, agreements, frameworks and handshakes that are representative of the timeframe in which they were constructed and how they have evolved in time. Collectively these vehicles attempt to keep political will, nuclear materials and technology in check. This paper captures only the briefest abstract of the more significant aspects on the Nonproliferation Regime. Of particular relevance to this discussion is the special nonproliferation sensitivity associated with the uranium isotope separation and spent fuel reprocessing aspects of the nuclear fuel cycle.

  3. Nuclear telemedicine

    NASA Astrophysics Data System (ADS)

    Morrison, R. T.; Szasz, I. J.

    1990-06-01

    Diagnostic nuclear medicine patient images have been transniitted for 8 years from a regional conununity hospital to a university teaching hospital 700 kiloinetres away employing slow scan TV and telephone. Transruission and interpretation were done at the end of each working day or as circumstances required in cases of emergencies. Referring physicians received the nuclear medicine procedure report at the end of the completion day or within few minutes of completion in case of emergency procedures. To date more than 25 patient studies have been transmitted for interpretation. Blinded reinterpretation of the original hard copy data of 350 patient studies resulted in 100 agreement with the interpretation of transmitted data. This technique provides high quality diagnostic and therapeutic nuclear medicine services in remote hospitals where the services of an on-site nuclear physician is not available. 2. HISTORY Eight years ago when the nuclear medicine physician at Trail Regional Hospital left the Trail area and an other could not be recruited we examined the feasibility of image transmission by phone for interpretation since closing the department would have imposed unacceptable physical and financial hardship and medical constraints on the patient population the nearest nuclear medicine facility was at some 8 hours drive away. In hospital patients would have to be treated either based purely on physical findings or flown to Vancouver at considerable cost to the health care system (estimated cost $1500.

  4. Dynamic Post-Transcriptional Regulation of HIV-1 Gene Expression

    PubMed Central

    Kula, Anna; Marcello, Alessandro

    2012-01-01

    Gene expression of the human immunodeficiency virus type 1 (HIV-1) is a highly regulated process. Basal transcription of the integrated provirus generates early transcripts that encode for the viral products Tat and Rev. Tat promotes the elongation of RNA polymerase while Rev mediates the nuclear export of viral RNAs that contain the Rev-responsive RNA element (RRE). These RNAs are exported from the nucleus to allow expression of Gag-Pol and Env proteins and for the production of full-length genomic RNAs. A balance exists between completely processed mRNAs and RRE-containing RNAs. Rev functions as an adaptor that recruits cellular factors to re-direct singly spliced and unspliced viral RNAs to nuclear export. The aim of this review is to address the dynamic regulation of this post-transcriptional pathway in light of recent findings that implicate several novel cellular cofactors of Rev function. PMID:24832221

  5. Methylation of an intragenic alternative promoter regulates transcription of GARP.

    PubMed

    Haupt, Sonja; Söntgerath, Viktoria Sophie Apollonia; Leipe, Jan; Schulze-Koops, Hendrik; Skapenko, Alla

    2016-02-01

    Alternative promoter usage has been proposed as a mechanism regulating transcriptional and translational diversity in highly elaborated systems like the immune system in humans. Here, we report that transcription of human glycoprotein A repetitions predominant (GARP) in regulatory CD4 T cells (Tregs) is tightly regulated by two alternative promoters. An intragenic promoter contains several CpGs and acts as a weak promoter that is demethylated and initiates transcription Treg-specifically. The strong up-stream promoter containing a CpG-island is, in contrast, fully demethylated throughout tissues. Transcriptional activity of the strong promoter was surprisingly down-regulated upon demethylation of the weak promoter. This demethylation-induced transcriptional attenuation regulated the magnitude of GARP expression and correlated with disease activity in rheumatoid arthritis. Treg-specific GARP transcription was initiated by synergistic interaction of forkhead box protein 3 (Foxp3) with nuclear factor of activated T cells (NFAT) and was underpinned by permissive chromatin remodeling caused by release of the H3K4 demethylase, PLU-1. Our findings describe a novel function of alternative promoters in regulating the extent of transcription. Moreover, since GARP functions as a transporter of transforming growth factor β (TGFβ), a cytokine with broad pleiotropic traits, GARP transcriptional attenuation by alternative promoters might provide a mechanism regulating peripheral TGFβ to avoid unwanted harmful effects.

  6. Transcript isoforms of promyelocytic leukemia in mouse male and female gametes.

    PubMed

    Ebrahimian, Mahboobeh; Mojtahedzadeh, Mahsa; Bazett-Jones, David; Dehghani, Hesam

    2010-01-01

    Promyelocytic leukemia (PML) nuclear bodies and proteins have been implicated in many functions of the nucleus. It is not known whether the PML gene is transcribed and expressed as PML nuclear bodies in gamete cells or in the early mammalian embryo. In this study using reverse transcription-polymerase chain reaction and immunocytochemistry we show the presence of PML transcripts and identify their variants in the mature mouse gametes. Mature sperm contains isoform II; however, oocyte contains transcript isoforms I, II, and possibly other unknown isoforms of PML. This indicates that the mature gametes may carry the transcripts to the newly created embryo. We also show that sperm and oocyte cells do not contain PML nuclear bodies. We find that the first appearance of PML nuclear bodies is in the 2-cell-stage mouse embryo. Appearance of PML nuclear bodies in the 2-cell-stage embryo may correspond to the major transcriptional activity of the embryonic genome. In summary, this report emphasizes the necessity to perform further experiments to investigate the presence and function of PML transcripts and nuclear bodies in earlier stages of germ cell and also later stages of the preimplantation development.

  7. Mapping yeast transcriptional networks.

    PubMed

    Hughes, Timothy R; de Boer, Carl G

    2013-09-01

    The term "transcriptional network" refers to the mechanism(s) that underlies coordinated expression of genes, typically involving transcription factors (TFs) binding to the promoters of multiple genes, and individual genes controlled by multiple TFs. A multitude of studies in the last two decades have aimed to map and characterize transcriptional networks in the yeast Saccharomyces cerevisiae. We review the methodologies and accomplishments of these studies, as well as challenges we now face. For most yeast TFs, data have been collected on their sequence preferences, in vivo promoter occupancy, and gene expression profiles in deletion mutants. These systematic studies have led to the identification of new regulators of numerous cellular functions and shed light on the overall organization of yeast gene regulation. However, many yeast TFs appear to be inactive under standard laboratory growth conditions, and many of the available data were collected using techniques that have since been improved. Perhaps as a consequence, comprehensive and accurate mapping among TF sequence preferences, promoter binding, and gene expression remains an open challenge. We propose that the time is ripe for renewed systematic efforts toward a complete mapping of yeast transcriptional regulatory mechanisms.

  8. Fungal CSL transcription factors

    PubMed Central

    Převorovský, Martin; Půta, František; Folk, Petr

    2007-01-01

    Background The CSL (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) transcription factor family members are well-known components of the transmembrane receptor Notch signaling pathway, which plays a critical role in metazoan development. They function as context-dependent activators or repressors of transcription of their responsive genes, the promoters of which harbor the GTG(G/A)GAA consensus elements. Recently, several studies described Notch-independent activities of the CSL proteins. Results We have identified putative CSL genes in several fungal species, showing that this family is not confined to metazoans. We have analyzed their sequence conservation and identified the presence of well-defined domains typical of genuine CSL proteins. Furthermore, we have shown that the candidate fungal protein sequences contain highly conserved regions known to be required for sequence-specific DNA binding in their metazoan counterparts. The phylogenetic analysis of the newly identified fungal CSL proteins revealed the existence of two distinct classes, both of which are present in all the species studied. Conclusion Our findings support the evolutionary origin of the CSL transcription factor family in the last common ancestor of fungi and metazoans. We hypothesize that the ancestral CSL function involved DNA binding and Notch-independent regulation of transcription and that this function may still be shared, to a certain degree, by the present CSL family members from both fungi and metazoans. PMID:17629904

  9. Focus on Refugees. Transcript.

    ERIC Educational Resources Information Center

    Brandel, Sarah; And Others

    This is the transcript of the "Focus on Refugees," proqram conducted by the Overseas Development Council. Remarks from the following participants are included: (1) Sarah Brandel, Associate Fellow at the Overseas Development Council; (2) Gary Perkins, Chief of Mission of the Washington Office of the United Nations High Commissioner for Refugees…

  10. 10 CFR 1704.8 - Transcripts, recordings, or minutes of closed meetings.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 4 2012-01-01 2012-01-01 false Transcripts, recordings, or minutes of closed meetings. 1704.8 Section 1704.8 Energy DEFENSE NUCLEAR FACILITIES SAFETY BOARD RULES IMPLEMENTING THE GOVERNMENT IN THE SUNSHINE ACT § 1704.8 Transcripts, recordings, or minutes of closed meetings. Along with...

  11. 10 CFR 1704.8 - Transcripts, recordings, or minutes of closed meetings.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 4 2011-01-01 2011-01-01 false Transcripts, recordings, or minutes of closed meetings. 1704.8 Section 1704.8 Energy DEFENSE NUCLEAR FACILITIES SAFETY BOARD RULES IMPLEMENTING THE GOVERNMENT IN THE SUNSHINE ACT § 1704.8 Transcripts, recordings, or minutes of closed meetings. Along with...

  12. 10 CFR 1704.8 - Transcripts, recordings, or minutes of closed meetings.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 4 2010-01-01 2010-01-01 false Transcripts, recordings, or minutes of closed meetings. 1704.8 Section 1704.8 Energy DEFENSE NUCLEAR FACILITIES SAFETY BOARD RULES IMPLEMENTING THE GOVERNMENT IN THE SUNSHINE ACT § 1704.8 Transcripts, recordings, or minutes of closed meetings. Along with...

  13. 10 CFR 1704.9 - Availability and retention of transcripts, recordings, and minutes, and applicable fees.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 4 2010-01-01 2010-01-01 false Availability and retention of transcripts, recordings, and minutes, and applicable fees. 1704.9 Section 1704.9 Energy DEFENSE NUCLEAR FACILITIES SAFETY BOARD RULES IMPLEMENTING THE GOVERNMENT IN THE SUNSHINE ACT § 1704.9 Availability and retention of transcripts,...

  14. 10 CFR 1704.8 - Transcripts, recordings, or minutes of closed meetings.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 4 2014-01-01 2014-01-01 false Transcripts, recordings, or minutes of closed meetings. 1704.8 Section 1704.8 Energy DEFENSE NUCLEAR FACILITIES SAFETY BOARD RULES IMPLEMENTING THE GOVERNMENT IN THE SUNSHINE ACT § 1704.8 Transcripts, recordings, or minutes of closed meetings. Along with...

  15. Children's (Pediatric) Nuclear Medicine

    MedlinePlus Videos and Cool Tools

    ... Professions Site Index A-Z Children's (Pediatric) Nuclear Medicine Children’s (pediatric) nuclear medicine imaging uses small amounts ... Children's Nuclear Medicine? What is Children's (Pediatric) Nuclear Medicine? Nuclear medicine is a branch of medical imaging ...

  16. COMBINING PROTEIN AND mRNA QUANTIFICATION TO DECIPHER TRANSCRIPTIONAL REGULATION

    PubMed Central

    Xu, Heng; Sepúlveda, Leonardo A.; Figard, Lauren; Sokac, Anna Marie; Golding, Ido

    2015-01-01

    We combine immunofluorescence and single-molecule fluorescence in situ hybridization (smFISH), followed by automated image analysis, to quantify the concentration of nuclear transcription factors, number of transcription factors bound, and number of nascent mRNAs synthesized at individual gene loci. A theoretical model is used to decipher how transcription-factor binding modulates the stochastic kinetics of mRNA production. We demonstrate this approach by examining the regulation of hunchback in the early Drosophila embryo. PMID:26098021

  17. MOF Acetyl Transferase Regulates Transcription and Respiration in Mitochondria.

    PubMed

    Chatterjee, Aindrila; Seyfferth, Janine; Lucci, Jacopo; Gilsbach, Ralf; Preissl, Sebastian; Böttinger, Lena; Mårtensson, Christoph U; Panhale, Amol; Stehle, Thomas; Kretz, Oliver; Sahyoun, Abdullah H; Avilov, Sergiy; Eimer, Stefan; Hein, Lutz; Pfanner, Nikolaus; Becker, Thomas; Akhtar, Asifa

    2016-10-20

    A functional crosstalk between epigenetic regulators and metabolic control could provide a mechanism to adapt cellular responses to environmental cues. We report that the well-known nuclear MYST family acetyl transferase MOF and a subset of its non-specific lethal complex partners reside in mitochondria. MOF regulates oxidative phosphorylation by controlling expression of respiratory genes from both nuclear and mtDNA in aerobically respiring cells. MOF binds mtDNA, and this binding is dependent on KANSL3. The mitochondrial pool of MOF, but not a catalytically deficient mutant, rescues respiratory and mtDNA transcriptional defects triggered by the absence of MOF. Mof conditional knockout has catastrophic consequences for tissues with high-energy consumption, triggering hypertrophic cardiomyopathy and cardiac failure in murine hearts; cardiomyocytes show severe mitochondrial degeneration and deregulation of mitochondrial nutrient metabolism and oxidative phosphorylation pathways. Thus, MOF is a dual-transcriptional regulator of nuclear and mitochondrial genomes connecting epigenetics and metabolism.

  18. Nuclear risk

    SciTech Connect

    Levenson, M.

    1989-01-01

    The title of our session, Nuclear Risk Versus Other Power Options, is provocative. It is also a title with different meanings to different people. To the utility chief executive officer, nuclear power is a high-risk financial undertaking because of political and economic barriers to cost recovery. To the utility dispatcher, it is a high-risk future power source since plant completion and start-up dates can be delayed for very long times due to uncertain legal and political issues. To the environmentalist, concerned about global effects such as greenhouse and acid rain, nuclear power is a relatively low risk energy source. To the financial people, nuclear power is a cash cow turned sour because of uncertainties as to what new plants will cost and whether they will even be allowed to operate. The statistics on risk are known and the results of probability risk assessment calculations of risks are known. The challenge is not to make nuclear power safer, it is already one of the safest, if not the safest, source of power currently available. The challenge is to find a way to communicate this to the public.

  19. Nuclear receptors and nonalcoholic fatty liver disease.

    PubMed

    Cave, Matthew C; Clair, Heather B; Hardesty, Josiah E; Falkner, K Cameron; Feng, Wenke; Clark, Barbara J; Sidey, Jennifer; Shi, Hongxue; Aqel, Bashar A; McClain, Craig J; Prough, Russell A

    2016-09-01

    Nuclear receptors are transcription factors which sense changing environmental or hormonal signals and effect transcriptional changes to regulate core life functions including growth, development, and reproduction. To support this function, following ligand-activation by xenobiotics, members of subfamily 1 nuclear receptors (NR1s) may heterodimerize with the retinoid X receptor (RXR) to regulate transcription of genes involved in energy and xenobiotic metabolism and inflammation. Several of these receptors including the peroxisome proliferator-activated receptors (PPARs), the pregnane and xenobiotic receptor (PXR), the constitutive androstane receptor (CAR), the liver X receptor (LXR) and the farnesoid X receptor (FXR) are key regulators of the gut:liver:adipose axis and serve to coordinate metabolic responses across organ systems between the fed and fasting states. Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and may progress to cirrhosis and even hepatocellular carcinoma. NAFLD is associated with inappropriate nuclear receptor function and perturbations along the gut:liver:adipose axis including obesity, increased intestinal permeability with systemic inflammation, abnormal hepatic lipid metabolism, and insulin resistance. Environmental chemicals may compound the problem by directly interacting with nuclear receptors leading to metabolic confusion and the inability to differentiate fed from fasting conditions. This review focuses on the impact of nuclear receptors in the pathogenesis and treatment of NAFLD. Clinical trials including PIVENS and FLINT demonstrate that nuclear receptor targeted therapies may lead to the paradoxical dissociation of steatosis, inflammation, fibrosis, insulin resistance, dyslipidemia and obesity. Novel strategies currently under development (including tissue-specific ligands and dual receptor agonists) may be required to separate the beneficial effects of nuclear receptor activation from unwanted metabolic

  20. Nuclear waste

    SciTech Connect

    Not Available

    1988-05-01

    This paper discusses how, as part of the Department of Energy's implementation of the Nuclear Waste Policy Act of 1982, DOE is required to investigate a site at Yucca Mountain, Nevada and, if it determines that the site is suitable, recommend to the President its selection for a nuclear waste repository. The Nuclear Regulatory Commission, in considering development of the plan, issued five objections, one of which is DOE's failure to recognize the range of alternative conceptual models of the Yucca Mountain site that can be supported by the limited existing technical data. At the end of the quarter DOE directed its project offices in Washington and Texas to begin orderly phase-out of all site-specific repository activities. Costs for this phase-out are $53 million for the Deaf Smith site and $85 million for the Hanford site.

  1. Transcriptionally active genome regions are preferred targets for retrovirus integration.

    PubMed Central

    Scherdin, U; Rhodes, K; Breindl, M

    1990-01-01

    We have analyzed the transcriptional activity of cellular target sequences for Moloney murine leukemia virus integration in mouse fibroblasts. At least five of the nine random, unselected integration target sequences studied showed direct evidence for transcriptional activity by hybridization to nuclear run-on transcripts prepared from uninfected cells. At least four of the sequences contained multiple recognition sites for several restriction enzymes that cut preferentially in CpG-rich islands, indicating integration into 5' or 3' ends or flanking regions of genes. Assuming that only a minor fraction (less than 20%) of the genome is transcribed in mammalian cells, we calculated the probability that this association of retroviral integration sites with transcribed sequences is due to chance to be very low (1.6 x 10(-2]. Thus, our results strongly suggest that transcriptionally active genome regions are preferred targets for retrovirus integration. Images PMID:2296087

  2. 7SK-BAF axis controls pervasive transcription at enhancers

    PubMed Central

    Flynn, Ryan A.; Do, Brian T.; Rubin, Adam J.; Calo, Eliezer; Lee, Byron; Kuchelmeister, Hannes; Rale, Michael; Chu, Ci; Kool, Eric T.; Wysocka, Joanna; Khavari, Paul A.

    2016-01-01

    RNA functions at enhancers remain mysterious. Here we show that the 7SK small nuclear RNA (snRNA) inhibits enhancer transcription by modulating nucleosome position. 7SK occupies enhancers and super enhancers genome-wide in mouse and human cells, and 7SK is required to limit eRNA initiation and synthesis in a manner distinct from promoter pausing. Clustered elements at super enhancers uniquely require 7SK to prevent convergent transcription and DNA damage signaling. 7SK physically interacts with the BAF chromatin remodeling complex, recruit BAF to enhancers, and inhibits enhancer transcription by modulating chromatin structure. In turn, 7SK occupancy at enhancers coincides with Brd4 and is exquisitely sensitive to the bromodomain inhibitor JQ1. Thus, 7SK employs distinct mechanisms to counteract diverse consequences of pervasive transcription that distinguish super enhancers, enhancers, and promoters. PMID:26878240

  3. Nuclear Models

    NASA Astrophysics Data System (ADS)

    Fossión, Rubén

    2010-09-01

    The atomic nucleus is a typical example of a many-body problem. On the one hand, the number of nucleons (protons and neutrons) that constitute the nucleus is too large to allow for exact calculations. On the other hand, the number of constituent particles is too small for the individual nuclear excitation states to be explained by statistical methods. Another problem, particular for the atomic nucleus, is that the nucleon-nucleon (n-n) interaction is not one of the fundamental forces of Nature, and is hard to put in a single closed equation. The nucleon-nucleon interaction also behaves differently between two free nucleons (bare interaction) and between two nucleons in the nuclear medium (dressed interaction). Because of the above reasons, specific nuclear many-body models have been devised of which each one sheds light on some selected aspects of nuclear structure. Only combining the viewpoints of different models, a global insight of the atomic nucleus can be gained. In this chapter, we revise the the Nuclear Shell Model as an example of the microscopic approach, and the Collective Model as an example of the geometric approach. Finally, we study the statistical properties of nuclear spectra, basing on symmetry principles, to find out whether there is quantum chaos in the atomic nucleus. All three major approaches have been rewarded with the Nobel Prize of Physics. In the text, we will stress how each approach introduces its own series of approximations to reduce the prohibitingly large number of degrees of freedom of the full many-body problem to a smaller manageable number of effective degrees of freedom.

  4. Nuclear pursuits

    SciTech Connect

    Not Available

    1993-05-01

    This table lists quantities of warheads (in stockpile, peak number per year, total number built, number of known test explosions), weapon development milestones (developers of the atomic bomb and hydrogen bomb, date of first operational ICBM, first nuclear-powered naval SSN in service, first MIRVed missile deployed), and testing milestones (first fission test, type of boosted fission weapon, multistage thermonuclear test, number of months from fission bomb to multistage thermonuclear bomb, etc.), and nuclear infrastructure (assembly plants, plutonium production reactors, uranium enrichment plants, etc.). Countries included in the tally are the United States, Soviet Union, Britain, France, and China.

  5. Nuclear Receptors, RXR, and the Big Bang.

    PubMed

    Evans, Ronald M; Mangelsdorf, David J

    2014-03-27

    Isolation of genes encoding the receptors for steroids, retinoids, vitamin D, and thyroid hormone and their structural and functional analysis revealed an evolutionarily conserved template for nuclear hormone receptors. This discovery sparked identification of numerous genes encoding related proteins, termed orphan receptors. Characterization of these orphan receptors and, in particular, of the retinoid X receptor (RXR) positioned nuclear receptors at the epicenter of the "Big Bang" of molecular endocrinology. This Review provides a personal perspective on nuclear receptors and explores their integrated and coordinated signaling networks that are essential for multicellular life, highlighting the RXR heterodimer and its associated ligands and transcriptional mechanism.

  6. Nuclear Receptors, RXR & the Big Bang

    PubMed Central

    Evans, Ronald M.; Mangelsdorf, David J.

    2014-01-01

    Summary Isolation of genes encoding the receptors for steroids, retinoids, vitamin D and thyroid hormone, and their structural and functional analysis revealed an evolutionarily conserved template for nuclear hormone receptors. This discovery sparked identification of numerous genes encoding related proteins, termed orphan receptors. Characterization of these orphan receptors, and in particular of the retinoid X receptor (RXR), positioned nuclear receptors at the epicenter of the “Big Bang” of molecular endocrinology. This review provides a personal perspective on nuclear receptors and explores their integrated and coordinated signaling networks that are essential for multi-cellular life, highlighting the RXR heterodimer and its associated ligands and transcriptional mechanism. PMID:24679540

  7. Nuclear Dynamics of BRCA1-Dependent Transcription Regulation

    DTIC Science & Technology

    2006-08-01

    are ligand- and domain-dependent Z. Dave Sharp1,*, Maureen G. Mancini2,*, Cruz A. Hinojos2,*, Fangyan Dai2, Valeria Berno2, Adam T. Szafran2, Kelly P... Barron et al., 1989; Cao et al., 1987; Crenshaw et al., 1989; Day et al., 1990; Howard and Maurer, 1995; Ingraham et al., 1990; Mangalam et al., 1989...References Barron , E. A., Cao, Z., Schneider, B. G., Kraig, E., Carrillo, A. J. and Sharp, Z. D. (1989). Dual functions of a cis-acting element within the

  8. Machine Transcription--Practically Speaking.

    ERIC Educational Resources Information Center

    Clippinger, Dorinda A.

    1984-01-01

    Draws transcription teaching principles from Gagne's theories about learning. Recommends 12-16 weeks of instruction, pre-transcription development of related skills, frequent feedback, and use of teaching materials that are arranged to take advantage of learning cycles. (SK)

  9. Using Nuclear Receptor Activity to Stratify Hepatocarcinogens

    EPA Science Inventory

    Nuclear receptors (NR) are a superfamily of ligand-activated transcription factors that control a range of cellular processes. Persistent stimulation of some NR is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. Here we report on a systematic an...

  10. Non-transcriptional regulatory processes shape transcriptional network dynamics.

    PubMed

    Ray, J Christian J; Tabor, Jeffrey J; Igoshin, Oleg A

    2011-10-11

    Information about the extra- or intracellular environment is often captured as biochemical signals that propagate through regulatory networks. These signals eventually drive phenotypic changes, typically by altering gene expression programmes in the cell. Reconstruction of transcriptional regulatory networks has given a compelling picture of bacterial physiology, but transcriptional network maps alone often fail to describe phenotypes. Cellular response dynamics are ultimately determined by interactions between transcriptional and non-transcriptional networks, with dramatic implications for physiology and evolution. Here, we provide an overview of non-transcriptional interactions that can affect the performance of natural and synthetic bacterial regulatory networks.

  11. Transcriptional Regulation of Tetrapyrrole Biosynthesis in Arabidopsis thaliana

    PubMed Central

    Kobayashi, Koichi; Masuda, Tatsuru

    2016-01-01

    Biosynthesis of chlorophyll (Chl) involves many enzymatic reactions that share several first steps for biosynthesis of other tetrapyrroles such as heme, siroheme, and phycobilins. Chl allows photosynthetic organisms to capture light energy for photosynthesis but with simultaneous threat of photooxidative damage to cells. To prevent photodamage by Chl and its highly photoreactive intermediates, photosynthetic organisms have developed multiple levels of regulatory mechanisms to coordinate tetrapyrrole biosynthesis (TPB) with the formation of photosynthetic and photoprotective systems and to fine-tune the metabolic flow with the varying needs of Chl and other tetrapyrroles under various developmental and environmental conditions. Among a wide range of regulatory mechanisms of TPB, this review summarizes transcriptional regulation of TPB genes during plant development, with focusing on several transcription factors characterized in Arabidopsis thaliana. Key TPB genes are tightly coexpressed with other photosynthesis-associated nuclear genes and are induced by light, oscillate in a diurnal and circadian manner, are coordinated with developmental and nutritional status, and are strongly downregulated in response to arrested chloroplast biogenesis. LONG HYPOCOTYL 5 and PHYTOCHROME-INTERACTING FACTORs, which are positive and negative transcription factors with a wide range of light signaling, respectively, target many TPB genes for light and circadian regulation. GOLDEN2-LIKE transcription factors directly regulate key TPB genes to fine-tune the formation of the photosynthetic apparatus with chloroplast functionality. Some transcription factors such as FAR-RED ELONGATED HYPOCOTYL3, REVEILLE1, and scarecrow-like transcription factors may directly regulate some specific TPB genes, whereas other factors such as GATA transcription factors are likely to regulate TPB genes in an indirect manner. Comprehensive transcriptional analyses of TPB genes and detailed characterization of

  12. Nuclear orbiting

    SciTech Connect

    Shapira, D.

    1988-01-01

    Nuclear orbiting following collisions between sd and p shell nuclei is discussed. The dependence of this process on the real and imaginary parts of the nucleus-nucleus potential is discussed, as well as the evolution of the dinucleus toward a fully equilibrated fused system. 26 refs., 15 figs.

  13. Nuclear Misinformation

    ERIC Educational Resources Information Center

    Ford, Daniel F.; Kendall, Henry W.

    1975-01-01

    Many scientists feel that research into nuclear safety has been diverted or distorted, and the results of the research concealed or inaccurately reported on a large number of occasions. Of particular concern have been the emergency cooling systems which have not, as yet, been adequately tested. (Author/MA)

  14. Nuclear Terrorism.

    SciTech Connect

    Hecker, Siegfried S.

    2001-01-01

    As pointed out by several speakers, the level of violence and destruction in terrorist attacks has increased significantly during the past decade. Fortunately, few have involved weapons of mass destruction, and none have achieved mass casualties. The Aum Shinrikyo release of lethal nerve agent, sarin, in the Tokyo subway on March 20, 1995 clearly broke new ground by crossing the threshold in attempting mass casualties with chemical weapons. However, of all weapons of mass destruction, nuclear weapons still represent the most frightening threat to humankind. Nuclear weapons possess an enormous destructive force. The immediacy and scale of destruction are unmatched. In addition to destruction, terrorism also aims to create fear among the public and governments. Here also, nuclear weapons are unmatched. The public's fear of nuclear weapons or, for that matter, of all radioactivity is intense. To some extent, this fear arises from a sense of unlimited vulnerability. That is, radioactivity is seen as unbounded in three dimensions - distance, it is viewed as having unlimited reach; quantity, it is viewed as having deadly consequences in the smallest doses (the public is often told - incorrectly, of course - that one atom of plutonium will kill); and time, if it does not kill you immediately, then it will cause cancer decades hence.

  15. Nuclear medicine

    SciTech Connect

    Wagner, H.N. Jr.

    1986-10-17

    In 1985 and 1986 nuclear medicine became more and more oriented toward in vov chemistry, chiefly as a result of advances in positron emission tomography (PET). The most important trend was the extension of PET technology into the care of patients with brain tumors, epilepsy, and heart disease. A second trend was the increasing use of single-photon emission computed tomography (SPECT).

  16. NUCLEAR REACTOR

    DOEpatents

    Treshow, M.

    1961-09-01

    A boiling-water nuclear reactor is described wherein control is effected by varying the moderator-to-fuel ratio in the reactor core. This is accomplished by providing control tubes containing a liquid control moderator in the reactor core and providing means for varying the amount of control moderatcr within the control tubes.

  17. Nuclear Science.

    ERIC Educational Resources Information Center

    Pennsylvania State Dept. of Education, Harrisburg. Bureau of Curriculum Services.

    This document is a report on a course in nuclear science for the high school curriculum. The course is designed to provide a basic but comprehensive understanding of the atom in the light of modern knowledge, and to show how people attempt to harness the tremendous energy liberated through fission and fusion reactions. The course crosses what are…

  18. Nuclear energy.

    PubMed

    Wilson, Peter D

    2010-01-01

    The technical principles and practices of the civil nuclear industry are described with particular reference to fission and its products, natural and artificial radioactivity elements principally concerned and their relationships, main types of reactor, safety issues, the fuel cycle, waste management, issues related to weapon proliferation, environmental considerations and possible future developments.

  19. Transcriptional activation of Xenopus class III genes in chromatin isolated from sperm and somatic nuclei.

    PubMed Central

    Wolffe, A P

    1989-01-01

    Xenopus sperm chromatin lacks class III transcription complexes and somatic histone H1. Inactive class III genes in sperm chromatin are easily programmed with transcription complexes de novo and transcribed in Xenopus oocyte nuclear extract. In contrast, repressed class III genes in somatic chromatin are not transcribed in the oocyte nuclear extract. Class III genes that are initially inactive or repressed in both types of chromatin can be efficiently transcribed in a cell free preparation of Xenopus eggs. Chromatin mediated repression of class III genes in somatic nuclei is reversible in Xenopus egg extract, but not in the oocyte nuclear extract. Any inhibition of transcription attributed to chromatin assembly onto a gene, will therefore depend on the extract in which transcription is assayed. Images PMID:2915929

  20. Transcriptional regulation of steroid hydroxylase genes by corticotropin.

    PubMed Central

    John, M E; John, M C; Boggaram, V; Simpson, E R; Waterman, M R

    1986-01-01

    Maintenance of optimal steroidogenic capacity in the adrenal cortex is the result of a cAMP-dependent response to the peptide hormone corticotropin (ACTH). The molecular mechanism of this action of ACTH has been examined by using five recombinant DNA clones specific for enzymes of the steroidogenic pathway (P-450scc, P-45011 beta, P-450C21, P-45017 alpha, and adrenodoxin). The presence of nuclear precursors in steady-state RNA samples derived from cultured bovine adrenocortical cells and moderate increases in the number of RNA chain initiations, as determined by in vitro nuclear run-off assays, indicate that ACTH controls the expression of the gene(s) for each of these proteins at the transcriptional level. The ACTH-mediated increase in accumulation of transcripts specific for steroid hydroxylases in nuclear RNA can be specifically blocked by inhibiting protein synthesis in bovine adrenocortical cell cultures. The steady-state concentrations of nuclear RNA for control genes show no decrease upon cycloheximide treatment. These studies suggest that a primary action of ACTH in the adrenal cortex is to activate (via cAMP) the synthesis of rapidly turning over protein factors that in turn mediate increased initiation of transcription of steroid hydroxylase genes. We propose that these protein factors impart specificity of induction to genes encoding components of this pathway in steroidogenic tissues. Images PMID:3014507

  1. The EDLL motif: a potent plant transcriptional activation domain from AP2/ERF transcription factors.

    PubMed

    Tiwari, Shiv B; Belachew, Alemu; Ma, Siu Fong; Young, Melinda; Ade, Jules; Shen, Yu; Marion, Colleen M; Holtan, Hans E; Bailey, Adina; Stone, Jeffrey K; Edwards, Leslie; Wallace, Andreah D; Canales, Roger D; Adam, Luc; Ratcliffe, Oliver J; Repetti, Peter P

    2012-06-01

    In plants, the ERF/EREBP family of transcriptional regulators plays a key role in adaptation to various biotic and abiotic stresses. These proteins contain a conserved AP2 DNA-binding domain and several uncharacterized motifs. Here, we describe a short motif, termed