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Sample records for regulatory dna elements

  1. Transcription regulatory elements are punctuation marks for DNA replication.

    PubMed

    Mirkin, Ekaterina V; Castro Roa, Daniel; Nudler, Evgeny; Mirkin, Sergei M

    2006-05-01

    Collisions between DNA replication and transcription significantly affect genome organization, regulation, and stability. Previous studies have described collisions between replication forks and elongating RNA polymerases. Although replication collisions with the transcription-initiation or -termination complexes are potentially even more important because most genes are not actively transcribed during DNA replication, their existence and mechanisms remained unproven. To address this matter, we have designed a bacterial promoter that binds RNA polymerase and maintains it in the initiating mode by precluding the transition into the elongation mode. By using electrophoretic analysis of replication intermediates, we have found that this steadfast transcription-initiation complex inhibits replication fork progression in an orientation-dependent manner during head-on collisions. Transcription terminators also appeared to attenuate DNA replication, but in the opposite, codirectional orientation. Thus, transcription regulatory signals may serve as "punctuation marks" for DNA replication in vivo. PMID:16670199

  2. Cotyledon nuclear proteins bind to DNA fragments harboring regulatory elements of phytohemagglutinin genes.

    PubMed Central

    Riggs, C D; Voelker, T A; Chrispeels, M J

    1989-01-01

    The effects of deleting DNA sequences upstream from the phytohemagglutinin-L gene of Phaseolus vulgaris have been examined with respect to the level of gene product produced in the seeds of transgenic tobacco. Our studies indicate that several upstream regions quantitatively modulate expression. Between -1000 and -675, a negative regulatory element reduces expression approximately threefold relative to shorter deletion mutants that do not contain this region. Positive regulatory elements lie between -550 and -125 and, compared with constructs containing only 125 base pairs of upstream sequences (-125), the presence of these two regions can be correlated with a 25-fold and a 200-fold enhancement of phytohemagglutinin-L levels. These experiments were complemented by gel retardation assays, which demonstrated that two of the three regions bind cotyledon nuclear proteins from mid-mature seeds. One of the binding sites maps near a DNA sequence that is highly homologous to protein binding domains located upstream from the soybean seed lectin and Kunitz trypsin inhibitor genes. Competition experiments demonstrated that the upstream regions of a bean beta-phaseolin gene, the soybean seed lectin gene, and an oligonucleotide from the upstream region of the trypsin inhibitor gene can compete differentially for factor binding. We suggest that these legume genes may be regulated in part by evolutionarily conserved protein/DNA interactions. PMID:2535513

  3. Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9

    PubMed Central

    Li, Jinhuan; Shou, Jia; Guo, Ya; Tang, Yuanxiao; Wu, Yonghu; Jia, Zhilian; Zhai, Yanan; Chen, Zhifeng; Xu, Quan; Wu, Qiang

    2015-01-01

    The human genome contains millions of DNA regulatory elements and a large number of gene clusters, most of which have not been tested experimentally. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) programed with a synthetic single-guide RNA (sgRNA) emerges as a method for genome editing in virtually any organisms. Here we report that targeted DNA fragment inversions and duplications could easily be achieved in human and mouse genomes by CRISPR with two sgRNAs. Specifically, we found that, in cultured human cells and mice, efficient precise inversions of DNA fragments ranging in size from a few tens of bp to hundreds of kb could be generated. In addition, DNA fragment duplications and deletions could also be generated by CRISPR through trans-allelic recombination between the Cas9-induced double-strand breaks (DSBs) on two homologous chromosomes (chromatids). Moreover, junctions of combinatorial inversions and duplications of the protocadherin (Pcdh) gene clusters induced by Cas9 with four sgRNAs could be detected. In mice, we obtained founders with alleles of precise inversions, duplications, and deletions of DNA fragments of variable sizes by CRISPR. Interestingly, we found that very efficient inversions were mediated by microhomology-mediated end joining (MMEJ) through short inverted repeats. We showed for the first time that DNA fragment inversions could be transmitted through germlines in mice. Finally, we applied this CRISPR method to a regulatory element of the Pcdhα cluster and found a new role in the regulation of members of the Pcdhγ cluster. This simple and efficient method should be useful in manipulating mammalian genomes to study millions of regulatory DNA elements as well as vast numbers of gene clusters. PMID:25757625

  4. Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9.

    PubMed

    Li, Jinhuan; Shou, Jia; Guo, Ya; Tang, Yuanxiao; Wu, Yonghu; Jia, Zhilian; Zhai, Yanan; Chen, Zhifeng; Xu, Quan; Wu, Qiang

    2015-08-01

    The human genome contains millions of DNA regulatory elements and a large number of gene clusters, most of which have not been tested experimentally. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) programed with a synthetic single-guide RNA (sgRNA) emerges as a method for genome editing in virtually any organisms. Here we report that targeted DNA fragment inversions and duplications could easily be achieved in human and mouse genomes by CRISPR with two sgRNAs. Specifically, we found that, in cultured human cells and mice, efficient precise inversions of DNA fragments ranging in size from a few tens of bp to hundreds of kb could be generated. In addition, DNA fragment duplications and deletions could also be generated by CRISPR through trans-allelic recombination between the Cas9-induced double-strand breaks (DSBs) on two homologous chromosomes (chromatids). Moreover, junctions of combinatorial inversions and duplications of the protocadherin (Pcdh) gene clusters induced by Cas9 with four sgRNAs could be detected. In mice, we obtained founders with alleles of precise inversions, duplications, and deletions of DNA fragments of variable sizes by CRISPR. Interestingly, we found that very efficient inversions were mediated by microhomology-mediated end joining (MMEJ) through short inverted repeats. We showed for the first time that DNA fragment inversions could be transmitted through germlines in mice. Finally, we applied this CRISPR method to a regulatory element of the Pcdhα cluster and found a new role in the regulation of members of the Pcdhγ cluster. This simple and efficient method should be useful in manipulating mammalian genomes to study millions of regulatory DNA elements as well as vast numbers of gene clusters. PMID:25757625

  5. De novo DNA demethylation and noncoding transcription define active intergenic regulatory elements.

    PubMed

    Schlesinger, Felix; Smith, Andrew D; Gingeras, Thomas R; Hannon, Gregory J; Hodges, Emily

    2013-10-01

    Deep sequencing of mammalian DNA methylomes has uncovered a previously unpredicted number of discrete hypomethylated regions in intergenic space (iHMRs). Here, we combined whole-genome bisulfite sequencing data with extensive gene expression and chromatin-state data to define functional classes of iHMRs, and to reconstruct the dynamics of their establishment in a developmental setting. Comparing HMR profiles in embryonic stem and primary blood cells, we show that iHMRs mark an exclusive subset of active DNase hypersensitive sites (DHS), and that both developmentally constitutive and cell-type-specific iHMRs display chromatin states typical of distinct regulatory elements. We also observe that iHMR changes are more predictive of nearby gene activity than the promoter HMR itself, and that expression of noncoding RNAs within the iHMR accompanies full activation and complete demethylation of mature B cell enhancers. Conserved sequence features corresponding to iHMR transcript start sites, including a discernible TATA motif, suggest a conserved, functional role for transcription in these regions. Similarly, we explored both primate-specific and human population variation at iHMRs, finding that while enhancer iHMRs are more variable in sequence and methylation status than any other functional class, conservation of the TATA box is highly predictive of iHMR maintenance, reflecting the impact of sequence plasticity and transcriptional signals on iHMR establishment. Overall, our analysis allowed us to construct a three-step timeline in which (1) intergenic DHS are pre-established in the stem cell, (2) partial demethylation of blood-specific intergenic DHSs occurs in blood progenitors, and (3) complete iHMR formation and transcription coincide with enhancer activation in lymphoid-specified cells. PMID:23811145

  6. De novo DNA demethylation and noncoding transcription define active intergenic regulatory elements

    PubMed Central

    Schlesinger, Felix; Smith, Andrew D.; Gingeras, Thomas R.; Hannon, Gregory J.; Hodges, Emily

    2013-01-01

    Deep sequencing of mammalian DNA methylomes has uncovered a previously unpredicted number of discrete hypomethylated regions in intergenic space (iHMRs). Here, we combined whole-genome bisulfite sequencing data with extensive gene expression and chromatin-state data to define functional classes of iHMRs, and to reconstruct the dynamics of their establishment in a developmental setting. Comparing HMR profiles in embryonic stem and primary blood cells, we show that iHMRs mark an exclusive subset of active DNase hypersensitive sites (DHS), and that both developmentally constitutive and cell-type-specific iHMRs display chromatin states typical of distinct regulatory elements. We also observe that iHMR changes are more predictive of nearby gene activity than the promoter HMR itself, and that expression of noncoding RNAs within the iHMR accompanies full activation and complete demethylation of mature B cell enhancers. Conserved sequence features corresponding to iHMR transcript start sites, including a discernible TATA motif, suggest a conserved, functional role for transcription in these regions. Similarly, we explored both primate-specific and human population variation at iHMRs, finding that while enhancer iHMRs are more variable in sequence and methylation status than any other functional class, conservation of the TATA box is highly predictive of iHMR maintenance, reflecting the impact of sequence plasticity and transcriptional signals on iHMR establishment. Overall, our analysis allowed us to construct a three-step timeline in which (1) intergenic DHS are pre-established in the stem cell, (2) partial demethylation of blood-specific intergenic DHSs occurs in blood progenitors, and (3) complete iHMR formation and transcription coincide with enhancer activation in lymphoid-specified cells. PMID:23811145

  7. Lead Exposure during Early Human Development and DNA Methylation of Imprinted Gene Regulatory Elements in Adulthood

    PubMed Central

    Li, Yue; Xie, Changchun; Murphy, Susan K.; Skaar, David; Nye, Monica; Vidal, Adriana C.; Cecil, Kim M.; Dietrich, Kim N.; Puga, Alvaro; Jirtle, Randy L.; Hoyo, Cathrine

    2015-01-01

    30 to 78 months. Conclusions: Our findings provide evidence that early childhood lead exposure results in sex-dependent and gene-specific DNA methylation differences in the DMRs of PEG3, IGF2/H19, and PLAGL1/HYMAI in adulthood. Citation: Li Y, Xie C, Murphy SK, Skaar D, Nye M, Vidal AC, Cecil KM, Dietrich KN, Puga A, Jirtle RL, Hoyo C. 2016. Lead exposure during early human development and DNA methylation of imprinted gene regulatory elements in adulthood. Environ Health Perspect 124:666–673; http://dx.doi.org/10.1289/ehp.1408577 PMID:26115033

  8. Identification of Regulatory DNA Elements Using Genome-wide Mapping of DNase I Hypersensitive Sites during Tomato Fruit Development.

    PubMed

    Qiu, Zhengkun; Li, Ren; Zhang, Shuaibin; Wang, Ketao; Xu, Meng; Li, Jiayang; Du, Yongchen; Yu, Hong; Cui, Xia

    2016-08-01

    Development and ripening of tomato fruit are precisely controlled by transcriptional regulation, which depends on the orchestrated accessibility of regulatory proteins to promoters and other cis-regulatory DNA elements. This accessibility and its effect on gene expression play a major role in defining the developmental process. To understand the regulatory mechanism and functional elements modulating morphological and anatomical changes during fruit development, we generated genome-wide high-resolution maps of DNase I hypersensitive sites (DHSs) from the fruit tissues of the tomato cultivar "Moneymaker" at 20 days post anthesis as well as break stage. By exploring variation of DHSs across fruit development stages, we pinpointed the most likely hypersensitive sites related to development-specific genes. By detecting binding motifs on DHSs of these development-specific genes or genes in the ascorbic acid biosynthetic pathway, we revealed the common regulatory elements contributing to coordinating gene transcription of plant ripening and specialized metabolic pathways. Our results contribute to a better understanding of the regulatory dynamics of genes involved in tomato fruit development and ripening. PMID:27250572

  9. Identification and characterization of methylation-dependent/independent DNA regulatory elements in the human SLC9B1 gene

    PubMed Central

    Kumar, Priya L.; James, Paul F.

    2015-01-01

    The human NHEDC1 (hNHEDC1) protein is thought to be essential for sperm motility and fertility however the mechanisms regulating its gene expression are largely unknown. In this study we have identified multiple DNA regulatory elements in the 5′ end of the gene encoding hNHEDC1 (SLC9B1) and have explored the role that DNA methylation at these elements plays in the regulation of its expression. We first show that the full-length hNHEDC1 protein is testis-specific for the tissues that we tested and that it localizes to the cells of the seminiferous tubules. In silico analysis of the SLC9B1 gene locus identified two putative promoters (P1 and P2) and two CpG islands - CpGI (overlapping with P1) and CpGII (intragenic) - at the 5′ end of the gene. By deletion analysis of P1, we show that the region from −23bp to +200bp relative to the transcription start site (TSS) is sufficient for optimal promoter activity in a germ cell line. Additionally, in vitro methylation of the P1 (the −500bp to +200bp region relative to the TSS) abolishes its activity in germ cells and somatic cells strongly suggesting that DNA methylation at this promoter could regulate SLC9B1 expression. Furthermore, bisulfite-sequencing analysis of the P1/CpGI uncovered reduced methylation in the testis vs. lung whereas CpGII displayed no differences in methylation between these two tissues. Additionally, treatment of HEK 293 cells with 5-Aza2-Deoxycytidine led to upregulation of NHEDC1 transcript and reduced methylation in the promoter CpGI. Finally, we have uncovered both enhancer and silencer functions of the intragenic SLC9B1 CpGII. In all, our data suggests that SLC9B1 gene expression could be regulated via a concerted action of DNA methylation-dependent and independent mechanisms mediated by these multiple DNA regulatory elements. PMID:25701605

  10. Ubiquitous and neuronal DNA-binding proteins interact with a negative regulatory element of the human hypoxanthine phosphoribosyltransferase gene.

    PubMed Central

    Rincón-Limas, D E; Amaya-Manzanares, F; Niño-Rosales, M L; Yu, Y; Yang, T P; Patel, P I

    1995-01-01

    The hypoxanthine phosphoribosyltransferase (HPRT) gene is constitutively expressed at low levels in all tissues but at higher levels in the brain; the significance and mechanism of this differential expression are unknown. We previously identified a 182-bp element (hHPRT-NE) within the 5'-flanking region of the human HPRT (hHPRT) gene, which is involved not only in conferring neuronal specificity but also in repressing gene expression in nonneuronal tissues. Here we report that this element interacts with different nuclear proteins, some of which are present specifically in neuronal cells (complex I) and others of which are present in cells showing constitutive expression of the gene (complex II). In addition, we found that complex I factors are expressed in human NT2/D1 cells following induction of neuronal differentiation by retinoic acid. This finding correlates with an increase of HPRT gene transcription following neuronal differentiation. We also mapped the binding sites for both complexes to a 60-bp region (Ff; positions -510 to -451) which, when analyzed in transfection assays, functioned as a repressor element analogous to the full-length hHPRT-NE sequence. Methylation interference footprintings revealed a minimal unique DNA motif, 5'-GGAAGCC-3', as the binding site for nuclear proteins from both neuronal and nonneuronal sources. However, site-directed mutagenesis of the footprinted region indicated that different nucleotides are essential for the associations of these two complexes. Moreover, UV cross-linking experiments showed that both complexes are formed by the association of several different proteins. Taken together, these data suggest that differential interaction of DNA-binding factors with this regulatory element plays a crucial role in the brain-preferential expression of the gene, and they should lead to the isolation of transcriptional regulators important in neuronal expression of the HPRT gene. PMID:8524221

  11. Hormone stimulation of androgen receptor mediates dynamic changes in DNA methylation patterns at regulatory elements

    PubMed Central

    Dhiman, Vineet K.; Attwood, Kristopher; Campbell, Moray J.; Smiraglia, Dominic J.

    2015-01-01

    DNA methylation is an epigenetic modification that contributes to stable gene silencing by interfering with the ability of transcriptional regulators to bind to DNA. Recent findings have revealed that hormone stimulation of certain nuclear receptors induces rapid, dynamic changes in DNA methylation patterns alongside transcriptional responses at a subset of target loci, over time. However, the ability of androgen receptor (AR) to dynamically regulate gene transcription is relatively under-studied and its role in the regulation of DNA methylation patterns remains to be elucidated. Here we demonstrate in normal prostate cells that hormone stimulated AR activity results in dynamic changes in the transcription rate and DNA methylation patterns at the AR target genes, TIPARP and SGK1. Time-resolved chromatin immunoprecipitation experiments on the SGK1 locus reveals dynamic recruitment of AR and RNA Polymerase II, as well as the recruitment of proteins involved in the DNA demethylation process, TET1 and TDG. Furthermore, the presence of DNA methylation at dynamic regions inhibits protein binding and transcriptional activity of SGK1. These findings establish AR activity as a contributing factor to the dynamic regulation of DNA methylation patterns at target genes in prostate biology and infer further complexity involved in nuclear receptor mediation of transcriptional regulation. PMID:26646795

  12. A novel regulatory element (E77) isolated from CHO-K1 genomic DNA enhances stable gene expression in Chinese hamster ovary cells.

    PubMed

    Kang, Shin-Young; Kim, Yeon-Gu; Kang, Seunghee; Lee, Hong Weon; Lee, Eun Gyo

    2016-05-01

    Vectors flanked by regulatory DNA elements have been used to generate stable cell lines with high productivity and transgene stability; however, regulatory elements in Chinese hamster ovary (CHO) cells, which are the most widely used mammalian cells in biopharmaceutical production, are still poorly understood. We isolated a novel gene regulatory element from CHO-K1 cells, designated E77, which was found to enhance the stable expression of a transgene. A genomic library was constructed by combining CHO-K1 genomic DNA fragments with a CMV promoter-driven GFP expression vector, and the E77 element was isolated by screening. The incorporation of the E77 regulatory element resulted in the generation of an increased number of clones with high expression, thereby enhancing the expression level of the transgene in the stable transfectant cell pool. Interestingly, the E77 element was found to consist of two distinct fragments derived from different locations in the CHO genome shotgun sequence. High and stable transgene expression was obtained in transfected CHO cells by combining these fragments. Additionally, the function of E77 was found to be dependent on its site of insertion and specific orientation in the vector construct. Our findings demonstrate that stable gene expression mediated by the CMV promoter in CHO cells may be improved by the isolated novel gene regulatory element E77 identified in the present study. PMID:26762773

  13. Epigenetic conservation at gene regulatory elements revealed by non-methylated DNA profiling in seven vertebrates.

    PubMed

    Long, Hannah K; Sims, David; Heger, Andreas; Blackledge, Neil P; Kutter, Claudia; Wright, Megan L; Grützner, Frank; Odom, Duncan T; Patient, Roger; Ponting, Chris P; Klose, Robert J

    2013-01-01

    Two-thirds of gene promoters in mammals are associated with regions of non-methylated DNA, called CpG islands (CGIs), which counteract the repressive effects of DNA methylation on chromatin. In cold-blooded vertebrates, computational CGI predictions often reside away from gene promoters, suggesting a major divergence in gene promoter architecture across vertebrates. By experimentally identifying non-methylated DNA in the genomes of seven diverse vertebrates, we instead reveal that non-methylated islands (NMIs) of DNA are a central feature of vertebrate gene promoters. Furthermore, NMIs are present at orthologous genes across vast evolutionary distances, revealing a surprising level of conservation in this epigenetic feature. By profiling NMIs in different tissues and developmental stages we uncover a unifying set of features that are central to the function of NMIs in vertebrates. Together these findings demonstrate an ancient logic for NMI usage at gene promoters and reveal an unprecedented level of epigenetic conservation across vertebrate evolution. DOI:http://dx.doi.org/10.7554/eLife.00348.001. PMID:23467541

  14. The TTSMI database: a catalog of triplex target DNA sites associated with genes and regulatory elements in the human genome

    PubMed Central

    Jenjaroenpun, Piroon; Chew, Chee Siang; Yong, Tai Pang; Choowongkomon, Kiattawee; Thammasorn, Wimada; Kuznetsov, Vladimir A.

    2015-01-01

    A triplex target DNA site (TTS), a stretch of DNA that is composed of polypurines, is able to form a triple-helix (triplex) structure with triplex-forming oligonucleotides (TFOs) and is able to influence the site-specific modulation of gene expression and/or the modification of genomic DNA. The co-localization of a genomic TTS with gene regulatory signals and functional genome structures suggests that TFOs could potentially be exploited in antigene strategies for the therapy of cancers and other genetic diseases. Here, we present the TTS Mapping and Integration (TTSMI; http://ttsmi.bii.a-star.edu.sg) database, which provides a catalog of unique TTS locations in the human genome and tools for analyzing the co-localization of TTSs with genomic regulatory sequences and signals that were identified using next-generation sequencing techniques and/or predicted by computational models. TTSMI was designed as a user-friendly tool that facilitates (i) fast searching/filtering of TTSs using several search terms and criteria associated with sequence stability and specificity, (ii) interactive filtering of TTSs that co-localize with gene regulatory signals and non-B DNA structures, (iii) exploration of dynamic combinations of the biological signals of specific TTSs and (iv) visualization of a TTS simultaneously with diverse annotation tracks via the UCSC genome browser. PMID:25324314

  15. The TTSMI database: a catalog of triplex target DNA sites associated with genes and regulatory elements in the human genome.

    PubMed

    Jenjaroenpun, Piroon; Chew, Chee Siang; Yong, Tai Pang; Choowongkomon, Kiattawee; Thammasorn, Wimada; Kuznetsov, Vladimir A

    2015-01-01

    A triplex target DNA site (TTS), a stretch of DNA that is composed of polypurines, is able to form a triple-helix (triplex) structure with triplex-forming oligonucleotides (TFOs) and is able to influence the site-specific modulation of gene expression and/or the modification of genomic DNA. The co-localization of a genomic TTS with gene regulatory signals and functional genome structures suggests that TFOs could potentially be exploited in antigene strategies for the therapy of cancers and other genetic diseases. Here, we present the TTS Mapping and Integration (TTSMI; http://ttsmi.bii.a-star.edu.sg) database, which provides a catalog of unique TTS locations in the human genome and tools for analyzing the co-localization of TTSs with genomic regulatory sequences and signals that were identified using next-generation sequencing techniques and/or predicted by computational models. TTSMI was designed as a user-friendly tool that facilitates (i) fast searching/filtering of TTSs using several search terms and criteria associated with sequence stability and specificity, (ii) interactive filtering of TTSs that co-localize with gene regulatory signals and non-B DNA structures, (iii) exploration of dynamic combinations of the biological signals of specific TTSs and (iv) visualization of a TTS simultaneously with diverse annotation tracks via the UCSC genome browser. PMID:25324314

  16. Regulatory Mechanisms That Prevent Re-initiation of DNA Replication Can Be Locally Modulated at Origins by Nearby Sequence Elements

    PubMed Central

    Richardson, Christopher D.; Li, Joachim J.

    2014-01-01

    Eukaryotic cells must inhibit re-initiation of DNA replication at each of the thousands of origins in their genome because re-initiation can generate genomic alterations with extraordinary frequency. To minimize the probability of re-initiation from so many origins, cells use a battery of regulatory mechanisms that reduce the activity of replication initiation proteins. Given the global nature of these mechanisms, it has been presumed that all origins are inhibited identically. However, origins re-initiate with diverse efficiencies when these mechanisms are disabled, and this diversity cannot be explained by differences in the efficiency or timing of origin initiation during normal S phase replication. This observation raises the possibility of an additional layer of replication control that can differentially regulate re-initiation at distinct origins. We have identified novel genetic elements that are necessary for preferential re-initiation of two origins and sufficient to confer preferential re-initiation on heterologous origins when the control of re-initiation is partially deregulated. The elements do not enhance the S phase timing or efficiency of adjacent origins and thus are specifically acting as re-initiation promoters (RIPs). We have mapped the two RIPs to ∼60 bp AT rich sequences that act in a distance- and sequence-dependent manner. During the induction of re-replication, Mcm2-7 reassociates both with origins that preferentially re-initiate and origins that do not, suggesting that the RIP elements can overcome a block to re-initiation imposed after Mcm2-7 associates with origins. Our findings identify a local level of control in the block to re-initiation. This local control creates a complex genomic landscape of re-replication potential that is revealed when global mechanisms preventing re-replication are compromised. Hence, if re-replication does contribute to genomic alterations, as has been speculated for cancer cells, some regions of the genome

  17. Transcription factor trapping by RNA in gene regulatory elements

    PubMed Central

    Sigova, Alla A.; Abraham, Brian J.; Ji, Xiong; Molinie, Benoit; Hannett, Nancy M.; Eric Guo, Yang; Jangi, Mohini; Giallourakis, Cosmas C.; Sharp, Phillip A.; Young, Richard A.

    2016-01-01

    Transcription factors (TFs) bind specific sequences in promoter-proximal and distal DNA elements in order to regulate gene transcription. RNA is transcribed from both of these DNA elements, and some DNA-binding TFs bind RNA. Hence, RNA transcribed from regulatory elements may contribute to stable TF occupancy at these sites. We show that the ubiquitously expressed TF YY1 binds to both gene regulatory elements and also to their associated RNA species genome-wide. Reduced transcription of regulatory elements diminishes YY1 occupancy whereas artificial tethering of RNA enhances YY1 occupancy at these elements. We propose that RNA makes a modest but important contribution to the maintenance of certain TFs at gene regulatory elements and suggest that transcription of regulatory elements produces a positive feedback loop that contributes to the stability of gene expression programs. PMID:26516199

  18. Transcription factor trapping by RNA in gene regulatory elements.

    PubMed

    Sigova, Alla A; Abraham, Brian J; Ji, Xiong; Molinie, Benoit; Hannett, Nancy M; Guo, Yang Eric; Jangi, Mohini; Giallourakis, Cosmas C; Sharp, Phillip A; Young, Richard A

    2015-11-20

    Transcription factors (TFs) bind specific sequences in promoter-proximal and -distal DNA elements to regulate gene transcription. RNA is transcribed from both of these DNA elements, and some DNA binding TFs bind RNA. Hence, RNA transcribed from regulatory elements may contribute to stable TF occupancy at these sites. We show that the ubiquitously expressed TF Yin-Yang 1 (YY1) binds to both gene regulatory elements and their associated RNA species across the entire genome. Reduced transcription of regulatory elements diminishes YY1 occupancy, whereas artificial tethering of RNA enhances YY1 occupancy at these elements. We propose that RNA makes a modest but important contribution to the maintenance of certain TFs at gene regulatory elements and suggest that transcription of regulatory elements produces a positive-feedback loop that contributes to the stability of gene expression programs. PMID:26516199

  19. 3D-Trajectories Adopted by Coding and Regulatory DNA Elements: First-Passage Times for Genomic Interactions

    PubMed Central

    Lucas, Joseph S.; Zhang, Yaojun; Dudko, Olga K.; Murre, Cornelis

    2014-01-01

    SUMMARY During B lymphocyte development, immunoglobulin heavy chain variable (VH), diversity (DH) and joining (JH) segments assemble to generate a diverse antigen receptor repertoire. Here we have marked the distal VH and DH-JH-Eμ regions with Tet-operator binding sites and traced their 3D-trajectories in pro-B cells transduced with a retrovirus encoding Tet-repressor-EGFP. We found that these elements displayed fractional Langevin motion (fLm) due to the viscoelastic hindrance from the surrounding network of proteins and chromatin fibers. Using fractional Langevin dynamics modeling, we found that, with high probability, DHJH elements reach a VH element within minutes. Spatial confinement emerged as the dominant parameter that determined the frequency of such encounters. We propose that the viscoelastic nature of the nuclear environment causes coding elements and regulatory elements to bounce back and forth in a spring-like fashion until specific genomic interactions are established and that spatial confinement of topological domains largely controls first-passage times for genomic interactions. PMID:24998931

  20. A G/C-rich DNA-regulatory element controls positive expression of the sea urchin Lytechinus pictus aboral ectoderm-specific LpS1 gene.

    PubMed

    Wang, W; Klein, W H

    1996-02-01

    The LpS1 beta gene of Lytechinus pictus is activated at the late cleavage stage about 12 hr after fertilization. LpS1 beta transcripts accumulate exclusively in aboral ectoderm lineages. LpS1 beta is thus a classic example of a gene whose expression is tightly controlled both temporally and spatially during early development. Previous studies on the LpS1 beta promoter identified two G-string DNA elements, one proximal and one distal to the LpS1 beta transcriptional start site, which bind to an ectoderm-enriched nuclear factor. In this report, we show that the ectoderm G-string factor binds to a G/C-rich region larger than the G-string itself and that the binding of the G-string factor requires sequences immediately downstream from the G-string. These downstream sequences are essential for full promoter activity. Two regions of 5'-flanking DNA are required for positive control of LpS1 beta, region I from base pairs -762 to -511, and region II, which includes the G/C-rich element, from base pairs -108 to -61. Region I also contains a mesenchyme cell repressor element. The results indicate that LpS1 beta expression is regulated positively in ectoderm cells and negatively in mesenchyme cells. Similar positive and negative control mechanisms regulate the expression of the related Spec genes of Strongylocentrotus purpuratus, but in this gene family the DNA elements are entirely different. We hypothesize that cis-regulatory elements are evolutionarily dynamic and that many different combinations of DNA elements are capable of given rise to aboral ectoderm-specific expression. PMID:8634141

  1. Codon optimization and woodchuck hepatitis virus posttranscriptional regulatory element enhance the immune responses of DNA vaccines against infectious bursal disease virus in chickens.

    PubMed

    Li, Kai; Gao, Li; Gao, Honglei; Qi, Xiaole; Gao, Yulong; Qin, Liting; Wang, Yongqiang; Wang, Xiaomei

    2013-08-01

    The present study was undertaken to evaluate the protective efficacy of DNA vaccines against infectious bursal disease virus (IBDV) in chickens and to determine whether codon optimization and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) could improve the immunogenicity of the DNA vaccines. The VP2, VP243 and codon-optimized VP243 genes of IBDV were cloned into pCAGGS vector, and designated as pCAGVP2, pCAGVP243 and pCAGoptiVP243, respectively. Plasmids pCAGWVP243 and pCAGWoptiVP243 carrying the WPRE elements were also constructed as DNA vaccines. To evaluate vaccine efficacy, 2-week-old chickens were injected intramuscularly with the constructed plasmids twice at 2-week intervals and challenged with very virulent IBDV 2 weeks post-boost. Plasmid pCAGVP243 induced better immune responses than pCAGVP2. Chickens immunized with pCAGoptiVP243 and pCAGWVP243 had higher levels of antibody titers, lymphoproliferation responses and cytokine production compared with pCAGVP243. Furthermore, plasmid pCAGWoptiVP243 induced the highest levels of immune responses among the groups. After challenged, DNA vaccines pCAGVP2, pCAGVP243, pCAGoptiVP243, pCAGWVP243 and pCAGWoptiVP243 conferred protection for 33%, 60%, 80%, 87% and 100% of chickens, respectively, as evidenced by the absence of clinical signs, mortality, and bursal atrophy. These results indicate that codon optimization and WPRE could enhance the protective efficacy of DNA vaccines against IBDV and these two approaches could work together synergistically in a single DNA vaccine. PMID:23631937

  2. Decitabine alters the expression of Mecp2 isoforms via dynamic DNA methylation at the Mecp2 regulatory elements in neural stem cells

    PubMed Central

    2013-01-01

    Background Aberrant MeCP2 expression in brain is associated with neurodevelopmental disorders including autism. In the brain of stressed mouse and autistic human patients, reduced MeCP2 expression is correlated with Mecp2/MECP2 promoter hypermethylation. Altered expression of MeCP2 isoforms (MeCP2E1 and MeCP2E2) is associated with neurological disorders, highlighting the importance of proper regulation of both isoforms. While known regulatory elements (REs) within the MECP2/Mecp2 promoter and intron 1 are involved in MECP2/Mecp2 regulation, Mecp2 isoform-specific regulatory mechanisms are unknown. We hypothesized that DNA methylation at these REs may impact the expression of Mecp2 isoforms. Methods We used a previously characterized in vitro differentiating neural stem cell (NSC) system to investigate the interplay between Mecp2 isoform-specific expression and DNA methylation at the Mecp2 REs. We studied altered expression of Mecp2 isoforms, affected by global DNA demethylation and remethylation, induced by exposure and withdrawal of decitabine (5-Aza-2′-deoxycytidine). Further, we performed correlation analysis between DNA methylation at the Mecp2 REs and the expression of Mecp2 isoforms after decitabine exposure and withdrawal. Results At different stages of NSC differentiation, Mecp2 isoforms showed reciprocal expression patterns associated with minor, but significant changes in DNA methylation at the Mecp2 REs. Decitabine treatment induced Mecp2e1/MeCP2E1 (but not Mecp2e2) expression at day (D) 2, associated with DNA demethylation at the Mecp2 REs. In contrast, decitabine withdrawal downregulated both Mecp2 isoforms to different extents at D8, without affecting DNA methylation at the Mecp2 REs. NSC cell fate commitment was minimally affected by decitabine under tested conditions. Expression of both isoforms negatively correlated with methylation at specific regions of the Mecp2 promoter, both at D2 and D8. The correlation between intron 1 methylation and Mecp

  3. Transposable Elements and DNA Methylation Create in Embryonic Stem Cells Human-Specific Regulatory Sequences Associated with Distal Enhancers and Noncoding RNAs.

    PubMed

    Glinsky, Gennadi V

    2015-06-01

    Despite significant progress in the structural and functional characterization of the human genome, understanding of the mechanisms underlying the genetic basis of human phenotypic uniqueness remains limited. Here, I report that transposable element-derived sequences, most notably LTR7/HERV-H, LTR5_Hs, and L1HS, harbor 99.8% of the candidate human-specific regulatory loci (HSRL) with putative transcription factor-binding sites in the genome of human embryonic stem cells (hESC). A total of 4,094 candidate HSRL display selective and site-specific binding of critical regulators (NANOG [Nanog homeobox], POU5F1 [POU class 5 homeobox 1], CCCTC-binding factor [CTCF], Lamin B1), and are preferentially located within the matrix of transcriptionally active DNA segments that are hypermethylated in hESC. hESC-specific NANOG-binding sites are enriched near the protein-coding genes regulating brain size, pluripotency long noncoding RNAs, hESC enhancers, and 5-hydroxymethylcytosine-harboring regions immediately adjacent to binding sites. Sequences of only 4.3% of hESC-specific NANOG-binding sites are present in Neanderthals' genome, suggesting that a majority of these regulatory elements emerged in Modern Humans. Comparisons of estimated creation rates of novel TF-binding sites revealed that there was 49.7-fold acceleration of creation rates of NANOG-binding sites in genomes of Chimpanzees compared with the mouse genomes and further 5.7-fold acceleration in genomes of Modern Humans compared with the Chimpanzees genomes. Preliminary estimates suggest that emergence of one novel NANOG-binding site detectable in hESC required 466 years of evolution. Pathway analysis of coding genes that have hESC-specific NANOG-binding sites within gene bodies or near gene boundaries revealed their association with physiological development and functions of nervous and cardiovascular systems, embryonic development, behavior, as well as development of a diverse spectrum of pathological conditions

  4. Transposable Elements and DNA Methylation Create in Embryonic Stem Cells Human-Specific Regulatory Sequences Associated with Distal Enhancers and Noncoding RNAs

    PubMed Central

    Glinsky, Gennadi V.

    2015-01-01

    Despite significant progress in the structural and functional characterization of the human genome, understanding of the mechanisms underlying the genetic basis of human phenotypic uniqueness remains limited. Here, I report that transposable element-derived sequences, most notably LTR7/HERV-H, LTR5_Hs, and L1HS, harbor 99.8% of the candidate human-specific regulatory loci (HSRL) with putative transcription factor-binding sites in the genome of human embryonic stem cells (hESC). A total of 4,094 candidate HSRL display selective and site-specific binding of critical regulators (NANOG [Nanog homeobox], POU5F1 [POU class 5 homeobox 1], CCCTC-binding factor [CTCF], Lamin B1), and are preferentially located within the matrix of transcriptionally active DNA segments that are hypermethylated in hESC. hESC-specific NANOG-binding sites are enriched near the protein-coding genes regulating brain size, pluripotency long noncoding RNAs, hESC enhancers, and 5-hydroxymethylcytosine-harboring regions immediately adjacent to binding sites. Sequences of only 4.3% of hESC-specific NANOG-binding sites are present in Neanderthals’ genome, suggesting that a majority of these regulatory elements emerged in Modern Humans. Comparisons of estimated creation rates of novel TF-binding sites revealed that there was 49.7-fold acceleration of creation rates of NANOG-binding sites in genomes of Chimpanzees compared with the mouse genomes and further 5.7-fold acceleration in genomes of Modern Humans compared with the Chimpanzees genomes. Preliminary estimates suggest that emergence of one novel NANOG-binding site detectable in hESC required 466 years of evolution. Pathway analysis of coding genes that have hESC-specific NANOG-binding sites within gene bodies or near gene boundaries revealed their association with physiological development and functions of nervous and cardiovascular systems, embryonic development, behavior, as well as development of a diverse spectrum of pathological conditions

  5. Expression of 5 S rRNA genes linked to 35 S rDNA in plants, their epigenetic modification and regulatory element divergence

    PubMed Central

    2012-01-01

    Background In plants, the 5 S rRNA genes usually occur as separate tandems (S-type arrangement) or, less commonly, linked to 35 S rDNA units (L-type). The activity of linked genes remains unknown so far. We studied the homogeneity and expression of 5 S genes in several species from family Asteraceae known to contain linked 35 S-5 S units. Additionally, their methylation status was determined using bisulfite sequencing. Fluorescence in situ hybridization was applied to reveal the sub-nuclear positions of rDNA arrays. Results We found that homogenization of L-type units went to completion in most (4/6) but not all species. Two species contained major L-type and minor S-type units (termed Ls-type). The linked genes dominate 5 S rDNA expression while the separate tandems do not seem to be expressed. Members of tribe Anthemideae evolved functional variants of the polymerase III promoter in which a residing C-box element differs from the canonical angiosperm motif by as much as 30%. On this basis, a more relaxed consensus sequence of a plant C-box: (5’-RGSWTGGGTG-3’) is proposed. The 5 S paralogs display heavy DNA methylation similarly as to their unlinked counterparts. FISH revealed the close association of 35 S-5 S arrays with nucleolar periphery indicating that transcription of 5 S genes may occur in this territory. Conclusions We show that the unusual linked arrangement of 5 S genes, occurring in several plant species, is fully compatible with their expression and functionality. This extraordinary 5 S gene dynamics is manifested at different levels, such as variation in intrachromosomal positions, unit structure, epigenetic modification and considerable divergence of regulatory motifs. PMID:22716941

  6. Global Analysis of DNA Methylation Variation in Adipose Tissue from Twins Reveals Links to Disease-Associated Variants in Distal Regulatory Elements

    PubMed Central

    Grundberg, Elin; Meduri, Eshwar; Sandling, Johanna K.; Hedman, Åsa K.; Keildson, Sarah; Buil, Alfonso; Busche, Stephan; Yuan, Wei; Nisbet, James; Sekowska, Magdalena; Wilk, Alicja; Barrett, Amy; Small, Kerrin S.; Ge, Bing; Caron, Maxime; Shin, So-Youn; Ahmadi, Kourosh R.; Ainali, Chrysanthi; Barrett, Amy; Bataille, Veronique; Bell, Jordana T.; Buil, Alfonso; Deloukas, Panos; Dermitzakis, Emmanouil T.; Dimas, Antigone S.; Durbin, Richard; Glass, Daniel; Grundberg, Elin; Hassanali, Neelam; Hedman, Åsa K.; Ingle, Catherine; Knowles, David; Krestyaninova, Maria; Lindgren, Cecilia M.; Lowe, Christopher E.; McCarthy, Mark I.; Meduri, Eshwar; di Meglio, Paola; Min, Josine L.; Montgomery, Stephen B.; Nestle, Frank O.; Nica, Alexandra C.; Nisbet, James; O’Rahilly, Stephen; Parts, Leopold; Potter, Simon; Sandling, Johanna; Sekowska, Magdalena; Shin, So-Youn; Small, Kerrin S.; Soranzo, Nicole; Spector, Tim D.; Surdulescu, Gabriela; Travers, Mary E.; Tsaprouni, Loukia; Tsoka, Sophia; Wilk, Alicja; Yang, Tsun-Po; Zondervan, Krina T.; Lathrop, Mark; Dermitzakis, Emmanouil T.; McCarthy, Mark I.; Spector, Timothy D.; Bell, Jordana T.; Deloukas, Panos

    2013-01-01

    Epigenetic modifications such as DNA methylation play a key role in gene regulation and disease susceptibility. However, little is known about the genome-wide frequency, localization, and function of methylation variation and how it is regulated by genetic and environmental factors. We utilized the Multiple Tissue Human Expression Resource (MuTHER) and generated Illumina 450K adipose methylome data from 648 twins. We found that individual CpGs had low variance and that variability was suppressed in promoters. We noted that DNA methylation variation was highly heritable (h2median = 0.34) and that shared environmental effects correlated with metabolic phenotype-associated CpGs. Analysis of methylation quantitative-trait loci (metQTL) revealed that 28% of CpGs were associated with nearby SNPs, and when overlapping them with adipose expression quantitative-trait loci (eQTL) from the same individuals, we found that 6% of the loci played a role in regulating both gene expression and DNA methylation. These associations were bidirectional, but there were pronounced negative associations for promoter CpGs. Integration of metQTL with adipose reference epigenomes and disease associations revealed significant enrichment of metQTL overlapping metabolic-trait or disease loci in enhancers (the strongest effects were for high-density lipoprotein cholesterol and body mass index [BMI]). We followed up with the BMI SNP rs713586, a cg01884057 metQTL that overlaps an enhancer upstream of ADCY3, and used bisulphite sequencing to refine this region. Our results showed widespread population invariability yet sequence dependence on adipose DNA methylation but that incorporating maps of regulatory elements aid in linking CpG variation to gene regulation and disease risk in a tissue-dependent manner. PMID:24183450

  7. A method for using direct injection of plasmid DNA to study cis-regulatory element activity in F0 Xenopus embryos and tadpoles.

    PubMed

    Wang, Chen; Szaro, Ben G

    2015-02-01

    The ability to express exogenous reporter genes in intact, externally developing embryos, such as Xenopus, is a powerful tool for characterizing the activity of cis-regulatory gene elements during development. Although methods exist for generating transgenic Xenopus lines, more simplified methods for use with F0 animals would significantly speed the characterization of these elements. We discovered that injecting 2-cell stage embryos with a plasmid bearing a ϕC31 integrase-targeted attB element and two dual β-globin HS4 insulators flanking a reporter transgene in opposite orientations relative to each other yielded persistent expression with sufficiently high penetrance for characterizing the activity of the promoter without having to coinject integrase RNA. Expression began appropriately during development and persisted into swimming tadpole stages without perturbing the expression of the cognate endogenous gene. Coinjected plasmids having the same elements but expressing different reporter proteins were reliably coexpressed within the same cells, providing a useful control for variations in injections between animals. To overcome the high propensity of these plasmids to undergo recombination, we developed a method for generating them using conventional cloning methods and DH5α cells for propagation. We conclude that this method offers a convenient and reliable way to evaluate the activity of cis-regulatory gene elements in the intact F0 embryo. PMID:25448690

  8. Identification of DNA methylation changes at cis-regulatory elements during early steps of HSC differentiation using tagmentation-based whole genome bisulfite sequencing

    PubMed Central

    Lipka, Daniel B; Wang, Qi; Cabezas-Wallscheid, Nina; Klimmeck, Daniel; Weichenhan, Dieter; Herrmann, Carl; Lier, Amelie; Brocks, David; von Paleske, Lisa; Renders, Simon; Wünsche, Peer; Zeisberger, Petra; Gu, Lei; Haas, Simon; Essers, Marieke Ag; Brors, Benedikt; Eils, Roland; Trumpp, Andreas; Milsom, Michael D; Plass, Christoph

    2014-01-01

    Epigenetic alterations during cellular differentiation are a key molecular mechanism which both instructs and reinforces the process of lineage commitment. Within the haematopoietic system, progressive changes in the DNA methylome of haematopoietic stem cells (HSCs) are essential for the effective production of mature blood cells. Inhibition or loss of function of the cellular DNA methylation machinery has been shown to lead to a severe perturbation in blood production and is also an important driver of malignant transformation. HSCs constitute a very rare cell population in the bone marrow, capable of life-long self-renewal and multi-lineage differentiation. The low abundance of HSCs has been a major technological barrier to the global analysis of the CpG methylation status within both HSCs and their immediate progeny, the multipotent progenitors (MPPs). Within this Extra View article, we review the current understanding of how the DNA methylome regulates normal and malignant hematopoiesis. We also discuss the current methodologies that are available for interrogating the DNA methylation status of HSCs and MPPs and describe a new data set that was generated using tagmentation-based whole genome bisulfite sequencing (TWGBS) in order to comprehensively map methylated cytosines using the limited amount of genomic DNA that can be harvested from rare cell populations. Extended analysis of this data set clearly demonstrates the added value of genome-wide sequencing of methylated cytosines and identifies novel important cis-acting regulatory regions that are dynamically remodeled during the first steps of haematopoietic differentiation. PMID:25483069

  9. Evolutionary conservation of regulatory elements in vertebrate HOX gene clusters

    SciTech Connect

    Santini, Simona; Boore, Jeffrey L.; Meyer, Axel

    2003-12-31

    Due to their high degree of conservation, comparisons of DNA sequences among evolutionarily distantly-related genomes permit to identify functional regions in noncoding DNA. Hox genes are optimal candidate sequences for comparative genome analyses, because they are extremely conserved in vertebrates and occur in clusters. We aligned (Pipmaker) the nucleotide sequences of HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark, human and mouse (over 500 million years of evolutionary distance). We identified several highly conserved intergenic sequences, likely to be important in gene regulation. Only a few of these putative regulatory elements have been previously described as being involved in the regulation of Hox genes, while several others are new elements that might have regulatory functions. The majority of these newly identified putative regulatory elements contain short fragments that are almost completely conserved and are identical to known binding sites for regulatory proteins (Transfac). The conserved intergenic regions located between the most rostrally expressed genes in the developing embryo are longer and better retained through evolution. We document that presumed regulatory sequences are retained differentially in either A or A clusters resulting from a genome duplication in the fish lineage. This observation supports both the hypothesis that the conserved elements are involved in gene regulation and the Duplication-Deletion-Complementation model.

  10. Uncovering drug-responsive regulatory elements

    PubMed Central

    Luizon, Marcelo R; Ahituv, Nadav

    2016-01-01

    Nucleotide changes in gene regulatory elements can have a major effect on interindividual differences in drug response. For example, by reviewing all published pharmacogenomic genome-wide association studies, we show here that 96.4% of the associated single nucleotide polymorphisms reside in noncoding regions. We discuss how sequencing technologies are improving our ability to identify drug response-associated regulatory elements genome-wide and to annotate nucleotide variants within them. We highlight specific examples of how nucleotide changes in these elements can affect drug response and illustrate the techniques used to find them and functionally characterize them. Finally, we also discuss challenges in the field of drug-responsive regulatory elements that need to be considered in order to translate these findings into the clinic. PMID:26555224

  11. Transcriptional Regulatory Elements in Fungal Secondary Metabolism

    PubMed Central

    Yin, Wenbing; Keller, Nancy P.

    2013-01-01

    Filamentous fungi produce a variety of secondary metabolites of diverse beneficial and detrimental activities to humankind. The genes encoding the enzymatic machinery required to make these metabolites are typically clustered in fungal genomes. There is considerable evidence that secondary metabolite gene regulation is, in part, by transcriptional control through hierarchical levels of transcriptional regulatory elements involved in secondary metabolite cluster regulation. Identification of secondary metabolism regulatory elements could potentially provide a means of increasing production of beneficial metabolites, decreasing production of detrimental metabolites, aid in the identification of ‘silent’ natural products and also contribute to a broader understanding of molecular mechanisms by which secondary metabolites are produced. This review summarizes regulation of secondary metabolism associated on transcriptional regulatory elements from a broad view as well as tremendous advances in discovery of cryptic or novel secondary metabolites by genomic mining in the basis of this knowledge. PMID:21717315

  12. Constitutive transcription of the osteocalcin gene in osteosarcoma cells is reflected by altered protein-DNA interactions at promoter regulatory elements.

    PubMed Central

    Bortell, R; Owen, T A; Shalhoub, V; Heinrichs, A; Aronow, M A; Rochette-Egly, C; Lutz, Y; Stein, J L; Lian, J B; Stein, G S

    1993-01-01

    The bone-specific osteocalcin (OC) gene is transcribed only after completion of proliferation in normal diploid calvarial-derived osteoblasts during extracellular matrix mineralization. In contrast, the OC gene is expressed constitutively in both proliferating and nonproliferating ROS 17/2.8 osteosarcoma cells. To address molecular mechanisms associated with these tumor-related modifications in transcriptional control, we examined sequence-specific interactions of transactivation factors at key basal and hormone-responsive elements in the OC gene promoter. In ROS 17/2.8 cells compared to normal diploid osteoblasts, the absence of a stringent requirement for cessation of proliferation to support both induction of OC transcription and steroid hormone-mediated transcriptional modulation is reflected by modifications in transcription factor binding at (i) the two primary basal regulatory elements, the OC box (which contains a CCAAT motif as a central core) and the TATA/glucocorticoid-responsive element domain, and (ii) the vitamin D-responsive element. Particularly striking are two forms of the vitamin D receptor complex that are present in proliferating osteoblasts and osteosarcoma cells. Both forms of the complex are sensitive to vitamin D receptor antibody and retinoic X receptor antibody. After the down-regulation of proliferation, only the lower molecular weight complex is found in normal diploid osteoblasts. Both forms of the complex are present in nonproliferating ROS 17/2.8 cells with increased representation of the complex exhibiting reduced electrophoretic mobility that is phosphorylation-dependent. Images Fig. 3 Fig. 5 Fig. 6 PMID:8460137

  13. Effects of species combination on comparative analyses of conserved regulatory elements

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cross-species DNA sequence comparison is the primary approach to discover regulatory elements by identifying highly conserved sequences due to evolutionary constraints. Previously, we reported that a systematic approach, combining position-specific weight matrixes (JASPAR) and phylogenetic footprint...

  14. Systematic identification of conserved regulatory elements in upstream promoter regions of the cattle genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cross-species DNA sequence comparison is the primary approach to discover regulatory elements by identifying highly conserved sequences due to evolutionary constraints. Previously, we reported that a systematic approach, combining position-specific weight matrixes (JASPAR) and phylogenetic footprint...

  15. Prediction of conserved regulatory elements in promoter regions of the cattle genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cross-species DNA sequence comparison is the primary approach to discover regulatory elements by identifying highly conserved sequences due to evolutionary constraints. Previously, we reported that a systematic approach, combining position-specific weight matrixes (JASPAR) and phylogenetic footprint...

  16. Identifying Synonymous Regulatory Elements in Vertebrate Genomes

    SciTech Connect

    Ovcharenko, I; Nobrega, M A

    2005-02-07

    Synonymous gene regulation, defined as driving shared temporal and/or spatial expression of groups of genes, is likely predicated on genomic elements that contain similar modules of certain transcription factor binding sites (TFBS). We have developed a method to scan vertebrate genomes for evolutionary conserved modules of TFBS in a predefined configuration, and created a tool, named SynoR that identify synonymous regulatory elements (SREs) in vertebrate genomes. SynoR performs de novo identification of SREs utilizing known patterns of TFBS in active regulatory elements (REs) as seeds for genome scans. Layers of multiple-species conservation allow the use of differential phylogenetic sequence conservation filters in the search of SREs and the results are displayed as to provide an extensive annotation of genes containing detected REs. Gene Ontology categories are utilized to further functionally classify the identified genes, and integrated GNF Expression Atlas 2 data allow the cataloging of tissue-specificities of the predicted SREs. We illustrate how this new tool can be used to establish a linkage between human diseases and noncoding genomic content. SynoR is publicly available at http://synor.dcode.org.

  17. Dynamic SPR monitoring of yeast nuclear protein binding to a cis-regulatory element

    SciTech Connect

    Mao, Grace; Brody, James P.

    2007-11-09

    Gene expression is controlled by protein complexes binding to short specific sequences of DNA, called cis-regulatory elements. Expression of most eukaryotic genes is controlled by dozens of these elements. Comprehensive identification and monitoring of these elements is a major goal of genomics. In pursuit of this goal, we are developing a surface plasmon resonance (SPR) based assay to identify and monitor cis-regulatory elements. To test whether we could reliably monitor protein binding to a regulatory element, we immobilized a 16 bp region of Saccharomyces cerevisiae chromosome 5 onto a gold surface. This 16 bp region of DNA is known to bind several proteins and thought to control expression of the gene RNR1, which varies through the cell cycle. We synchronized yeast cell cultures, and then sampled these cultures at a regular interval. These samples were processed to purify nuclear lysate, which was then exposed to the sensor. We found that nuclear protein binds this particular element of DNA at a significantly higher rate (as compared to unsynchronized cells) during G1 phase. Other time points show levels of DNA-nuclear protein binding similar to the unsynchronized control. We also measured the apparent association complex of the binding to be 0.014 s{sup -1}. We conclude that (1) SPR-based assays can monitor DNA-nuclear protein binding and that (2) for this particular cis-regulatory element, maximum DNA-nuclear protein binding occurs during G1 phase.

  18. Bioinformatic analysis reveals an evolutional selection for DNA:RNA hybrid G-quadruplex structures as putative transcription regulatory elements in warm-blooded animals.

    PubMed

    Xiao, Shan; Zhang, Jia-Yu; Zheng, Ke-Wei; Hao, Yu-Hua; Tan, Zheng

    2013-12-01

    Recently, we reported the co-transcriptional formation of DNA:RNA hybrid G-quadruplex (HQ) structure by the non-template DNA strand and nascent RNA transcript, which in turn modulates transcription under both in vitro and in vivo conditions. Here we present bioinformatic analysis on putative HQ-forming sequences (PHQS) in the genomes of eukaryotic organisms. Starting from amphibian, PHQS motifs are concentrated in the immediate 1000-nt region downstream of transcription start sites, implying their potential role in transcription regulation. Moreover, their occurrence shows a strong bias toward the non-template versus the template strand. PHQS has become constitutional in genes in warm-blooded animals, and the magnitude of the strand bias correlates with the ability of PHQS to form HQ, suggesting a selection based on HQ formation. This strand bias is reversed in lower species, implying that the selection of PHQS/HQ depended on the living temperature of the organisms. In comparison with the putative intramolecular G-quadruplex-forming sequences (PQS), PHQS motifs are far more prevalent and abundant in the transcribed regions, making them the dominant candidates in the formation of G-quadruplexes in transcription. Collectively, these results suggest that the HQ structures are evolutionally selected to function in transcription and other transcription-mediated processes that involve guanine-rich non-template strand. PMID:23999096

  19. Massive contribution of transposable elements to mammalian regulatory sequences.

    PubMed

    Rayan, Nirmala Arul; Del Rosario, Ricardo C H; Prabhakar, Shyam

    2016-09-01

    Barbara McClintock discovered the existence of transposable elements (TEs) in the late 1940s and initially proposed that they contributed to the gene regulatory program of higher organisms. This controversial idea gained acceptance only much later in the 1990s, when the first examples of TE-derived promoter sequences were uncovered. It is now known that half of the human genome is recognizably derived from TEs. It is thus important to understand the scope and nature of their contribution to gene regulation. Here, we provide a timeline of major discoveries in this area and discuss how transposons have revolutionized our understanding of mammalian genomes, with a special emphasis on the massive contribution of TEs to primate evolution. Our analysis of primate-specific functional elements supports a simple model for the rate at which new functional elements arise in unique and TE-derived DNA. Finally, we discuss some of the challenges and unresolved questions in the field, which need to be addressed in order to fully characterize the impact of TEs on gene regulation, evolution and disease processes. PMID:27174439

  20. Isolation of a non-genomic origin fluoroquinolone responsive regulatory element using a combinatorial bioengineering approach

    PubMed Central

    Srivastava, Santosh Kumar; Iyer, V. Rajesh; Ghosh, Tamoghna; Lambadi, Paramesh Ramulu; Pathania, Ranjana; Navani, Naveen Kumar

    2016-01-01

    Advances in chemical biology have led to selection of synthetic functional nucleic acids for in vivo applications. Discovery of synthetic nucleic acid regulatory elements has been a long-standing goal of chemical biologists. Availability of vast genome level genetic resources has motivated efforts for discovery and understanding of inducible synthetic genetic regulatory elements. Such elements can lead to custom-design of switches and sensors, oscillators, digital logic evaluators and cell–cell communicators. Here, we describe a simple, robust and universally applicable module for discovery of inducible gene regulatory elements. The distinguishing feature is the use of a toxic peptide as a reporter to suppress the background of unwanted bacterial recombinants. Using this strategy, we show that it is possible to isolate genetic elements of non-genomic origin which specifically get activated in the presence of DNA gyrase A inhibitors belonging to fluoroquinolone (FQ) group of chemicals. Further, using a system level genetic resource, we prove that the genetic regulation is exerted through histone-like nucleoid structuring (H-NS) repressor protein. Till date, there are no reports of in vivo selection of non-genomic origin inducible regulatory promoter like elements. Our strategy opens an uncharted route to discover inducible synthetic regulatory elements from biologically-inspired nucleic acid sequences. PMID:26837578

  1. Isolation of a non-genomic origin fluoroquinolone responsive regulatory element using a combinatorial bioengineering approach.

    PubMed

    Srivastava, Santosh Kumar; Iyer, V Rajesh; Ghosh, Tamoghna; Lambadi, Paramesh Ramulu; Pathania, Ranjana; Navani, Naveen Kumar

    2016-03-18

    Advances in chemical biology have led to selection of synthetic functional nucleic acids for in vivo applications. Discovery of synthetic nucleic acid regulatory elements has been a long-standing goal of chemical biologists. Availability of vast genome level genetic resources has motivated efforts for discovery and understanding of inducible synthetic genetic regulatory elements. Such elements can lead to custom-design of switches and sensors, oscillators, digital logic evaluators and cell-cell communicators. Here, we describe a simple, robust and universally applicable module for discovery of inducible gene regulatory elements. The distinguishing feature is the use of a toxic peptide as a reporter to suppress the background of unwanted bacterial recombinants. Using this strategy, we show that it is possible to isolate genetic elements of non-genomic origin which specifically get activated in the presence of DNA gyrase A inhibitors belonging to fluoroquinolone (FQ) group of chemicals. Further, using a system level genetic resource, we prove that the genetic regulation is exerted through histone-like nucleoid structuring (H-NS) repressor protein. Till date, there are no reports of in vivo selection of non-genomic origin inducible regulatory promoter like elements. Our strategy opens an uncharted route to discover inducible synthetic regulatory elements from biologically-inspired nucleic acid sequences. PMID:26837578

  2. A User's Guide to the Encyclopedia of DNA Elements (ENCODE)

    PubMed Central

    2011-01-01

    The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome. PMID:21526222

  3. A user's guide to the encyclopedia of DNA elements (ENCODE).

    PubMed

    2011-04-01

    The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome. PMID:21526222

  4. EMERGE: a flexible modelling framework to predict genomic regulatory elements from genomic signatures

    PubMed Central

    van Duijvenboden, Karel; de Boer, Bouke A.; Capon, Nicolas; Ruijter, Jan M.; Christoffels, Vincent M.

    2016-01-01

    Regulatory DNA elements, short genomic segments that regulate gene expression, have been implicated in developmental disorders and human disease. Despite this clinical urgency, only a small fraction of the regulatory DNA repertoire has been confirmed through reporter gene assays. The overall success rate of functional validation of candidate regulatory elements is low. Moreover, the number and diversity of datasets from which putative regulatory elements can be identified is large and rapidly increasing. We generated a flexible and user-friendly tool to integrate the information from different types of genomic datasets, e.g. ATAC-seq, ChIP-seq, conservation, aiming to increase the ease and success rate of functional prediction. To this end, we developed the EMERGE program that merges all datasets that the user considers informative and uses a logistic regression framework, based on validated functional elements, to set optimal weights to these datasets. ROC curve analysis shows that a combination of datasets leads to improved prediction of tissue-specific enhancers in human, mouse and Drosophila genomes. Functional assays based on this prediction can be expected to have substantially higher success rates. The resulting integrated signal for prediction of functional elements can be plotted in a build-in genome browser or exported for further analysis. PMID:26531828

  5. Study of Cis-regulatory Elements in the Ascidian Ciona intestinalis.

    PubMed

    Irvine, Steven Q

    2013-03-01

    The ascidian (sea squirt) C. intestinalis has become an important model organism for the study of cis-regulation. This is largely due to the technology that has been developed for assessing cis-regulatory activity through the use of transient reporter transgenes introduced into fertilized eggs. This technique allows the rapid and inexpensive testing of endogenous or altered DNA for regulatory activity in vivo. This review examines evidence that C. intestinalis cis-regulatory elements are located more closely to coding regions than in other model organisms. I go on to compare the organization of cis-regulatory elements and conserved non-coding sequences in Ciona, mammals, and other deuterostomes for three representative C.intestinalis genes, Pax6, FoxAa, and the DlxA-B cluster, along with homologs in the other species. These comparisons point out some of the similarities and differences between cis-regulatory elements and their study in the various model organisms. Finally, I provide illustrations of how C. intestinalis lends itself to detailed study of the structure of cis-regulatory elements, which have led, and promise to continue to lead, to important insights into the fundamentals of transcriptional regulation. PMID:23997651

  6. Regulatory elements responsible for inducible expression of the granulocyte colony-stimulating factor gene in macrophages.

    PubMed Central

    Nishizawa, M; Nagata, S

    1990-01-01

    Granulocyte colony-stimulating factor (G-CSF) plays an essential role in granulopoiesis during bacterial infection. Macrophages produce G-CSF in response to bacterial endotoxins such as lipopolysaccharide (LPS). To elucidate the mechanism of the induction of G-CSF gene in macrophages or macrophage-monocytes, we have examined regulatory cis elements in the promoter of mouse G-CSF gene. Analyses of linker-scanning and internal deletion mutants of the G-CSF promoter by the chloramphenicol acetyltransferase assay have indicated that at least three regulatory elements are indispensable for the LPS-induced expression of the G-CSF gene in macrophages. When one of the three elements was reiterated and placed upstream of the TATA box of the G-CSF promoter, it mediated inducibility as a tissue-specific and orientation-independent enhancer. Although this element contains a conserved NF-kappa B-like binding site, the gel retardation assay and DNA footprint analysis with nuclear extracts from macrophage cell lines demonstrated that nuclear proteins bind to the DNA sequence downstream of the NF-kappa B-like element, but not to the conserved element itself. The DNA sequence of the binding site was found to have some similarities to the LPS-responsive element which was recently identified in the promoter of the mouse class II major histocompatibility gene. Images PMID:1691438

  7. The identification of cis-regulatory elements: A review from a machine learning perspective.

    PubMed

    Li, Yifeng; Chen, Chih-Yu; Kaye, Alice M; Wasserman, Wyeth W

    2015-12-01

    The majority of the human genome consists of non-coding regions that have been called junk DNA. However, recent studies have unveiled that these regions contain cis-regulatory elements, such as promoters, enhancers, silencers, insulators, etc. These regulatory elements can play crucial roles in controlling gene expressions in specific cell types, conditions, and developmental stages. Disruption to these regions could contribute to phenotype changes. Precisely identifying regulatory elements is key to deciphering the mechanisms underlying transcriptional regulation. Cis-regulatory events are complex processes that involve chromatin accessibility, transcription factor binding, DNA methylation, histone modifications, and the interactions between them. The development of next-generation sequencing techniques has allowed us to capture these genomic features in depth. Applied analysis of genome sequences for clinical genetics has increased the urgency for detecting these regions. However, the complexity of cis-regulatory events and the deluge of sequencing data require accurate and efficient computational approaches, in particular, machine learning techniques. In this review, we describe machine learning approaches for predicting transcription factor binding sites, enhancers, and promoters, primarily driven by next-generation sequencing data. Data sources are provided in order to facilitate testing of novel methods. The purpose of this review is to attract computational experts and data scientists to advance this field. PMID:26499213

  8. An integrated encyclopedia of DNA elements in the human genome.

    PubMed

    2012-09-01

    The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall, the project provides new insights into the organization and regulation of our genes and genome, and is an expansive resource of functional annotations for biomedical research. PMID:22955616

  9. An Integrated Encyclopedia of DNA Elements in the Human Genome

    PubMed Central

    2012-01-01

    Summary The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure, and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall the project provides new insights into the organization and regulation of our genes and genome, and an expansive resource of functional annotations for biomedical research. PMID:22955616

  10. Evolution of Cis-Regulatory Elements and Regulatory Networks in Duplicated Genes of Arabidopsis1[OPEN

    PubMed Central

    Guo, Xu Qiu; Adams, Keith L.

    2015-01-01

    Plant genomes contain large numbers of duplicated genes that contribute to the evolution of new functions. Following duplication, genes can exhibit divergence in their coding sequence and their expression patterns. Changes in the cis-regulatory element landscape can result in changes in gene expression patterns. High-throughput methods developed recently can identify potential cis-regulatory elements on a genome-wide scale. Here, we use a recent comprehensive data set of DNase I sequencing-identified cis-regulatory binding sites (footprints) at single-base-pair resolution to compare binding sites and network connectivity in duplicated gene pairs in Arabidopsis (Arabidopsis thaliana). We found that duplicated gene pairs vary greatly in their cis-regulatory element architecture, resulting in changes in regulatory network connectivity. Whole-genome duplicates (WGDs) have approximately twice as many footprints in their promoters left by potential regulatory proteins than do tandem duplicates (TDs). The WGDs have a greater average number of footprint differences between paralogs than TDs. The footprints, in turn, result in more regulatory network connections between WGDs and other genes, forming denser, more complex regulatory networks than shown by TDs. When comparing regulatory connections between duplicates, WGDs had more pairs in which the two genes are either partially or fully diverged in their network connections, but fewer genes with no network connections than the TDs. There is evidence of younger TDs and WGDs having fewer unique connections compared with older duplicates. This study provides insights into cis-regulatory element evolution and network divergence in duplicated genes. PMID:26474639

  11. Two cis-DNA elements involved in myeloid-cell-specific expression and gamma interferon (IFN-gamma) activation of the human high-affinity Fc gamma receptor gene: a novel IFN regulatory mechanism.

    PubMed Central

    Perez, C; Wietzerbin, J; Benech, P D

    1993-01-01

    The human high-affinity receptor for the constant region of immunoglobulin G (human Fc gamma R1) is encoded by two mRNAs induced selectively by gamma interferon (IFN-gamma) and expressed in cells of myeloid lineage. The cis-DNA element (GRR) previously found to confer IFN-gamma responsiveness to this gene acts as an inducible enhancer and is the target of an IFN-gamma-activated factor(s) (GIRE-BP) in cells of different origins. Although the GRR motif is not related to the DNA elements involved in the regulation of other IFN-stimulated genes, GIRE-BP binding depends on the IFN-gamma-dependent activation of the 91-kDa protein known to be one of the factors of a transcriptional complex activated by IFN-alpha. Deletions of the Fc gamma R1 promoter allowed us to identify a 25-bp element, downstream from the GRR motif, conferring cell-type-specific expression. This element, called MATE (myeloid activating transcription element), is the DNA target for constitutive factors forming two complexes, MATE-BP1 and MATE-BP2. In accordance with the functional analysis, MATE-BP binding activities were detected in extracts prepared from myeloid cell lines such as THP-1, HL-60, and U-937 but not in HeLa cell extracts. The MATE motif is present not only in the promoter of other Fc receptor genes but also in several promoters of genes whose expression is restricted to monocytic cells. Our results suggest that human Fc gamma R1 gene expression in myeloid cells is initiated by the interaction of IFN-gamma-activated factors with cell-type-specific factors through their binding to the GRR and MATE motifs. Images PMID:8455606

  12. Modeling DNA sequence-based cis-regulatory gene networks.

    PubMed

    Bolouri, Hamid; Davidson, Eric H

    2002-06-01

    Gene network analysis requires computationally based models which represent the functional architecture of regulatory interactions, and which provide directly testable predictions. The type of model that is useful is constrained by the particular features of developmentally active cis-regulatory systems. These systems function by processing diverse regulatory inputs, generating novel regulatory outputs. A computational model which explicitly accommodates this basic concept was developed earlier for the cis-regulatory system of the endo16 gene of the sea urchin. This model represents the genetically mandated logic functions that the system executes, but also shows how time-varying kinetic inputs are processed in different circumstances into particular kinetic outputs. The same basic design features can be utilized to construct models that connect the large number of cis-regulatory elements constituting developmental gene networks. The ultimate aim of the network models discussed here is to represent the regulatory relationships among the genomic control systems of the genes in the network, and to state their functional meaning. The target site sequences of the cis-regulatory elements of these genes constitute the physical basis of the network architecture. Useful models for developmental regulatory networks must represent the genetic logic by which the system operates, but must also be capable of explaining the real time dynamics of cis-regulatory response as kinetic input and output data become available. Most importantly, however, such models must display in a direct and transparent manner fundamental network design features such as intra- and intercellular feedback circuitry; the sources of parallel inputs into each cis-regulatory element; gene battery organization; and use of repressive spatial inputs in specification and boundary formation. Successful network models lead to direct tests of key architectural features by targeted cis-regulatory analysis. PMID

  13. A saturation screen for cis-acting regulatory DNA in the Hox genes of Ciona intestinalis

    SciTech Connect

    Keys, David N.; Lee, Byung-in; Di Gregorio, Anna; Harafuji, Naoe; Detter, Chris; Wang, Mei; Kahsai, Orsalem; Ahn, Sylvia; Arellano, Andre; Zhang, Quin; Trong, Stephan; Doyle, Sharon A.; Satoh, Noriyuki; Satou, Yutaka; Saiga, Hidetoshi; Christian, Allen; Rokhsar, Dan; Hawkins, Trevor L.; Levine, Mike; Richardson, Paul

    2005-01-05

    A screen for the systematic identification of cis-regulatory elements within large (>100 kb) genomic domains containing Hox genes was performed by using the basal chordate Ciona intestinalis. Randomly generated DNA fragments from bacterial artificial chromosomes containing two clusters of Hox genes were inserted into a vector upstream of a minimal promoter and lacZ reporter gene. A total of 222 resultant fusion genes were separately electroporated into fertilized eggs, and their regulatory activities were monitored in larvae. In sum, 21 separable cis-regulatory elements were found. These include eight Hox linked domains that drive expression in nested anterior-posterior domains of ectodermally derived tissues. In addition to vertebrate-like CNS regulation, the discovery of cis-regulatory domains that drive epidermal transcription suggests that C. intestinalis has arthropod-like Hox patterning in the epidermis.

  14. Comprehensive identification and analysis of human accelerated regulatory DNA

    PubMed Central

    Gittelman, Rachel M.; Hun, Enna; Ay, Ferhat; Madeoy, Jennifer; Pennacchio, Len; Noble, William S.; Hawkins, R. David; Akey, Joshua M.

    2015-01-01

    It has long been hypothesized that changes in gene regulation have played an important role in human evolution, but regulatory DNA has been much more difficult to study compared with protein-coding regions. Recent large-scale studies have created genome-scale catalogs of DNase I hypersensitive sites (DHSs), which demark potentially functional regulatory DNA. To better define regulatory DNA that has been subject to human-specific adaptive evolution, we performed comprehensive evolutionary and population genetics analyses on over 18 million DHSs discovered in 130 cell types. We identified 524 DHSs that are conserved in nonhuman primates but accelerated in the human lineage (haDHS), and estimate that 70% of substitutions in haDHSs are attributable to positive selection. Through extensive computational and experimental analyses, we demonstrate that haDHSs are often active in brain or neuronal cell types; play an important role in regulating the expression of developmentally important genes, including many transcription factors such as SOX6, POU3F2, and HOX genes; and identify striking examples of adaptive regulatory evolution that may have contributed to human-specific phenotypes. More generally, our results reveal new insights into conserved and adaptive regulatory DNA in humans and refine the set of genomic substrates that distinguish humans from their closest living primate relatives. PMID:26104583

  15. Comprehensive identification and analysis of human accelerated regulatory DNA.

    PubMed

    Gittelman, Rachel M; Hun, Enna; Ay, Ferhat; Madeoy, Jennifer; Pennacchio, Len; Noble, William S; Hawkins, R David; Akey, Joshua M

    2015-09-01

    It has long been hypothesized that changes in gene regulation have played an important role in human evolution, but regulatory DNA has been much more difficult to study compared with protein-coding regions. Recent large-scale studies have created genome-scale catalogs of DNase I hypersensitive sites (DHSs), which demark potentially functional regulatory DNA. To better define regulatory DNA that has been subject to human-specific adaptive evolution, we performed comprehensive evolutionary and population genetics analyses on over 18 million DHSs discovered in 130 cell types. We identified 524 DHSs that are conserved in nonhuman primates but accelerated in the human lineage (haDHS), and estimate that 70% of substitutions in haDHSs are attributable to positive selection. Through extensive computational and experimental analyses, we demonstrate that haDHSs are often active in brain or neuronal cell types; play an important role in regulating the expression of developmentally important genes, including many transcription factors such as SOX6, POU3F2, and HOX genes; and identify striking examples of adaptive regulatory evolution that may have contributed to human-specific phenotypes. More generally, our results reveal new insights into conserved and adaptive regulatory DNA in humans and refine the set of genomic substrates that distinguish humans from their closest living primate relatives. PMID:26104583

  16. Close Sequence Comparisons are Sufficient to Identify Humancis-Regulatory Elements

    SciTech Connect

    Prabhakar, Shyam; Poulin, Francis; Shoukry, Malak; Afzal, Veena; Rubin, Edward M.; Couronne, Olivier; Pennacchio, Len A.

    2005-12-01

    Cross-species DNA sequence comparison is the primary method used to identify functional noncoding elements in human and other large genomes. However, little is known about the relative merits of evolutionarily close and distant sequence comparisons, due to the lack of a universal metric for sequence conservation, and also the paucity of empirically defined benchmark sets of cis-regulatory elements. To address this problem, we developed a general-purpose algorithm (Gumby) that detects slowly-evolving regions in primate, mammalian and more distant comparisons without requiring adjustment of parameters, and ranks conserved elements by P-value using Karlin-Altschul statistics. We benchmarked Gumby predictions against previously identified cis-regulatory elements at diverse genomic loci, and also tested numerous extremely conserved human-rodent sequences for transcriptional enhancer activity using reporter-gene assays in transgenic mice. Human regulatory elements were identified with acceptable sensitivity and specificity by comparison with 1-5 other eutherian mammals or 6 other simian primates. More distant comparisons (marsupial, avian, amphibian and fish) failed to identify many of the empirically defined functional noncoding elements. We derived an intuitive relationship between ancient and recent noncoding sequence conservation from whole genome comparative analysis, which explains some of these findings. Lastly, we determined that, in addition to strength of conservation, genomic location and/or density of surrounding conserved elements must also be considered in selecting candidate enhancers for testing at embryonic time points.

  17. Evolution of anterior Hox regulatory elements among chordates

    PubMed Central

    2011-01-01

    Background The Hox family of transcription factors has a fundamental role in segmentation pathways and axial patterning of embryonic development and their clustered organization is linked with the regulatory mechanisms governing their coordinated expression along embryonic axes. Among chordates, of particular interest are the Hox paralogous genes in groups 1-4 since their expression is coupled to the control of regional identity in the anterior nervous system, where the highest structural diversity is observed. Results To investigate the degree of conservation in cis-regulatory components that form the basis of Hox expression in the anterior nervous system, we have used assays for transcriptional activity in ascidians and vertebrates to compare and contrast regulatory potential. We identified four regulatory sequences located near the CiHox1, CiHox2 and CiHox4 genes of the ascidian Ciona intestinalis which direct neural specific domains of expression. Using functional assays in Ciona and vertebrate embryos in combination with sequence analyses of enhancer fragments located in similar positions adjacent to Hox paralogy group genes, we compared the activity of these four Ciona cis-elements with a series of neural specific enhancers from the amphioxus Hox1-3 genes and from mouse Hox paralogous groups 1-4. Conclusions This analysis revealed that Kreisler and Krox20 dependent enhancers critical in segmental regulation of the hindbrain appear to be specific for the vertebrate lineage. In contrast, neural enhancers that function as Hox response elements through the action of Hox/Pbx binding motifs have been conserved during chordate evolution. The functional assays reveal that these Hox response cis-elements are recognized by the regulatory components of different and extant species. Together, our results indicate that during chordate evolution, cis-elements dependent upon Hox/Pbx regulatory complexes, are responsible for key aspects of segmental Hox expression in neural

  18. Conservation of DNA curvature signals in regulatory regions of prokaryotic genes

    PubMed Central

    Jáuregui, Ruy; Abreu-Goodger, Cei; Moreno-Hagelsieb, Gabriel; Collado-Vides, Julio; Merino, Enrique

    2003-01-01

    DNA curvature plays a well-characterized role in many transcriptional regulation mechanisms. We present evidence for the conservation of curvature signals in putative regulatory regions of several archaeal and eubacterial genomes. Genes with highly curved upstream regions were identified in orthologous groups, based on the annotations of the Cluster of Orthologous Groups of proteins (COG) database. COGs possessing a significant number of genes with curvature signals were analyzed, and conserved properties were found in several cases. Curvature signals related to regulatory sites, previously described in single organisms, were located in a broad spectrum of bacterial genomes. Global regulatory proteins, such as HU, IHF and FIS, known to bind to curved DNA and to be autoregulated, were found to present conserved DNA curvature signals in their regulatory regions, emphasizing the fact that structural parameters of the DNA molecule are conserved elements in the process of transcriptional regulation of some systems. It is currently an open question whether these diverse systems are part of an integrated global regulatory response in different microorganisms. PMID:14627810

  19. Transcriptional Targeting in the Airway Using Novel Gene Regulatory Elements

    PubMed Central

    Burnight, Erin R.; Wang, Guoshun; McCray, Paul B.

    2012-01-01

    The delivery of cystic fibrosis transmembrane conductance regulator (CFTR) to airway epithelia is a goal of many gene therapy strategies to treat cystic fibrosis. Because the native regulatory elements of the CFTR are not well characterized, the development of vectors with heterologous promoters of varying strengths and specificity would aid in our selection of optimal reagents for the appropriate expression of the vector-delivered CFTR gene. Here we contrasted the performance of several novel gene-regulatory elements. Based on airway expression analysis, we selected putative regulatory elements from BPIFA1 and WDR65 to investigate. In addition, we selected a human CFTR promoter region (∼ 2 kb upstream of the human CFTR transcription start site) to study. Using feline immunodeficiency virus vectors containing the candidate elements driving firefly luciferase, we transduced murine nasal epithelia in vivo. Luciferase expression persisted for 30 weeks, which was the duration of the experiment. Furthermore, when the nasal epithelium was ablated using the detergent polidocanol, the mice showed a transient loss of luciferase expression that returned 2 weeks after administration, suggesting that our vectors transduced a progenitor cell population. Importantly, the hWDR65 element drove sufficient CFTR expression to correct the anion transport defect in CFTR-null epithelia. These results will guide the development of optimal vectors for sufficient, sustained CFTR expression in airway epithelia. PMID:22447971

  20. Identification of germline transcriptional regulatory elements in Aedes aegypti

    NASA Astrophysics Data System (ADS)

    Akbari, Omar S.; Papathanos, Philippos A.; Sandler, Jeremy E.; Kennedy, Katie; Hay, Bruce A.

    2014-02-01

    The mosquito Aedes aegypti is the principal vector for the yellow fever and dengue viruses, and is also responsible for recent outbreaks of the alphavirus chikungunya. Vector control strategies utilizing engineered gene drive systems are being developed as a means of replacing wild, pathogen transmitting mosquitoes with individuals refractory to disease transmission, or bringing about population suppression. Several of these systems, including Medea, UDMEL, and site-specific nucleases, which can be used to drive genes into populations or bring about population suppression, utilize transcriptional regulatory elements that drive germline-specific expression. Here we report the identification of multiple regulatory elements able to drive gene expression specifically in the female germline, or in the male and female germline, in the mosquito Aedes aegypti. These elements can also be used as tools with which to probe the roles of specific genes in germline function and in the early embryo, through overexpression or RNA interference.

  1. Identification of a novel cis-regulatory element essential for immune tolerance.

    PubMed

    LaFlam, Taylor N; Seumois, Grégory; Miller, Corey N; Lwin, Wint; Fasano, Kayla J; Waterfield, Michael; Proekt, Irina; Vijayanand, Pandurangan; Anderson, Mark S

    2015-11-16

    Thymic central tolerance is essential to preventing autoimmunity. In medullary thymic epithelial cells (mTECs), the Autoimmune regulator (Aire) gene plays an essential role in this process by driving the expression of a diverse set of tissue-specific antigens (TSAs), which are presented and help tolerize self-reactive thymocytes. Interestingly, Aire has a highly tissue-restricted pattern of expression, with only mTECs and peripheral extrathymic Aire-expressing cells (eTACs) known to express detectable levels in adults. Despite this high level of tissue specificity, the cis-regulatory elements that control Aire expression have remained obscure. Here, we identify a highly conserved noncoding DNA element that is essential for Aire expression. This element shows enrichment of enhancer-associated histone marks in mTECs and also has characteristics of being an NF-κB-responsive element. Finally, we find that this element is essential for Aire expression in vivo and necessary to prevent spontaneous autoimmunity, reflecting the importance of this regulatory DNA element in promoting immune tolerance. PMID:26527800

  2. Taking DNA to market and regulatory defaults.

    PubMed

    Flower, Michael J

    1981-01-01

    Forums for public participation in guiding the direction of recombinant DNA research and development are being closed off as the commercialization of research proceeds in conjunction with a government move to deregulation and a dying down of public debate on the potential hazards of gene-splicing technology. It appears that the private sector alone will decide such issues of public concern as the question of risks, the withholding of research results for proprietary reasons, joint university-industrial ventures, return on public investment in research, and the possibility of genetic intervention in humans. PMID:11645466

  3. Computational discovery of regulatory elements in a continuous expression space

    PubMed Central

    2012-01-01

    Approaches for regulatory element discovery from gene expression data usually rely on clustering algorithms to partition the data into clusters of co-expressed genes. Gene regulatory sequences are then mined to find overrepresented motifs in each cluster. However, this ad hoc partition rarely fits the biological reality. We propose a novel method called RED2 that avoids data clustering by estimating motif densities locally around each gene. We show that RED2 detects numerous motifs not detected by clustering-based approaches, and that most of these correspond to characterized motifs. RED2 can be accessed online through a user-friendly interface. PMID:23186104

  4. Bioinformatic identification of novel regulatory DNA sequence motifs in Streptomyces coelicolor

    PubMed Central

    Studholme, David J; Bentley, Stephen D; Kormanec, Jan

    2004-01-01

    Background Streptomyces coelicolor is a bacterium with a vast repertoire of metabolic functions and complex systems of cellular development. Its genome sequence is rich in genes that encode regulatory proteins to control these processes in response to its changing environment. We wished to apply a recently published bioinformatic method for identifying novel regulatory sequence signals to gain new insights into regulation in S. coelicolor. Results The method involved production of position-specific weight matrices from alignments of over-represented words of DNA sequence. We generated 2497 weight matrices, each representing a candidate regulatory DNA sequence motif. We scanned the genome sequence of S. coelicolor against each of these matrices. A DNA sequence motif represented by one of the matrices was found preferentially in non-coding sequences immediately upstream of genes involved in polysaccharide degradation, including several that encode chitinases. This motif (TGGTCTAGACCA) was also found upstream of genes encoding components of the phosphoenolpyruvate phosphotransfer system (PTS). We hypothesise that this DNA sequence motif represents a regulatory element that is responsive to availability of carbon-sources. Other motifs of potential biological significance were found upstream of genes implicated in secondary metabolism (TTAGGTtAGgCTaACCTAA), sigma factors (TGACN19TGAC), DNA replication and repair (ttgtCAGTGN13TGGA), nucleotide conversions (CTACgcNCGTAG), and ArsR (TCAGN12TCAG). A motif found upstream of genes involved in chromosome replication (TGTCagtgcN7Tagg) was similar to a previously described motif found in UV-responsive promoters. Conclusions We successfully applied a recently published in silico method to identify conserved sequence motifs in S. coelicolor that may be biologically significant as regulatory elements. Our data are broadly consistent with and further extend data from previously published studies. We invite experimental testing of

  5. An information transmission model for transcription factor binding at regulatory DNA sites

    PubMed Central

    2012-01-01

    Background Computational identification of transcription factor binding sites (TFBSs) is a rapid, cost-efficient way to locate unknown regulatory elements. With increased potential for high-throughput genome sequencing, the availability of accurate computational methods for TFBS prediction has never been as important as it currently is. To date, identifying TFBSs with high sensitivity and specificity is still an open challenge, necessitating the development of novel models for predicting transcription factor-binding regulatory DNA elements. Results Based on the information theory, we propose a model for transcription factor binding of regulatory DNA sites. Our model incorporates position interdependencies in effective ways. The model computes the information transferred (TI) between the transcription factor and the TFBS during the binding process and uses TI as the criterion to determine whether the sequence motif is a possible TFBS. Based on this model, we developed a computational method to identify TFBSs. By theoretically proving and testing our model using both real and artificial data, we found that our model provides highly accurate predictive results. Conclusions In this study, we present a novel model for transcription factor binding regulatory DNA sites. The model can provide an increased ability to detect TFBSs. PMID:22672438

  6. Identifying splicing regulatory elements with de Bruijn graphs.

    PubMed

    Badr, Eman; Heath, Lenwood S

    2014-12-01

    Splicing regulatory elements (SREs) are short, degenerate sequences on pre-mRNA molecules that enhance or inhibit the splicing process via the binding of splicing factors, proteins that regulate the functioning of the spliceosome. Existing methods for identifying SREs in a genome are either experimental or computational. Here, we propose a formalism based on de Bruijn graphs that combines genomic structure, word count enrichment analysis, and experimental evidence to identify SREs found in exons. In our approach, SREs are not restricted to a fixed length (i.e., k-mers, for a fixed k). As a result, we identify 2001 putative exonic enhancers and 3080 putative exonic silencers for human genes, with lengths varying from 6 to 15 nucleotides. Many of the predicted SREs overlap with experimentally verified binding sites. Our model provides a novel method to predict variable length putative regulatory elements computationally for further experimental investigation. PMID:25393830

  7. PREDetector: a new tool to identify regulatory elements in bacterial genomes.

    PubMed

    Hiard, Samuel; Marée, Raphaël; Colson, Séverine; Hoskisson, Paul A; Titgemeyer, Fritz; van Wezel, Gilles P; Joris, Bernard; Wehenkel, Louis; Rigali, Sébastien

    2007-06-15

    In the post-genomic area, the prediction of transcription factor regulons by position weight matrix-based programmes is a powerful approach to decipher biological pathways and to modelize regulatory networks in bacteria. The main difficulty once a regulon prediction is available is to estimate its reliability prior to start expensive experimental validations and therefore trying to find a way how to identify true positive hits from an endless list of potential target genes of a regulatory protein. Here we introduce PREDetector (Prokaryotic Regulatory Elements Detector), a tool developed for predicting regulons of DNA-binding proteins in bacterial genomes that, beside the automatic prediction, scoring and positioning of potential binding sites and their respective target genes in annotated bacterial genomes, it also provides an easy way to estimate the thresholds where to find reliable possible new target genes. PREDetector can be downloaded freely at http://www.montefiore.ulg.ac.be/~hiard/PreDetector/PreDetector.php. PMID:17451648

  8. Environment-induced epigenetic reprogramming in genomic regulatory elements in smoking mothers and their children.

    PubMed

    Bauer, Tobias; Trump, Saskia; Ishaque, Naveed; Thürmann, Loreen; Gu, Lei; Bauer, Mario; Bieg, Matthias; Gu, Zuguang; Weichenhan, Dieter; Mallm, Jan-Philipp; Röder, Stefan; Herberth, Gunda; Takada, Eiko; Mücke, Oliver; Winter, Marcus; Junge, Kristin M; Grützmann, Konrad; Rolle-Kampczyk, Ulrike; Wang, Qi; Lawerenz, Christian; Borte, Michael; Polte, Tobias; Schlesner, Matthias; Schanne, Michaela; Wiemann, Stefan; Geörg, Christina; Stunnenberg, Hendrik G; Plass, Christoph; Rippe, Karsten; Mizuguchi, Junichiro; Herrmann, Carl; Eils, Roland; Lehmann, Irina

    2016-03-01

    Epigenetic mechanisms have emerged as links between prenatal environmental exposure and disease risk later in life. Here, we studied epigenetic changes associated with maternal smoking at base pair resolution by mapping DNA methylation, histone modifications, and transcription in expectant mothers and their newborn children. We found extensive global differential methylation and carefully evaluated these changes to separate environment associated from genotype-related DNA methylation changes. Differential methylation is enriched in enhancer elements and targets in particular "commuting" enhancers having multiple, regulatory interactions with distal genes. Longitudinal whole-genome bisulfite sequencing revealed that DNA methylation changes associated with maternal smoking persist over years of life. Particularly in children prenatal environmental exposure leads to chromatin transitions into a hyperactive state. Combined DNA methylation, histone modification, and gene expression analyses indicate that differential methylation in enhancer regions is more often functionally translated than methylation changes in promoters or non-regulatory elements. Finally, we show that epigenetic deregulation of a commuting enhancer targeting c-Jun N-terminal kinase 2 (JNK2) is linked to impaired lung function in early childhood. PMID:27013061

  9. Combinatorial Gene Regulatory Functions Underlie Ultraconserved Elements in Drosophila

    PubMed Central

    Warnefors, Maria; Hartmann, Britta; Thomsen, Stefan; Alonso, Claudio R.

    2016-01-01

    Ultraconserved elements (UCEs) are discrete genomic elements conserved across large evolutionary distances. Although UCEs have been linked to multiple facets of mammalian gene regulation their extreme evolutionary conservation remains largely unexplained. Here, we apply a computational approach to investigate this question in Drosophila, exploring the molecular functions of more than 1,500 UCEs shared across the genomes of 12 Drosophila species. Our data indicate that Drosophila UCEs are hubs for gene regulatory functions and suggest that UCE sequence invariance originates from their combinatorial roles in gene control. We also note that the gene regulatory roles of intronic and intergenic UCEs (iUCEs) are distinct from those found in exonic UCEs (eUCEs). In iUCEs, transcription factor (TF) and epigenetic factor binding data strongly support iUCE roles in transcriptional and epigenetic regulation. In contrast, analyses of eUCEs indicate that they are two orders of magnitude more likely than the expected to simultaneously include protein-coding sequence, TF-binding sites, splice sites, and RNA editing sites but have reduced roles in transcriptional or epigenetic regulation. Furthermore, we use a Drosophila cell culture system and transgenic Drosophila embryos to validate the notion of UCE combinatorial regulatory roles using an eUCE within the Hox gene Ultrabithorax and show that its protein-coding region also contains alternative splicing regulatory information. Taken together our experiments indicate that UCEs emerge as a result of combinatorial gene regulatory roles and highlight common features in mammalian and insect UCEs implying that similar processes might underlie ultraconservation in diverse animal taxa. PMID:27247329

  10. Combinatorial Gene Regulatory Functions Underlie Ultraconserved Elements in Drosophila.

    PubMed

    Warnefors, Maria; Hartmann, Britta; Thomsen, Stefan; Alonso, Claudio R

    2016-09-01

    Ultraconserved elements (UCEs) are discrete genomic elements conserved across large evolutionary distances. Although UCEs have been linked to multiple facets of mammalian gene regulation their extreme evolutionary conservation remains largely unexplained. Here, we apply a computational approach to investigate this question in Drosophila, exploring the molecular functions of more than 1,500 UCEs shared across the genomes of 12 Drosophila species. Our data indicate that Drosophila UCEs are hubs for gene regulatory functions and suggest that UCE sequence invariance originates from their combinatorial roles in gene control. We also note that the gene regulatory roles of intronic and intergenic UCEs (iUCEs) are distinct from those found in exonic UCEs (eUCEs). In iUCEs, transcription factor (TF) and epigenetic factor binding data strongly support iUCE roles in transcriptional and epigenetic regulation. In contrast, analyses of eUCEs indicate that they are two orders of magnitude more likely than the expected to simultaneously include protein-coding sequence, TF-binding sites, splice sites, and RNA editing sites but have reduced roles in transcriptional or epigenetic regulation. Furthermore, we use a Drosophila cell culture system and transgenic Drosophila embryos to validate the notion of UCE combinatorial regulatory roles using an eUCE within the Hox gene Ultrabithorax and show that its protein-coding region also contains alternative splicing regulatory information. Taken together our experiments indicate that UCEs emerge as a result of combinatorial gene regulatory roles and highlight common features in mammalian and insect UCEs implying that similar processes might underlie ultraconservation in diverse animal taxa. PMID:27247329

  11. SERE, a widely dispersed bacterial repetitive DNA element.

    PubMed

    Rajashekara, G; Koeuth, T; Nevile, S; Back, A; Nagaraja, K V; Lupski, J R; Kapur, V

    1998-06-01

    The presence of a Salmonella serotype Enteritidis repeat element (SERE) located within the upstream regulatory region of the sefABCD operon encoding fimbrial proteins is reported. DNA dot-blot hybridisation analyses and computerised searches of genetic databases indicate that SERE is well conserved and widely distributed throughout the bacterial and archaeal kingdoms. A SERE-based polymerase chain reaction (SERE-PCR) assay was developed to fingerprint 54 isolates of Enteritidis representing nine distinct phage types and 54 isolates of other Salmonella serotypes. SERE-PCR identified five distinct fingerprint profiles among the 54 Enteritidis isolates; no correlation between phage types and SERE-PCR fingerprint patterns was noticed. SERE-PCR was reproducible, rapid and easy to perform. The results of this investigation suggest that the limited heterogeneity of SERE-PCR fingerprint patterns can be utilised to develop serotype- and serogroup-specific fingerprint patterns for isolates of Enteritidis. PMID:9879967

  12. Diverse regulatory circuits for transfer of conjugative elements.

    PubMed

    Singh, Praveen K; Meijer, Wilfried J J

    2014-09-01

    Conjugation systems are present on many plasmids as well as on chromosomally integrated elements. Conjugation, which is a major route by which bacteria exchange genetic material, is a complex and energy-consuming process. Hence, a shared feature of conjugation systems is that expression of the genes involved is strictly controlled in such a way that conjugation is kept in a default 'OFF' state and that the process is switched on only under conditions that favor the transfer of the conjugative element into a recipient cell. However, there is a remarkable diversity in the way by which conjugation genes present on different transferable elements are regulated. Here, we review these diverse regulatory circuits on the basis of several prototypes with a special focus on the recently discovered regulation of the conjugation genes present on the native Bacillus subtilis plasmid pLS20. PMID:24995588

  13. Transcriptional regulatory elements downstream of the JunB gene.

    PubMed Central

    Perez-Albuerne, E D; Schatteman, G; Sanders, L K; Nathans, D

    1993-01-01

    JunB is an immediate early transcription factor that is induced by a variety of extracellular signaling agents, including growth factors, phorbol esters, and agents that elevate cyclic AMP. The mechanism of activation of the gene encoding JunB by these agents is not well understood. By using the JunB gene together with flanking DNA in transfection experiments, we show that a serum response element (SRE) and/or a cAMP response element (CRE) downstream of the gene mediate the response of the gene in mouse NIH 3T3 cells to serum, platelet-derived growth factor, basic fibroblast growth factor, phorbol ester, and forskolin. In addition, a segment of DNA just upstream of the TATA box is required for optimal activation of the gene. Images Fig. 1 Fig. 5 PMID:8265655

  14. The structure and function of the regulatory elements of the Escherichia coli uvrB gene.

    PubMed Central

    van den Berg, E; Zwetsloot, J; Noordermeer, I; Pannekoek, H; Dekker, B; Dijkema, R; van Ormondt, H

    1981-01-01

    The construction and properties of recombinant plasmids carrying the Escherichia coli uvrB gene, including its transcriptional- and translational regulatory elements, is reported. The DNA sequence of the region, which governs the expression of the uvrB gene, has been determined. Within this sequence two non-overlapping DNA segments match the model sequence for Escherichia coli promoters (1). The '-10 regions' and the '-35 regions' of the proposed uvrB promoters are, respectively, 5'TAAAAT (P1), 5'TATAAT (P2) and 5'TTGGCA (P1), 5'GTGATG (P2). The existence and the position of these promoters has been established by elimination of one promoter (P2), using molecular cloning procedures, by length measurements of in vitro synthesized 'run-off' transcripts and by protection of the uvrB regulatory region for S1 nuclease digestion using in vivo made RNA. Potential sites of interaction within the uvrB regulatory region with regulatory proteins, such as the LexA protein (2) and the UvrC protein (3) are discussed. Images PMID:6273801

  15. BRCA1 EXON 11, a CERES (composite regulatory element of splicing) element involved in splice regulation.

    PubMed

    Tammaro, Claudia; Raponi, Michela; Wilson, David I; Baralle, Diana

    2014-01-01

    Unclassified variants (UV) of BRCA1 can affect normal pre-mRNA splicing. Here, we investigate the UV c.693G>A, a "silent" change in BRCA1 exon 11, which we have found induces aberrant splicing in patient carriers and in vitro. Using a minigene assay, we show that the UV c.693G>A has a strong effect on the splicing isoform ratio of BRCA1. Systematic site-directed mutagenesis of the area surrounding the nucleotide position c.693G>A induced variable changes in the level of exon 11 inclusion/exclusion in the mRNA, pointing to the presence of a complex regulatory element with overlapping enhancer and silencer functions. Accordingly, protein binding analysis in the region detected several splicing regulatory factors involved, including SRSF1, SRSF6 and SRSF9, suggesting that this sequence represents a composite regulatory element of splicing (CERES). PMID:25056543

  16. Analysis of long-range interactions in primary human cells identifies cooperative CFTR regulatory elements

    PubMed Central

    Moisan, Stéphanie; Berlivet, Soizik; Ka, Chandran; Gac, Gérald Le; Dostie, Josée; Férec, Claude

    2016-01-01

    A mechanism by which control DNA elements regulate transcription over large linear genomic distances is by achieving close physical proximity with genes, and looping of the intervening chromatin paths. Alterations of such regulatory ‘chromatin looping’ systems are likely to play a critical role in human genetic disease at large. Here, we studied the spatial organization of a ≈790 kb locus encompassing the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Dysregulation of CFTR is responsible for cystic fibrosis, which is the most common lethal genetic disorder in Caucasian populations. CFTR is a relatively large gene of 189 kb with a rather complex tissue-specific and temporal expression profile. We used chromatin conformation at the CFTR locus to identify new DNA sequences that regulate its transcription. By comparing 5C chromatin interaction maps of the CFTR locus in expressing and non-expressing human primary cells, we identified several new contact points between the CFTR promoter and its surroundings, in addition to regions featuring previously described regulatory elements. We demonstrate that two of these novel interacting regions cooperatively increase CFTR expression, and suggest that the new enhancer elements located on either side of the gene are brought together through chromatin looping via CTCF. PMID:26615198

  17. Analysis of long-range interactions in primary human cells identifies cooperative CFTR regulatory elements.

    PubMed

    Moisan, Stéphanie; Berlivet, Soizik; Ka, Chandran; Gac, Gérald Le; Dostie, Josée; Férec, Claude

    2016-04-01

    A mechanism by which control DNA elements regulate transcription over large linear genomic distances is by achieving close physical proximity with genes, and looping of the intervening chromatin paths. Alterations of such regulatory 'chromatin looping' systems are likely to play a critical role in human genetic disease at large. Here, we studied the spatial organization of a ≈790 kb locus encompassing the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Dysregulation ofCFTRis responsible for cystic fibrosis, which is the most common lethal genetic disorder in Caucasian populations.CFTRis a relatively large gene of 189 kb with a rather complex tissue-specific and temporal expression profile. We used chromatin conformation at theCFTRlocus to identify new DNA sequences that regulate its transcription. By comparing 5C chromatin interaction maps of theCFTRlocus in expressing and non-expressing human primary cells, we identified several new contact points between theCFTRpromoter and its surroundings, in addition to regions featuring previously described regulatory elements. We demonstrate that two of these novel interacting regions cooperatively increaseCFTRexpression, and suggest that the new enhancer elements located on either side of the gene are brought together through chromatin loopingviaCTCF. PMID:26615198

  18. Close sequence comparisons are sufficient to identify human cis-regulatory elements.

    PubMed

    Prabhakar, Shyam; Poulin, Francis; Shoukry, Malak; Afzal, Veena; Rubin, Edward M; Couronne, Olivier; Pennacchio, Len A

    2006-07-01

    Cross-species DNA sequence comparison is the primary method used to identify functional noncoding elements in human and other large genomes. However, little is known about the relative merits of evolutionarily close and distant sequence comparisons. To address this problem, we identified evolutionarily conserved noncoding regions in primate, mammalian, and more distant comparisons using a uniform approach (Gumby) that facilitates unbiased assessment of the impact of evolutionary distance on predictive power. We benchmarked computational predictions against previously identified cis-regulatory elements at diverse genomic loci and also tested numerous extremely conserved human-rodent sequences for transcriptional enhancer activity using an in vivo enhancer assay in transgenic mice. Human regulatory elements were identified with acceptable sensitivity (53%-80%) and true-positive rate (27%-67%) by comparison with one to five other eutherian mammals or six other simian primates. More distant comparisons (marsupial, avian, amphibian, and fish) failed to identify many of the empirically defined functional noncoding elements. Our results highlight the practical utility of close sequence comparisons, and the loss of sensitivity entailed by more distant comparisons. We derived an intuitive relationship between ancient and recent noncoding sequence conservation from whole-genome comparative analysis that explains most of the observations from empirical benchmarking. Lastly, we determined that, in addition to strength of conservation, genomic location and/or density of surrounding conserved elements must also be considered in selecting candidate enhancers for in vivo testing at embryonic time points. PMID:16769978

  19. Structural property of regulatory elements in human promoters

    NASA Astrophysics Data System (ADS)

    Cao, Xiao-Qin; Zeng, Jia; Yan, Hong

    2008-04-01

    The capacity of transcription factors to activate gene expression is encoded in the promoter sequences, which are composed of short regulatory motifs that function as transcription factor binding sites (TFBSs) for specific proteins. To the best of our knowledge, the structural property of TFBSs that controls transcription is still poorly understood. Rigidity is one of the important structural properties of DNA, and plays an important role in guiding DNA-binding proteins to the target sites efficiently. After analyzing the rigidity of 2897 TFBSs in 1871 human promoters, we show that TFBSs are generally more flexible than other genomic regions such as exons, introns, 3' untranslated regions, and TFBS-poor promoter regions. Furthermore, we find that the density of TFBSs is consistent with the average rigidity profile of human promoters upstream of the transcription start site, which implies that TFBSs directly influence the promoter structure. We also examine the local rigid regions probably caused by specific TFBSs such as the DNA sequence TATA(A/T)A(A/T) box, which may inhibit nucleosomes and thereby facilitate the access of transcription factors bound nearby. Our results suggest that the structural property of TFBSs accounts for the promoter structure as well as promoter activity.

  20. Single-stranded DNA-binding proteins PURalpha and PURbeta bind to a purine-rich negative regulatory element of the alpha-myosin heavy chain gene and control transcriptional and translational regulation of the gene expression. Implications in the repression of alpha-myosin heavy chain during heart failure.

    PubMed

    Gupta, Madhu; Sueblinvong, Viranuj; Raman, Jai; Jeevanandam, Valluvan; Gupta, Mahesh P

    2003-11-01

    The alpha-myosin heavy chain is a principal molecule of the thick filament of the sarcomere, expressed primarily in cardiac myocytes. The mechanism for its cardiac-restricted expression is not yet fully understood. We previously identified a purine-rich negative regulatory (PNR) element in the first intron of the gene, which is essential for its cardiac-specific expression (Gupta, M., Zak, R., Libermann, T. A., and Gupta, M. P. (1998) Mol. Cell. Biol. 18, 7243-7258). In this study we cloned and characterized muscle and non-muscle factors that bind to this element. We show that two single-stranded DNA-binding proteins of the PUR family, PURalpha and PURbeta, which are derived from cardiac myocytes, bind to the plus strand of the PNR element. In functional assays, PURalpha and PURbeta repressed alpha-myosin heavy chain (alpha-MHC) gene expression in the presence of upstream regulatory sequences of the gene. However, from HeLa cells an Ets family of protein, Ets-related protein (ERP), binds to double-stranded PNR element. The ERP.PNR complex inhibited the activity of the basal transcription complex from homologous as well as heterologous promoters in a PNR position-independent manner, suggesting that ERP acts as a silencer of alpha-MHC gene expression in non-muscle cells. We also show that PUR proteins are capable of binding to alpha-MHC mRNA and attenuate its translational efficiency. Furthermore, we show robust expression of PUR proteins in failing hearts where alpha-MHC mRNA levels are suppressed. Together, these results reveal that (i) PUR proteins participate in transcriptional as well as translational regulation of alpha-MHC expression in cardiac myocytes and (ii) ERP may be involved in cardiac-restricted expression of the alpha-MHC gene by preventing its expression in non-muscle cells. PMID:12933792

  1. Epistatic Interactions in the Arabinose Cis-Regulatory Element

    PubMed Central

    Lagator, Mato; Igler, Claudia; Moreno, Anaísa B.; Guet, Călin C.; Bollback, Jonathan P.

    2016-01-01

    Changes in gene expression are an important mode of evolution; however, the proximate mechanism of these changes is poorly understood. In particular, little is known about the effects of mutations within cis binding sites for transcription factors, or the nature of epistatic interactions between these mutations. Here, we tested the effects of single and double mutants in two cis binding sites involved in the transcriptional regulation of the Escherichia coli araBAD operon, a component of arabinose metabolism, using a synthetic system. This system decouples transcriptional control from any posttranslational effects on fitness, allowing a precise estimate of the effect of single and double mutations, and hence epistasis, on gene expression. We found that epistatic interactions between mutations in the araBAD cis-regulatory element are common, and that the predominant form of epistasis is negative. The magnitude of the interactions depended on whether the mutations are located in the same or in different operator sites. Importantly, these epistatic interactions were dependent on the presence of arabinose, a native inducer of the araBAD operon in vivo, with some interactions changing in sign (e.g., from negative to positive) in its presence. This study thus reveals that mutations in even relatively simple cis-regulatory elements interact in complex ways such that selection on the level of gene expression in one environment might perturb regulation in the other environment in an unpredictable and uncorrelated manner. PMID:26589997

  2. Epistatic Interactions in the Arabinose Cis-Regulatory Element.

    PubMed

    Lagator, Mato; Igler, Claudia; Moreno, Anaísa B; Guet, Călin C; Bollback, Jonathan P

    2016-03-01

    Changes in gene expression are an important mode of evolution; however, the proximate mechanism of these changes is poorly understood. In particular, little is known about the effects of mutations within cis binding sites for transcription factors, or the nature of epistatic interactions between these mutations. Here, we tested the effects of single and double mutants in two cis binding sites involved in the transcriptional regulation of the Escherichia coli araBAD operon, a component of arabinose metabolism, using a synthetic system. This system decouples transcriptional control from any posttranslational effects on fitness, allowing a precise estimate of the effect of single and double mutations, and hence epistasis, on gene expression. We found that epistatic interactions between mutations in the araBAD cis-regulatory element are common, and that the predominant form of epistasis is negative. The magnitude of the interactions depended on whether the mutations are located in the same or in different operator sites. Importantly, these epistatic interactions were dependent on the presence of arabinose, a native inducer of the araBAD operon in vivo, with some interactions changing in sign (e.g., from negative to positive) in its presence. This study thus reveals that mutations in even relatively simple cis-regulatory elements interact in complex ways such that selection on the level of gene expression in one environment might perturb regulation in the other environment in an unpredictable and uncorrelated manner. PMID:26589997

  3. [Regulatory potential of S/MAR elements in transient expression].

    PubMed

    Sass, A V; Ruda, V M; Akopov, S B; Snezhkov, E V; Nikolaev, L G; Sverdlov, E D

    2005-01-01

    S/MARs (scaffold/matrix attachment regions) are the DNA regions that are involved in the interaction with the nuclear matrix and are identified by in vitro methods. According to the available information, S/MARs possess an insulating activity, i.e., the ability to block the interaction between the enhancer and promoter in vivo, and are, probably, intact insulators or their fragments. Nevertheless, there is still no direct proof for this correspondence. To obtain additional information on the insulator activity of S/MARs, we selected five DNA fragments of different lengths and affinities for the nuclear matrix from the previously constructed library of S/MARs and tested their ability to serve as insulators. Two of five elements exhibited an insulator (enhancer-blocking) activity upon the transient transfection of CHO cells. None of the S/MARs displayed either promoter or enhancer/silencer activities in these cells. PMID:15787217

  4. Cell-type-specific enrichment of risk-associated regulatory elements at ovarian cancer susceptibility loci

    PubMed Central

    Coetzee, Simon G.; Shen, Howard C.; Hazelett, Dennis J.; Lawrenson, Kate; Kuchenbaecker, Karoline; Tyrer, Jonathan; Rhie, Suhn K.; Levanon, Keren; Karst, Alison; Drapkin, Ronny; Ramus, Susan J.; Couch, Fergus J.; Offit, Kenneth; Chenevix-Trench, Georgia; Monteiro, Alvaro N.A.; Antoniou, Antonis; Freedman, Matthew; Coetzee, Gerhard A.; Pharoah, Paul D.P.; Noushmehr, Houtan; Gayther, Simon A.

    2015-01-01

    Understanding the regulatory landscape of the human genome is a central question in complex trait genetics. Most single-nucleotide polymorphisms (SNPs) associated with cancer risk lie in non-protein-coding regions, implicating regulatory DNA elements as functional targets of susceptibility variants. Here, we describe genome-wide annotation of regions of open chromatin and histone modification in fallopian tube and ovarian surface epithelial cells (FTSECs, OSECs), the debated cellular origins of high-grade serous ovarian cancers (HGSOCs) and in endometriosis epithelial cells (EECs), the likely precursor of clear cell ovarian carcinomas (CCOCs). The regulatory architecture of these cell types was compared with normal human mammary epithelial cells and LNCaP prostate cancer cells. We observed similar positional patterns of global enhancer signatures across the three different ovarian cancer precursor cell types, and evidence of tissue-specific regulatory signatures compared to non-gynecological cell types. We found significant enrichment for risk-associated SNPs intersecting regulatory biofeatures at 17 known HGSOC susceptibility loci in FTSECs (P = 3.8 × 10−30), OSECs (P = 2.4 × 10−23) and HMECs (P = 6.7 × 10−15) but not for EECs (P = 0.45) or LNCaP cells (P = 0.88). Hierarchical clustering of risk SNPs conditioned on the six different cell types indicates FTSECs and OSECs are highly related (96% of samples using multi-scale bootstrapping) suggesting both cell types may be precursors of HGSOC. These data represent the first description of regulatory catalogues of normal precursor cells for different ovarian cancer subtypes, and provide unique insights into the tissue specific regulatory variation with respect to the likely functional targets of germline genetic susceptibility variants for ovarian cancer. PMID:25804953

  5. Novel green tissue-specific synthetic promoters and cis-regulatory elements in rice

    PubMed Central

    Wang, Rui; Zhu, Menglin; Ye, Rongjian; Liu, Zuoxiong; Zhou, Fei; Chen, Hao; Lin, Yongjun

    2015-01-01

    As an important part of synthetic biology, synthetic promoter has gradually become a hotspot in current biology. The purposes of the present study were to synthesize green tissue-specific promoters and to discover green tissue-specific cis-elements. We first assembled several regulatory sequences related to tissue-specific expression in different combinations, aiming to obtain novel green tissue-specific synthetic promoters. GUS assays of the transgenic plants indicated 5 synthetic promoters showed green tissue-specific expression patterns and different expression efficiencies in various tissues. Subsequently, we scanned and counted the cis-elements in different tissue-specific promoters based on the plant cis-elements database PLACE and the rice cDNA microarray database CREP for green tissue-specific cis-element discovery, resulting in 10 potential cis-elements. The flanking sequence of one potential core element (GEAT) was predicted by bioinformatics. Then, the combination of GEAT and its flanking sequence was functionally identified with synthetic promoter. GUS assays of the transgenic plants proved its green tissue-specificity. Furthermore, the function of GEAT flanking sequence was analyzed in detail with site-directed mutagenesis. Our study provides an example for the synthesis of rice tissue-specific promoters and develops a feasible method for screening and functional identification of tissue-specific cis-elements with their flanking sequences at the genome-wide level in rice. PMID:26655679

  6. Rat beta 1-adrenergic receptor regulatory region containing consensus AP-2 elements recognizes novel transactivator proteins.

    PubMed

    Kirigiti, P; Yang, Y F; Li, X; Li, B; Midson, C N; Machida, C A

    2000-03-01

    beta 1-Adrenergic receptors (beta1-ARs) serve as important regulators of central nervous system (CNS)-mediated behavior and several neural functions, including mood, memory, neuroendocrine control, and stimulation of autonomic function. Using beta 1-AR-luciferase reporter recombinants, we have previously determined that important beta 1-AR genetic elements controlling expression within the C6 glioma cell line are contained within the region -396 to -299, relative to the translational start site. By conducting progressive internal deletions of the rat beta 1-AR 5' flanking region and with the use of beta 1-AR-luciferase recombinants, we have verified that this region contains the primary beta 1-AR promoter and/or major regulatory elements. To begin the identification of protein factors involved in beta 1-AR transcriptional activity conferred by this beta 1-AR region and flanking sequences, we conducted electrophoretic mobility shift assays using defined beta 1-AR DNA subregion probes. One probe (GS-1), encompassing the region -396 to -367, was found to produce two major and two minor mobility shift complexes when bound to nuclear extracts from the beta 1-AR expresser C6 cell line. UV-crosslinking of DNA-protein complexes, coupled with DNase I digestion, indicated that this beta 1-AR region interacts with one major protein of approximately 117 kDa molecular weight and additional minor proteins. GS-1 DNA-protein complexes were observed using beta 1-AR expresser tissues in the CNS, including cortex, hippocampus, and olfactory bulb. No DNA-protein complexes were observed when using nuclear extracts from beta 1-AR nonexpresser tissues; in some cases, using L6 cells, previously characterized to express little or no beta1-ARs, a reduction in intensities of the DNA-protein complexes was observed. Competition experiments indicate that nuclear protein binds to one of two subregions within the GS-1 sequence that contain AP-2-like consensus elements. Recombinant AP-2 protein

  7. Regulatory Elements of the Staphylococcus aureus Protein A (Spa) Promoter†

    PubMed Central

    Gao, Jinxin; Stewart, George C.

    2004-01-01

    Staphylococcal protein A (Spa) is an important virulence factor of Staphylococcus aureus. Transcription of the spa determinant occurs during the exponential growth phase and is repressed when the cells enter the postexponential growth phase. Regulation of spa expression has been found to be complicated, with regulation involving multiple factors, including Agr, SarA, SarS, SarT, Rot, and MgrA. Our understanding of how these factors work on the spa promoter to regulate spa expression is incomplete. To identify regulatory sites within the spa promoter, analysis of deletion derivatives of the promoter in host strains deficient in one or more of the regulatory factors was undertaken, and several critical features of spa regulation were revealed. The transcriptional start sites of spa were determined by primer extension. The spa promoter sequences were subcloned in front of a promoterless chloramphenicol acetyltransferase reporter gene. Various lengths of spa truncations with the same 3′ end were constructed, and the resultant plasmids were transduced into strains with different regulatory genetic backgrounds. Our results identified upstream promoter sequences necessary for Agr system regulation of spa expression. The cis elements for SarS activity, an activator of spa expression, and for SarA activity, a repressor of spa expression, were identified. The well-characterized SarA consensus sequence on the spa promoter was found to be insufficient for SarA repression of the spa promoter. Full repression required the presence of a second consensus site adjacent to the SarS binding site. Sequences directly upstream of the core promoter sequence were found to stimulate transcription. PMID:15175287

  8. MicroRNAs as regulatory elements in immune system logic.

    PubMed

    Mehta, Arnav; Baltimore, David

    2016-04-28

    MicroRNAs (miRNAs) are crucial post-transcriptional regulators of haematopoietic cell fate decisions. They act by negatively regulating the expression of key immune development genes, thus contributing important logic elements to the regulatory circuitry. Deletion studies have made it increasingly apparent that they confer robustness to immune cell development, especially under conditions of environmental stress such as infectious challenge and ageing. Aberrant expression of certain miRNAs can lead to pathological consequences, such as autoimmunity and haematological cancers. In this Review, we discuss the mechanisms by which several miRNAs influence immune development and buffer normal haematopoietic output, first at the level of haematopoietic stem cells, then in innate and adaptive immune cells. We then discuss the pathological consequences of dysregulation of these miRNAs. PMID:27121651

  9. BLSSpeller: exhaustive comparative discovery of conserved cis-regulatory elements

    PubMed Central

    De Witte, Dieter; Van de Velde, Jan; Decap, Dries; Van Bel, Michiel; Audenaert, Pieter; Demeester, Piet; Dhoedt, Bart; Vandepoele, Klaas; Fostier, Jan

    2015-01-01

    Motivation: The accurate discovery and annotation of regulatory elements remains a challenging problem. The growing number of sequenced genomes creates new opportunities for comparative approaches to motif discovery. Putative binding sites are then considered to be functional if they are conserved in orthologous promoter sequences of multiple related species. Existing methods for comparative motif discovery usually rely on pregenerated multiple sequence alignments, which are difficult to obtain for more diverged species such as plants. As a consequence, misaligned regulatory elements often remain undetected. Results: We present a novel algorithm that supports both alignment-free and alignment-based motif discovery in the promoter sequences of related species. Putative motifs are exhaustively enumerated as words over the IUPAC alphabet and screened for conservation using the branch length score. Additionally, a confidence score is established in a genome-wide fashion. In order to take advantage of a cloud computing infrastructure, the MapReduce programming model is adopted. The method is applied to four monocotyledon plant species and it is shown that high-scoring motifs are significantly enriched for open chromatin regions in Oryza sativa and for transcription factor binding sites inferred through protein-binding microarrays in O.sativa and Zea mays. Furthermore, the method is shown to recover experimentally profiled ga2ox1-like KN1 binding sites in Z.mays. Availability and implementation: BLSSpeller was written in Java. Source code and manual are available at http://bioinformatics.intec.ugent.be/blsspeller Contact: Klaas.Vandepoele@psb.vib-ugent.be or jan.fostier@intec.ugent.be Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26254488

  10. Satellite DNA-like elements associated with genes within euchromatin of the beetle Tribolium castaneum.

    PubMed

    Brajković, Josip; Feliciello, Isidoro; Bruvo-Mađarić, Branka; Ugarković, Durđica

    2012-08-01

    In the red flour beetle Tribolium castaneum the major TCAST satellite DNA accounts for 35% of the genome and encompasses the pericentromeric regions of all chromosomes. Because of the presence of transcriptional regulatory elements and transcriptional activity in these sequences, TCAST satellite DNAs also have been proposed to be modulators of gene expression within euchromatin. Here, we analyze the distribution of TCAST homologous repeats in T. castaneum euchromatin and study their association with genes as well as their potential gene regulatory role. We identified 68 arrays composed of TCAST-like elements distributed on all chromosomes. Based on sequence characteristics the arrays were composed of two types of TCAST-like elements. The first type consists of TCAST satellite-like elements in the form of partial monomers or tandemly arranged monomers, up to tetramers, whereas the second type consists of TCAST-like elements embedded with a complex unit that resembles a DNA transposon. TCAST-like elements were also found in the 5' untranslated region (UTR) of the CR1-3_TCa retrotransposon, and therefore retrotransposition may have contributed to their dispersion throughout the genome. No significant difference in the homogenization of dispersed TCAST-like elements was found either at the level of local arrays or chromosomes nor among different chromosomes. Of 68 TCAST-like elements, 29 were located within introns, with the remaining elements flanked by genes within a 262 to 404,270 nt range. TCAST-like elements are statistically overrepresented near genes with immunoglobulin-like domains attesting to their nonrandom distribution and a possible gene regulatory role. PMID:22908042

  11. FootprintDB: Analysis of Plant Cis-Regulatory Elements, Transcription Factors, and Binding Interfaces.

    PubMed

    Contreras-Moreira, Bruno; Sebastian, Alvaro

    2016-01-01

    FootprintDB is a database and search engine that compiles regulatory sequences from open access libraries of curated DNA cis-elements and motifs, and their associated transcription factors (TFs). It systematically annotates the binding interfaces of the TFs by exploiting protein-DNA complexes deposited in the Protein Data Bank. Each entry in footprintDB is thus a DNA motif linked to the protein sequence of the TF(s) known to recognize it, and in most cases, the set of predicted interface residues involved in specific recognition. This chapter explains step-by-step how to search for DNA motifs and protein sequences in footprintDB and how to focus the search to a particular organism. Two real-world examples are shown where this software was used to analyze transcriptional regulation in plants. Results are described with the aim of guiding users on their interpretation, and special attention is given to the choices users might face when performing similar analyses. PMID:27557773

  12. Widespread contribution of transposable elements to the innovation of gene regulatory networks.

    PubMed

    Sundaram, Vasavi; Cheng, Yong; Ma, Zhihai; Li, Daofeng; Xing, Xiaoyun; Edge, Peter; Snyder, Michael P; Wang, Ting

    2014-12-01

    Transposable elements (TEs) have been shown to contain functional binding sites for certain transcription factors (TFs). However, the extent to which TEs contribute to the evolution of TF binding sites is not well known. We comprehensively mapped binding sites for 26 pairs of orthologous TFs in two pairs of human and mouse cell lines (representing two cell lineages), along with epigenomic profiles, including DNA methylation and six histone modifications. Overall, we found that 20% of binding sites were embedded within TEs. This number varied across different TFs, ranging from 2% to 40%. We further identified 710 TF-TE relationships in which genomic copies of a TE subfamily contributed a significant number of binding peaks for a TF, and we found that LTR elements dominated these relationships in human. Importantly, TE-derived binding peaks were strongly associated with open and active chromatin signatures, including reduced DNA methylation and increased enhancer-associated histone marks. On average, 66% of TE-derived binding events were cell type-specific with a cell type-specific epigenetic landscape. Most of the binding sites contributed by TEs were species-specific, but we also identified binding sites conserved between human and mouse, the functional relevance of which was supported by a signature of purifying selection on DNA sequences of these TEs. Interestingly, several TFs had significantly expanded binding site landscapes only in one species, which were linked to species-specific gene functions, suggesting that TEs are an important driving force for regulatory innovation. Taken together, our data suggest that TEs have significantly and continuously shaped gene regulatory networks during mammalian evolution. PMID:25319995

  13. Widespread contribution of transposable elements to the innovation of gene regulatory networks

    PubMed Central

    Sundaram, Vasavi; Cheng, Yong; Ma, Zhihai; Li, Daofeng; Xing, Xiaoyun; Edge, Peter

    2014-01-01

    Transposable elements (TEs) have been shown to contain functional binding sites for certain transcription factors (TFs). However, the extent to which TEs contribute to the evolution of TF binding sites is not well known. We comprehensively mapped binding sites for 26 pairs of orthologous TFs in two pairs of human and mouse cell lines (representing two cell lineages), along with epigenomic profiles, including DNA methylation and six histone modifications. Overall, we found that 20% of binding sites were embedded within TEs. This number varied across different TFs, ranging from 2% to 40%. We further identified 710 TF–TE relationships in which genomic copies of a TE subfamily contributed a significant number of binding peaks for a TF, and we found that LTR elements dominated these relationships in human. Importantly, TE-derived binding peaks were strongly associated with open and active chromatin signatures, including reduced DNA methylation and increased enhancer-associated histone marks. On average, 66% of TE-derived binding events were cell type-specific with a cell type-specific epigenetic landscape. Most of the binding sites contributed by TEs were species-specific, but we also identified binding sites conserved between human and mouse, the functional relevance of which was supported by a signature of purifying selection on DNA sequences of these TEs. Interestingly, several TFs had significantly expanded binding site landscapes only in one species, which were linked to species-specific gene functions, suggesting that TEs are an important driving force for regulatory innovation. Taken together, our data suggest that TEs have significantly and continuously shaped gene regulatory networks during mammalian evolution. PMID:25319995

  14. Cistrome and Epicistrome Features Shape the Regulatory DNA Landscape.

    PubMed

    O'Malley, Ronan C; Huang, Shao-Shan Carol; Song, Liang; Lewsey, Mathew G; Bartlett, Anna; Nery, Joseph R; Galli, Mary; Gallavotti, Andrea; Ecker, Joseph R

    2016-05-19

    The cistrome is the complete set of transcription factor (TF) binding sites (cis-elements) in an organism, while an epicistrome incorporates tissue-specific DNA chemical modifications and TF-specific chemical sensitivities into these binding profiles. Robust methods to construct comprehensive cistrome and epicistrome maps are critical for elucidating complex transcriptional networks that underlie growth, behavior, and disease. Here, we describe DNA affinity purification sequencing (DAP-seq), a high-throughput TF binding site discovery method that interrogates genomic DNA with in-vitro-expressed TFs. Using DAP-seq, we defined the Arabidopsis cistrome by resolving motifs and peaks for 529 TFs. Because genomic DNA used in DAP-seq retains 5-methylcytosines, we determined that >75% (248/327) of Arabidopsis TFs surveyed were methylation sensitive, a property that strongly impacts the epicistrome landscape. DAP-seq datasets also yielded insight into the biology and binding site architecture of numerous TFs, demonstrating the value of DAP-seq for cost-effective cistromic and epicistromic annotation in any organism. PMID:27203113

  15. The Y chromosome as a regulatory element shaping immune cell transcriptomes and susceptibility to autoimmune disease.

    PubMed

    Case, Laure K; Wall, Emma H; Dragon, Julie A; Saligrama, Naresha; Krementsov, Dimitry N; Moussawi, Mohamad; Zachary, James F; Huber, Sally A; Blankenhorn, Elizabeth P; Teuscher, Cory

    2013-09-01

    Understanding the DNA elements that constitute and control the regulatory genome is critical for the appropriate therapeutic management of complex diseases. Here, using chromosome Y (ChrY) consomic mouse strains on the C57BL/6J (B6) background, we show that susceptibility to two diverse animal models of autoimmune disease, experimental allergic encephalomyelitis (EAE) and experimental myocarditis, correlates with the natural variation in copy number of Sly and Rbmy multicopy ChrY genes. On the B6 background, ChrY possesses gene regulatory properties that impact genome-wide gene expression in pathogenic CD4(+) T cells. Using a ChrY consomic strain on the SJL background, we discovered a preference for ChrY-mediated gene regulation in macrophages, the immune cell subset underlying the EAE sexual dimorphism in SJL mice, rather than CD4(+) T cells. Importantly, in both genetic backgrounds, an inverse correlation exists between the number of Sly and Rbmy ChrY gene copies and the number of significantly up-regulated genes in immune cells, thereby supporting a link between copy number variation of Sly and Rbmy with the ChrY genetic element exerting regulatory properties. Additionally, we show that ChrY polymorphism can determine the sexual dimorphism in EAE and myocarditis. In humans, an analysis of the CD4(+) T cell transcriptome from male multiple sclerosis patients versus healthy controls provides further evidence for an evolutionarily conserved mechanism of gene regulation by ChrY. Thus, as in Drosophila, these data establish the mammalian ChrY as a member of the regulatory genome due to its ability to epigenetically regulate genome-wide gene expression in immune cells. PMID:23800453

  16. Characterization of Putative cis-Regulatory Elements in Genes Preferentially Expressed in Arabidopsis Male Meiocytes

    PubMed Central

    Li, Mingjun

    2014-01-01

    Meiosis is essential for plant reproduction because it is the process during which homologous chromosome pairing, synapsis, and meiotic recombination occur. The meiotic transcriptome is difficult to investigate because of the size of meiocytes and the confines of anther lobes. The recent development of isolation techniques has enabled the characterization of transcriptional profiles in male meiocytes of Arabidopsis. Gene expression in male meiocytes shows unique features. The direct interaction of transcription factors (TFs) with DNA regulatory sequences forms the basis for the specificity of transcriptional regulation. Here, we identified putative cis-regulatory elements (CREs) associated with male meiocyte-expressed genes using in silico tools. The upstream regions (1 kb) of the top 50 genes preferentially expressed in Arabidopsis meiocytes possessed conserved motifs. These motifs are putative binding sites of TFs, some of which share common functions, such as roles in cell division. In combination with cell-type-specific analysis, our findings could be a substantial aid for the identification and experimental verification of the protein-DNA interactions for the specific TFs that drive gene expression in meiocytes. PMID:25250331

  17. Transcriptional regulatory elements in the noncoding region of human papillomavirus type 6

    SciTech Connect

    Wu, Tzyy-Choou.

    1989-01-01

    The structure and function of the transcriptional regulatory region of human papillomavirus type 6 (HPV-6) has been investigated. To investigate tissue specific gene expression, a sensitive method to detect and localize HPV-6 viral DNA, mRNA and protein in plastic-embedded tissue sections of genital and respiratory tract papillomata by using in situ hybridization and immunoperoxidase assays has been developed. This method, using ultrathin sections and strand-specific {sup 3}H labeled riboprobes, offers the advantages of superior morphological preservation and detection of viral genomes at low copy number with good resolution, and the modified immunocytochemistry provides better sensitivity. The results suggest that genital tract epithelium is more permissive for HPV-6 replication than respiratory tract epithelium. To study the tissue tropism of HPV-6 at the level of regulation of viral gene expression, the polymerase chain reaction was used to isolate the noncoding region (NCR) of HPV-6 in independent isolates. Nucleotide sequence analysis of molecularly cloned DNA identified base substitutions, deletions/insertions and tandem duplications. Transcriptional regulatory elements in the NCR were assayed in recombinant plasmids containing the bacterial gene for chloramphenicol acetyl transferase.

  18. Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells

    PubMed Central

    Laurette, Patrick; Strub, Thomas; Koludrovic, Dana; Keime, Céline; Le Gras, Stéphanie; Seberg, Hannah; Van Otterloo, Eric; Imrichova, Hana; Siddaway, Robert; Aerts, Stein; Cornell, Robert A; Mengus, Gabrielle; Davidson, Irwin

    2015-01-01

    Microphthalmia-associated transcription factor (MITF) is the master regulator of the melanocyte lineage. To understand how MITF regulates transcription, we used tandem affinity purification and mass spectrometry to define a comprehensive MITF interactome identifying novel cofactors involved in transcription, DNA replication and repair, and chromatin organisation. We show that MITF interacts with a PBAF chromatin remodelling complex comprising BRG1 and CHD7. BRG1 is essential for melanoma cell proliferation in vitro and for normal melanocyte development in vivo. MITF and SOX10 actively recruit BRG1 to a set of MITF-associated regulatory elements (MAREs) at active enhancers. Combinations of MITF, SOX10, TFAP2A, and YY1 bind between two BRG1-occupied nucleosomes thus defining both a signature of transcription factors essential for the melanocyte lineage and a specific chromatin organisation of the regulatory elements they occupy. BRG1 also regulates the dynamics of MITF genomic occupancy. MITF-BRG1 interplay thus plays an essential role in transcription regulation in melanoma. DOI: http://dx.doi.org/10.7554/eLife.06857.001 PMID:25803486

  19. Engineering Synthetic cis-Regulatory Elements for Simultaneous Recognition of Three Transcriptional Factors in Bacteria.

    PubMed

    Amores, Gerardo Ruiz; Guazzaroni, María-Eugenia; Silva-Rocha, Rafael

    2015-12-18

    Recognition of cis-regulatory elements by transcription factors (TF) at target promoters is crucial to gene regulation in bacteria. In this process, binding of TFs to their cognate sequences depends on a set of physical interactions between these proteins and specific nucleotides in the operator region. Previously, we showed that in silico optimization algorithms are able to generate short sequences that are recognized by two different TFs of Escherichia coli, namely, CRP and IHF, thus generating an AND logic gate. Here, we expanded this approach in order to engineer DNA sequences that can be simultaneously recognized by three unrelated TFs (CRP, IHF, and Fis). Using in silico optimization and experimental validation strategies, we were able to obtain a candidate promoter (Plac-CFI1) regulated by only two TFs with an AND logic, thus demonstrating a limitation in the design. Subsequently, we modified the algorithm to allow the optimization of extended sequences, and were able to design two synthetic promoters (PCFI20-1 and PCFI22-5) that were functional in vivo. Expression assays in E. coli mutant strains for each TF revealed that while CRP positively regulates the promoter activities, IHF and Fis are strong repressors of both the promoter variants. Taken together, our results demonstrate the potential of in silico strategies in bacterial synthetic promoter engineering. Furthermore, the study also shows how small modifications in cis-regulatory elements can drastically affect the final logic of the resulting promoter. PMID:26305598

  20. Mapping open chromatin with formaldehyde-assisted isolation of regulatory elements.

    PubMed

    Nammo, Takao; Rodríguez-Seguí, Santiago A; Ferrer, Jorge

    2011-01-01

    Noncoding regulatory genomic elements are central for cellular function, differentiation, and disease, but remain poorly characterized. FAIRE (formaldehyde-assisted isolation of regulatory elements) has emerged as a simple method to identify and analyze active regulatory sequences based on their decreased nucleosomal content. More recently FAIRE was combined with high-throughput sequencing (FAIRE-seq) to locate tissue-specific regulatory elements at a genome scale in purified human pancreatic islets. Here we describe the implementation of the FAIRE method in human pancreatic islet cells. PMID:21913087

  1. Human polyomavirus JCV late leader peptide region contains important regulatory elements

    SciTech Connect

    Akan, Ilhan; Sariyer, Ilker Kudret; Biffi, Renato; Palermo, Victoria; Woolridge, Stefanie; White, Martyn K.; Amini, Shohreh |; Khalili, Kamel; Safak, Mahmut . E-mail: msafak@temple.edu

    2006-05-25

    Transcription is a complex process that relies on the cooperative interaction between sequence-specific factors and the basal transcription machinery. The strength of a promoter depends on upstream or downstream cis-acting DNA elements, which bind transcription factors. In this study, we investigated whether DNA elements located downstream of the JCV late promoter, encompassing the late leader peptide region, which encodes agnoprotein, play regulatory roles in the JCV lytic cycle. For this purpose, the entire coding region of the leader peptide was deleted and the functional consequences of this deletion were analyzed. We found that viral gene expression and replication were drastically reduced. Gene expression also decreased from a leader peptide point mutant but to a lesser extent. This suggested that the leader peptide region of JCV might contain critical cis-acting DNA elements to which transcription factors bind and regulate viral gene expression and replication. We analyzed the entire coding region of the late leader peptide by a footprinting assay and identified three major regions (region I, II and III) that were protected by nuclear proteins. Further investigation of the first two protected regions by band shift assays revealed a new band that appeared in new infection cycles, suggesting that viral infection induces new factors that interact with the late leader peptide region of JCV. Analysis of the effect of the leader peptide region on the promoter activity of JCV by transfection assays demonstrated that this region has a positive and negative effect on the large T antigen (LT-Ag)-mediated activation of the viral early and late promoters, respectively. Furthermore, a partial deletion analysis of the leader peptide region encompassing the protected regions I and II demonstrated a significant down-regulation of viral gene expression and replication. More importantly, these results were similar to that obtained from a complete deletion of the late leader

  2. Lessons from Domestication: Targeting Cis-Regulatory Elements for Crop Improvement.

    PubMed

    Swinnen, Gwen; Goossens, Alain; Pauwels, Laurens

    2016-06-01

    Domestication of wild plant species has provided us with crops that serve our human nutritional needs. Advanced DNA sequencing has propelled the unveiling of underlying genetic changes associated with domestication. Interestingly, many changes reside in cis-regulatory elements (CREs) that control the expression of an unmodified coding sequence. Sequence variation in CREs can impact gene expression levels, but also developmental timing and tissue specificity of expression. When genes are involved in multiple pathways or active in several organs and developmental stages CRE modifications are favored in contrast to mutations in coding regions, due to the lack of detrimental pleiotropic effects. Therefore, learning from domestication, we propose that CREs are interesting targets for genome editing to create new alleles for plant breeding. PMID:26876195

  3. The bacterial DnaA-trio replication origin element specifies single-stranded DNA initiator binding.

    PubMed

    Richardson, Tomas T; Harran, Omar; Murray, Heath

    2016-06-16

    DNA replication is tightly controlled to ensure accurate inheritance of genetic information. In all organisms, initiator proteins possessing AAA+ (ATPases associated with various cellular activities) domains bind replication origins to license new rounds of DNA synthesis. In bacteria the master initiator protein, DnaA, is highly conserved and has two crucial DNA binding activities. DnaA monomers recognize the replication origin (oriC) by binding double-stranded DNA sequences (DnaA-boxes); subsequently, DnaA filaments assemble and promote duplex unwinding by engaging and stretching a single DNA strand. While the specificity for duplex DnaA-boxes by DnaA has been appreciated for over 30 years, the sequence specificity for single-strand DNA binding has remained unknown. Here we identify a new indispensable bacterial replication origin element composed of a repeating trinucleotide motif that we term the DnaA-trio. We show that the function of the DnaA-trio is to stabilize DnaA filaments on a single DNA strand, thus providing essential precision to this binding mechanism. Bioinformatic analysis detects DnaA-trios in replication origins throughout the bacterial kingdom, indicating that this element is part of the core oriC structure. The discovery and characterization of the novel DnaA-trio extends our fundamental understanding of bacterial DNA replication initiation, and because of the conserved structure of AAA+ initiator proteins these findings raise the possibility of specific recognition motifs within replication origins of higher organisms. PMID:27281207

  4. Exaptation of Transposable Elements into Novel Cis-Regulatory Elements: Is the Evidence Always Strong?

    PubMed Central

    de Souza, Flávio S.J.; Franchini, Lucía F.; Rubinstein, Marcelo

    2013-01-01

    Transposable elements (TEs) are mobile genetic sequences that can jump around the genome from one location to another, behaving as genomic parasites. TEs have been particularly effective in colonizing mammalian genomes, and such heavy TE load is expected to have conditioned genome evolution. Indeed, studies conducted both at the gene and genome levels have uncovered TE insertions that seem to have been co-opted—or exapted—by providing transcription factor binding sites (TFBSs) that serve as promoters and enhancers, leading to the hypothesis that TE exaptation is a major factor in the evolution of gene regulation. Here, we critically review the evidence for exaptation of TE-derived sequences as TFBSs, promoters, enhancers, and silencers/insulators both at the gene and genome levels. We classify the functional impact attributed to TE insertions into four categories of increasing complexity and argue that so far very few studies have conclusively demonstrated exaptation of TEs as transcriptional regulatory regions. We also contend that many genome-wide studies dealing with TE exaptation in recent lineages of mammals are still inconclusive and that the hypothesis of rapid transcriptional regulatory rewiring mediated by TE mobilization must be taken with caution. Finally, we suggest experimental approaches that may help attributing higher-order functions to candidate exapted TEs. PMID:23486611

  5. Variation in vertebrate cis-regulatory elements in evolution and disease.

    PubMed

    Douglas, Adam Thomas; Hill, Robert D

    2014-01-01

    Much of the genetic information that drives animal diversity lies within the vast non-coding regions of the genome. Multi-species sequence conservation in non-coding regions of the genome flags important regulatory elements and more recently, techniques that look for functional signatures predicted for regulatory sequences have added to the identification of thousands more. For some time, biologists have argued that changes in cis-regulatory sequences creates the basic genetic framework for evolutionary change. Recent advances support this notion and show that there is extensive genomic variability in non-coding regulatory elements associated with trait variation, speciation and disease. PMID:25764334

  6. Variation in Vertebrate Cis-Regulatory Elements in Evolution and Disease.

    PubMed

    Douglas, Adam T; Hill, Robert E

    2014-05-01

    Much of the genetic information that drives animal diversity lies within the vast non-coding regions of the genome. Multi-species sequence conservation in non-coding regions of the genome flags important regulatory elements and more recently, techniques that look for functional signatures predicted for regulatory sequences have added to the identification of thousands more. For some time, biologists have argued that changes in cis-regulatory sequences creates the basic genetic framework for evolutionary change. Recent advances support this notion and show that there is extensive genomic variability in non-coding regulatory elements associated with trait variation, speciation and disease. PMID:24802895

  7. Variation in Vertebrate Cis-Regulatory Elements in Evolution and Disease

    PubMed Central

    Douglas, Adam Thomas; Hill, Robert E

    2014-01-01

    Much of the genetic information that drives animal diversity lies within the vast non-coding regions of the genome. Multi-species sequence conservation in non-coding regions of the genome flags important regulatory elements and more recently, techniques that look for functional signatures predicted for regulatory sequences have added to the identification of thousands more. For some time, biologists have argued that changes in cis-regulatory sequences creates the basic genetic framework for evolutionary change. Recent advances support this notion and show that there is extensive genomic variability in non-coding regulatory elements associated with trait variation, speciation and disease. PMID:25764334

  8. Functional Annotation of Putative Regulatory Elements at Cancer Susceptibility Loci

    PubMed Central

    Rosse, Stephanie A; Auer, Paul L; Carlson, Christopher S

    2014-01-01

    Most cancer-associated genetic variants identified from genome-wide association studies (GWAS) do not obviously change protein structure, leading to the hypothesis that the associations are attributable to regulatory polymorphisms. Translating genetic associations into mechanistic insights can be facilitated by knowledge of the causal regulatory variant (or variants) responsible for the statistical signal. Experimental validation of candidate functional variants is onerous, making bioinformatic approaches necessary to prioritize candidates for laboratory analysis. Thus, a systematic approach for recognizing functional (and, therefore, likely causal) variants in noncoding regions is an important step toward interpreting cancer risk loci. This review provides a detailed introduction to current regulatory variant annotations, followed by an overview of how to leverage these resources to prioritize candidate functional polymorphisms in regulatory regions. PMID:25288875

  9. Functional antagonism between inhibitor of DNA binding (Id) and adipocyte determination and differentiation factor 1/sterol regulatory element-binding protein-1c (ADD1/SREBP-1c) trans-factors for the regulation of fatty acid synthase promoter in adipocytes.

    PubMed Central

    Moldes, M; Boizard, M; Liepvre, X L; Fève, B; Dugail, I; Pairault, J

    1999-01-01

    We show that Id (inhibitor of DNA binding) 2 and Id3, dominant negative members of the helix-loop-helix (HLH) family, interact with the adipocyte determination and differentiation factor 1 (ADD1)/sterol regulatory element-binding protein (SREBP) 1c, a transcription factor of the basic HLH-leucine zipper family that controls the expression of several key genes of adipose metabolism. Gel mobility-shift assays performed with in vitro-translated ADD1, Id2 or Id3 proteins and a fatty acid synthase (FAS) promoter oligonucleotide showed evidence for a marked inhibition of the formation of DNA-ADD1 complexes by Id2 or Id3 proteins. Co-immunoprecipitation studies using in vitro-translated proteins demonstrated further the physical interaction of Id and ADD1/SREBP-1c proteins in the absence of DNA. Using the FAS gene as a model of an ADD1-regulated promoter in transiently transfected isolated rat adipocytes or mature 3T3-L1 adipocytes, a potent inhibition of the activity of the FAS-chloramphenicol acetyltransferase reporter gene was observed by overexpression of Id2 or Id3. Reciprocally, co-transfection of Id3 antisense and ADD1 expression vectors in preadipocytes potentiated the ADD1/SREBP-1c effect on the FAS promoter activity. Finally, in the non adipogenic NIH-3T3 cell line, most of the ADD1-mediated trans-activation of the FAS promoter was counteracted by co-transfection of Id2 or Id3 expression vectors. Previous studies have indicated Id gene expression to be down-regulated during adipogenesis [Moldes, Lasnier, Fève, Pairault and Djian (1997) Mol. Cell. Biol. 17, 1796-1804]. We here demonstrated that there was a dramatic rise of Id2 and Id3 mRNA levels when 3T3-L1 adipocytes or isolated rat fat cells were exposed to lipolytic and anti-lipogenic agents, forskolin and isoproterenol. Taken together, our data show that Id products are functionally involved in modulating ADD1/SREBP-1c transcriptional activity, and thus lipogenesis in adipocytes. PMID:10585876

  10. A steganalysis-based approach to comprehensive identification and characterization of functional regulatory elements

    PubMed Central

    Wang, Guandong; Zhang, Weixiong

    2006-01-01

    The comprehensive identification of cis-regulatory elements on a genome scale is a challenging problem. We develop a novel, steganalysis-based approach for genome-wide motif finding, called WordSpy, by viewing regulatory regions as a stegoscript with cis-elements embedded in 'background' sequences. We apply WordSpy to the promoters of cell-cycle-related genes of Saccharomyces cerevisiae and Arabidopsis thaliana, identifying all known cell-cycle motifs with high ranking. WordSpy can discover a complete set of cis-elements and facilitate the systematic study of regulatory networks. PMID:16787547

  11. SeqGL Identifies Context-Dependent Binding Signals in Genome-Wide Regulatory Element Maps

    PubMed Central

    Setty, Manu; Leslie, Christina S.

    2015-01-01

    Genome-wide maps of transcription factor (TF) occupancy and regions of open chromatin implicitly contain DNA sequence signals for multiple factors. We present SeqGL, a novel de novo motif discovery algorithm to identify multiple TF sequence signals from ChIP-, DNase-, and ATAC-seq profiles. SeqGL trains a discriminative model using a k-mer feature representation together with group lasso regularization to extract a collection of sequence signals that distinguish peak sequences from flanking regions. Benchmarked on over 100 ChIP-seq experiments, SeqGL outperformed traditional motif discovery tools in discriminative accuracy. Furthermore, SeqGL can be naturally used with multitask learning to identify genomic and cell-type context determinants of TF binding. SeqGL successfully scales to the large multiplicity of sequence signals in DNase- or ATAC-seq maps. In particular, SeqGL was able to identify a number of ChIP-seq validated sequence signals that were not found by traditional motif discovery algorithms. Thus compared to widely used motif discovery algorithms, SeqGL demonstrates both greater discriminative accuracy and higher sensitivity for detecting the DNA sequence signals underlying regulatory element maps. SeqGL is available at http://cbio.mskcc.org/public/Leslie/SeqGL/. PMID:26016777

  12. Identification of functional elements and regulatory circuits by Drosophila modENCODE

    SciTech Connect

    Roy, Sushmita; Ernst, Jason; Kharchenko, Peter V.; Kheradpour, Pouya; Negre, Nicolas; Eaton, Matthew L.; Landolin, Jane M.; Bristow, Christopher A.; Ma, Lijia; Lin, Michael F.; Washietl, Stefan; Arshinoff, Bradley I.; Ay, Ferhat; Meyer, Patrick E.; Robine, Nicolas; Washington, Nicole L.; Stefano, Luisa Di; Berezikov, Eugene; Brown, Christopher D.; Candeias, Rogerio; Carlson, Joseph W.; Carr, Adrian; Jungreis, Irwin; Marbach, Daniel; Sealfon, Rachel; Tolstorukov, Michael Y.; Will, Sebastian; Alekseyenko, Artyom A.; Artieri, Carlo; Booth, Benjamin W.; Brooks, Angela N.; Dai, Qi; Davis, Carrie A.; Duff, Michael O.; Feng, Xin; Gorchakov, Andrey A.; Gu, Tingting; Henikoff, Jorja G.; Kapranov, Philipp; Li, Renhua; MacAlpine, Heather K.; Malone, John; Minoda, Aki; Nordman, Jared; Okamura, Katsutomo; Perry, Marc; Powell, Sara K.; Riddle, Nicole C.; Sakai, Akiko; Samsonova, Anastasia; Sandler, Jeremy E.; Schwartz, Yuri B.; Sher, Noa; Spokony, Rebecca; Sturgill, David; van Baren, Marijke; Wan, Kenneth H.; Yang, Li; Yu, Charles; Feingold, Elise; Good, Peter; Guyer, Mark; Lowdon, Rebecca; Ahmad, Kami; Andrews, Justen; Berger, Bonnie; Brenner, Steven E.; Brent, Michael R.; Cherbas, Lucy; Elgin, Sarah C. R.; Gingeras, Thomas R.; Grossman, Robert; Hoskins, Roger A.; Kaufman, Thomas C.; Kent, William; Kuroda, Mitzi I.; Orr-Weaver, Terry; Perrimon, Norbert; Pirrotta, Vincenzo; Posakony, James W.; Ren, Bing; Russell, Steven; Cherbas, Peter; Graveley, Brenton R.; Lewis, Suzanna; Micklem, Gos; Oliver, Brian; Park, Peter J.; Celniker, Susan E.; Henikoff, Steven; Karpen, Gary H.; Lai, Eric C.; MacAlpine, David M.; Stein, Lincoln D.; White, Kevin P.; Kellis, Manolis

    2010-12-22

    To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation. Several years after the complete genetic sequencing of many species, it is still unclear how to translate genomic information into a functional map of cellular and developmental programs. The Encyclopedia of DNA Elements (ENCODE) (1) and model organism ENCODE (modENCODE) (2) projects use diverse genomic assays to comprehensively annotate the Homo sapiens (human), Drosophila melanogaster (fruit fly), and Caenorhabditis elegans (worm) genomes, through systematic generation and computational integration of functional genomic data sets. Previous genomic studies in flies have made seminal contributions to our understanding of basic biological mechanisms and genome functions, facilitated by genetic, experimental, computational, and manual annotation of the euchromatic and heterochromatic genome (3), small genome size, short life cycle, and a deep knowledge of development, gene function, and chromosome biology. The functions

  13. Deciphering RNA Regulatory Elements Involved in the Developmental and Environmental Gene Regulation of Trypanosoma brucei

    PubMed Central

    Gazestani, Vahid H.; Salavati, Reza

    2015-01-01

    Trypanosoma brucei is a vector-borne parasite with intricate life cycle that can cause serious diseases in humans and animals. This pathogen relies on fine regulation of gene expression to respond and adapt to variable environments, with implications in transmission and infectivity. However, the involved regulatory elements and their mechanisms of actions are largely unknown. Here, benefiting from a new graph-based approach for finding functional regulatory elements in RNA (GRAFFER), we have predicted 88 new RNA regulatory elements that are potentially involved in the gene regulatory network of T. brucei. We show that many of these newly predicted elements are responsive to both transcriptomic and proteomic changes during the life cycle of the parasite. Moreover, we found that 11 of predicted elements strikingly resemble previously identified regulatory elements for the parasite. Additionally, comparison with previously predicted motifs on T. brucei suggested the superior performance of our approach based on the current limited knowledge of regulatory elements in T. brucei. PMID:26529602

  14. A small regulatory element from chromosome 19 enhances liver-specific gene expression

    PubMed Central

    Li, C; Hirsch, M; Carter, P; Asokan, A; Zhou, X; Wu, Z; Samulski, RJ

    2016-01-01

    Tissue-specific promoters for gene therapy are typically too big for adeno-associated virus (AAV) vectors; thus, the exploration of small effective non-viral regulatory elements is of particular interest. Wild-type AAV can specifically integrate into a region on human chromosome 19 termed AAVS1. Earlier work has determined that a 347 bp fragment (Chr19) of AAVS1 has promoter and transcriptional enhancer activities. In this study, we further characterized this genetic regulation and investigated its application to AAV gene therapy in vitro and in vivo. The Chr19 347 bp fragment was dissected into three regulatory elements in human embryonic kidney cells: (i) TATA-independent promoter activity distributed throughout the fragment regardless of orientation, (ii) an orientation-dependent insulator function near the 5′ end and (iii) a 107 bp enhancer region near the 3′ end. The small enhancer region, coupled to the mini-CMV promoter, was used to drive the expression of several reporters following transduction by AAV2. In vivo data demonstrated enhanced transgene expression from the Chr19-mini-CMV promoter cassette after tail vein injection primarily in the liver at levels comparable to the chicken β-actin promoter and higher than the liver-specific TTR promoter (>2-fold). However, we did not observe this increase after muscle injection, suggesting tissue-specific enhancement. All of the results support identification of a small DNA fragment (347 bp) from AAV Chr19 integration site capable of providing efficient and enhanced liver-specific transcription when used in recombinant AAV vectors. PMID:18701910

  15. FARE-CAFE: a database of functional and regulatory elements of cancer-associated fusion events

    PubMed Central

    Korla, Praveen Kumar; Cheng, Jack; Huang, Chien-Hung; Tsai, Jeffrey J. P.; Liu, Yu-Hsuan; Kurubanjerdjit, Nilubon; Hsieh, Wen-Tsong; Chen, Huey-Yi; Ng, Ka-Lok

    2015-01-01

    Chromosomal translocation (CT) is of enormous clinical interest because this disorder is associated with various major solid tumors and leukemia. A tumor-specific fusion gene event may occur when a translocation joins two separate genes. Currently, various CT databases provide information about fusion genes and their genomic elements. However, no database of the roles of fusion genes, in terms of essential functional and regulatory elements in oncogenesis, is available. FARE-CAFE is a unique combination of CTs, fusion proteins, protein domains, domain–domain interactions, protein–protein interactions, transcription factors and microRNAs, with subsequent experimental information, which cannot be found in any other CT database. Genomic DNA information including, for example, manually collected exact locations of the first and second break points, sequences and karyotypes of fusion genes are included. FARE-CAFE will substantially facilitate the cancer biologist’s mission of elucidating the pathogenesis of various types of cancer. This database will ultimately help to develop ‘novel’ therapeutic approaches. Database URL: http://ppi.bioinfo.asia.edu.tw/FARE-CAFE PMID:26384373

  16. VEZF1 Elements Mediate Protection from DNA Methylation

    PubMed Central

    Strogantsev, Ruslan; Gaszner, Miklos; Hair, Alan; Felsenfeld, Gary; West, Adam G.

    2010-01-01

    There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm β-globin HS4 insulator to block the range of enhancers or the spread of repressive histone modifications is well established. Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin state. PMID:20062523

  17. Characterization of CYP1A1 regulatory elements in Atlantic tomcod

    SciTech Connect

    Roy, N.; Wirgin, I.; Courtenay, S.

    1995-12-31

    Coplanar PCBs, TCDD, and PAHs induce cytochrome P4501A1 (CYP1A1) mRNA in Atlantic tomcod from the Miramichi River (MR), whereas only PAHs induce gene expression in tomcod from the Hudson River (HR). Relative to the highly industrialized HR, MR is relatively clean. The authors hypothesize that non-inducibility of CYP1A1 mRNA in PCB (TCB) or TCDD treated tomcod from the HR is due to prior exposure to environmentally-borne xenobiotics. To evaluate the mechanisms which selectively inhibit CYP1A1 inducibility, they isolated and characterized 5{tilde O}and intronic CYP1A1 regulatory elements from tomcod genomic DNA. Tomcod 5{tilde O} CYP1A1 contains four motifs with core sequences identical to the aromatic hydrocarbon receptor elements (AhREs) identified in mammals. Electrophoretic mobility shift assays (EMSAs) with nuclear extracts prepared form the livers of B[a]P treated HR tomcod showed protein binding to 142 and 156 bp tomcod DNA fragments each containing two tomcod AhREs. EMSAs with nuclear extracts prepared from DMBA treated rat livers and human MOLT4 cells also showed protein binding to the fish AhREs. Protein binding at individual tomcod AhREs was characterized with hepatic protein extracts prepared from TCB, B[a]P, and vehicle treated tomcod from the HR and MR. Preliminary studies showed a difference in protein binding between HR and MR tomcod i.p. injected with TCB 1d, 5d, or 15d previous, but not B[a]P 6 hr or 24 hr previous. These results suggest that the mechanisms of CYP1A1 transcription are similar tomcod and mammals and that variation in levels of gene inducibility among individual tomcod may be due to differences in inducible protein binding to CYP1A1 AhREs.

  18. [Identification and mapping of cis-regulatory elements within long genomic sequences].

    PubMed

    Akopov, S B; Chernov, I P; Vetchinova, A S; Bulanenkova, S S; Nikolaev, L G

    2007-01-01

    The publication of the human and other metazoan genome sequences opened up the possibility for mapping and analysis of genomic regulatory elements. Unfortunately, experimental data on genomic positions of such sequences as enhancers, silencers, insulators, transcription terminators, and replication origins are very limited, especially at the whole genome level. As most genomic regulatory elements (e.g., enhancers) are generally gene-, tissue-, or cell-specific, the prediction of these elements in silico is often ambiguous. Therefore, the development of high-throughput experimental approaches for identification and mapping of genomic functional elements is highly desirable. In this review we discuss novel approaches to high-throughput experimental identification of mammalian genomes cis-regulatory elements which is a necessary step toward the complete genome annotation. PMID:18240562

  19. Genome-Wide Profiling of PARP1 Reveals an Interplay with Gene Regulatory Regions and DNA Methylation

    PubMed Central

    Nalabothula, Narasimharao; Al-jumaily, Taha; Eteleeb, Abdallah M.; Flight, Robert M.; Xiaorong, Shao; Moseley, Hunter; Rouchka, Eric C.; Fondufe-Mittendorf, Yvonne N.

    2015-01-01

    Poly (ADP-ribose) polymerase-1 (PARP1) is a nuclear enzyme involved in DNA repair, chromatin remodeling and gene expression. PARP1 interactions with chromatin architectural multi-protein complexes (i.e. nucleosomes) alter chromatin structure resulting in changes in gene expression. Chromatin structure impacts gene regulatory processes including transcription, splicing, DNA repair, replication and recombination. It is important to delineate whether PARP1 randomly associates with nucleosomes or is present at specific nucleosome regions throughout the cell genome. We performed genome-wide association studies in breast cancer cell lines to address these questions. Our studies show that PARP1 associates with epigenetic regulatory elements genome-wide, such as active histone marks, CTCF and DNase hypersensitive sites. Additionally, the binding of PARP1 to chromatin genome-wide is mutually exclusive with DNA methylation pattern suggesting a functional interplay between PARP1 and DNA methylation. Indeed, inhibition of PARylation results in genome-wide changes in DNA methylation patterns. Our results suggest that PARP1 controls the fidelity of gene transcription and marks actively transcribed gene regions by selectively binding to transcriptionally active chromatin. These studies provide a platform for developing our understanding of PARP1’s role in gene regulation. PMID:26305327

  20. Characterization of oocyte-expressed GDF9 gene in buffalo and mapping of its TSS and putative regulatory elements.

    PubMed

    Roy, B; Rajput, S; Raghav, S; Kumar, P; Verma, A; Jain, A; Jain, T; Singh, D; De, S; Goswami, S L; Datta, T K

    2013-05-01

    Summary In spite of emerging evidence about the vital role of GDF9 in determination of oocyte competence, there is insufficient information about its regulation of oocyte-specific expression, particularly in livestock animals. Because of the distinct prominence of buffalo as a dairy animal, the present study was undertaken to isolate and characterize GDF9 cDNA using orthologous primers based on the bovine GDF9 sequence. GDF9 transcripts were found to be expressed in oocytes irrespective of their follicular origin, and shared a single transcription start site (TSS) at -57 base pairs (bp) upstream of ATG. Assignment of the TSS is consistent with the presence of a TATA element at -23 of the TSS mapped in this study. Localization of a buffalo-specific minimal promoter within 320 bp upstream of ATG was consolidated by identification of an E-box element at -113bp. Presence of putative transcription factor binding sites and other cis regulatory elements were analyzed at ~5 kb upstream of TSS. Various germ cell-specific cis-acting regulatory elements (BNCF, BRNF, NR2F, SORY, Foxh1, OCT1, LHXF etc.) have been identified in the 5' flanking region of the buffalo GDF9 gene, including NOBOX DNA binding elements and consensuses E-boxes (CANNTG). Presence of two conserved E-boxes found on buffalo sequence at -520 and -718 positions deserves attention in view of its sequence deviation from other species. Two NOBOX binding elements (NBE) were detected at the -3471 and -203 positions. The fall of the NBE within the putative minimal promoter territory of buffalo GDF9 and its unique non-core binding sequence could have a possible role in the control of the core promoter activity. PMID:22230197

  1. Transposable Elements: From DNA Parasites to Architects of Metazoan Evolution

    PubMed Central

    Piskurek, Oliver; Jackson, Daniel J.

    2012-01-01

    One of the most unexpected insights that followed from the completion of the human genome a decade ago was that more than half of our DNA is derived from transposable elements (TEs). Due to advances in high throughput sequencing technologies it is now clear that TEs comprise the largest molecular class within most metazoan genomes. TEs, once categorised as "junk DNA", are now known to influence genomic structure and function by increasing the coding and non-coding genetic repertoire of the host. In this way TEs are key elements that stimulate the evolution of metazoan genomes. This review highlights several lines of TE research including the horizontal transfer of TEs through host-parasite interactions, the vertical maintenance of TEs over long periods of evolutionary time, and the direct role that TEs have played in generating morphological novelty. PMID:24704977

  2. Predictive modelling of gene expression from transcriptional regulatory elements.

    PubMed

    Budden, David M; Hurley, Daniel G; Crampin, Edmund J

    2015-07-01

    Predictive modelling of gene expression provides a powerful framework for exploring the regulatory logic underpinning transcriptional regulation. Recent studies have demonstrated the utility of such models in identifying dysregulation of gene and miRNA expression associated with abnormal patterns of transcription factor (TF) binding or nucleosomal histone modifications (HMs). Despite the growing popularity of such approaches, a comparative review of the various modelling algorithms and feature extraction methods is lacking. We define and compare three methods of quantifying pairwise gene-TF/HM interactions and discuss their suitability for integrating the heterogeneous chromatin immunoprecipitation (ChIP)-seq binding patterns exhibited by TFs and HMs. We then construct log-linear and ϵ-support vector regression models from various mouse embryonic stem cell (mESC) and human lymphoblastoid (GM12878) data sets, considering both ChIP-seq- and position weight matrix- (PWM)-derived in silico TF-binding. The two algorithms are evaluated both in terms of their modelling prediction accuracy and ability to identify the established regulatory roles of individual TFs and HMs. Our results demonstrate that TF-binding and HMs are highly predictive of gene expression as measured by mRNA transcript abundance, irrespective of algorithm or cell type selection and considering both ChIP-seq and PWM-derived TF-binding. As we encourage other researchers to explore and develop these results, our framework is implemented using open-source software and made available as a preconfigured bootable virtual environment. PMID:25231769

  3. From Cis-Regulatory Elements to Complex RNPs and Back

    PubMed Central

    Gebauer, Fátima; Preiss, Thomas; Hentze, Matthias W.

    2012-01-01

    Messenger RNAs (mRNAs), the templates for translation, have evolved to harbor abundant cis-acting sequences that affect their posttranscriptional fates. These elements are frequently located in the untranslated regions and serve as binding sites for trans-acting factors, RNA-binding proteins, and/or small non-coding RNAs. This article provides a systematic synopsis of cis-acting elements, trans-acting factors, and the mechanisms by which they affect translation. It also highlights recent technical advances that have ushered in the era of transcriptome-wide studies of the ribonucleoprotein complexes formed by mRNAs and their trans-acting factors. PMID:22751153

  4. DNA sequence of the maize transposable element Dissociation.

    PubMed

    Döring, H P; Tillmann, E; Starlinger, P

    The DNA sequence of the terminal 4.2 kilobases (kb) of the 30-kb insertion in the endosperm sucrose synthase gene of maize mutant sh-m5933 shows that it comprises two identical 2,040-base pair (bp) segments, one inserted in the reverse direction into the other. We suggest that the 2,040-bp sequence is an example of the transposable element Dissociation described by Barbara McClintock. PMID:6318121

  5. Multiple Regulatory Systems Coordinate DNA Replication with Cell Growth in Bacillus subtilis

    PubMed Central

    Murray, Heath; Koh, Alan

    2014-01-01

    In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s) that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes. PMID:25340815

  6. Functional and Regulatory Biomolecular Networks Organized by DNA Nanostructures

    NASA Astrophysics Data System (ADS)

    Liu, Minghui

    DNA has recently emerged as an extremely promising material to organize molecules on nanoscale. The reliability of base recognition, self-assembling behavior, and attractive structural properties of DNA are of unparalleled value in systems of this size. DNA scaffolds have already been used to organize a variety of molecules including nanoparticles and proteins. New protein-DNA bio-conjugation chemistries make it possible to precisely position proteins and other biomolecules on underlying DNA scaffolds, generating multi-biomolecule pathways with the ability to modulate intermolecular interactions and the local environment. This dissertation focuses on studying the application of using DNA nanostructure to direct the self-assembly of other biomolecular networks to translate biochemical pathways to non-cellular environments. Presented here are a series of studies toward this application. First, a novel strategy utilized DNA origami as a scaffold to arrange spherical virus capsids into one-dimensional arrays with precise nanoscale positioning. This hierarchical self-assembly allows us to position the virus particles with unprecedented control and allows the future construction of integrated multi-component systems from biological scaffolds using the power of rationally engineered DNA nanostructures. Next, discrete glucose oxidase (GOx)/ horseradish peroxidase (HRP) enzyme pairs were organized on DNA origami tiles with controlled interenzyme spacing and position. This study revealed two different distance-dependent kinetic processes associated with the assembled enzyme pairs. Finally, a tweezer-like DNA nanodevice was designed and constructed to actuate the activity of an enzyme/cofactor pair. Using this approach, several cycles of externally controlled enzyme inhibition and activation were successfully demonstrated. This principle of responsive enzyme nanodevices may be used to regulate other types of enzymes and to introduce feedback or feed-forward control loops.

  7. DREAM (Downstream Regulatory Element Antagonist Modulator) contributes to synaptic depression and contextual fear memory

    PubMed Central

    2010-01-01

    The downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, binds specifically to DNA and several nucleoproteins regulating gene expression and with proteins outside the nucleus to regulate membrane excitability or calcium homeostasis. DREAM is highly expressed in the central nervous system including the hippocampus and cortex; however, the roles of DREAM in hippocampal synaptic transmission and plasticity have not been investigated. Taking advantage of transgenic mice overexpressing a Ca2+-insensitive DREAM mutant (TgDREAM), we used integrative methods including electrophysiology, biochemistry, immunostaining, and behavior tests to study the function of DREAM in synaptic transmission, long-term plasticity and fear memory in hippocampal CA1 region. We found that NMDA receptor but not AMPA receptor-mediated current was decreased in TgDREAM mice. Moreover, synaptic plasticity, such as long-term depression (LTD) but not long-term potentiation (LTP), was impaired in TgDREAM mice. Biochemical experiments found that DREAM interacts with PSD-95 and may inhibit NMDA receptor function through this interaction. Contextual fear memory was significantly impaired in TgDREAM mice. By contrast, sensory responses to noxious stimuli were not affected. Our results demonstrate that DREAM plays a novel role in postsynaptic modulation of the NMDA receptor, and contributes to synaptic plasticity and behavioral memory. PMID:20205763

  8. DREAM (downstream regulatory element antagonist modulator) contributes to synaptic depression and contextual fear memory.

    PubMed

    Wu, Long-Jun; Mellström, Britt; Wang, Hansen; Ren, Ming; Domingo, Sofia; Kim, Susan S; Li, Xiang-Yao; Chen, Tao; Naranjo, Jose R; Zhuo, Min

    2010-01-01

    The downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, binds specifically to DNA and several nucleoproteins regulating gene expression and with proteins outside the nucleus to regulate membrane excitability or calcium homeostasis. DREAM is highly expressed in the central nervous system including the hippocampus and cortex; however, the roles of DREAM in hippocampal synaptic transmission and plasticity have not been investigated. Taking advantage of transgenic mice overexpressing a Ca2+-insensitive DREAM mutant (TgDREAM), we used integrative methods including electrophysiology, biochemistry, immunostaining, and behavior tests to study the function of DREAM in synaptic transmission, long-term plasticity and fear memory in hippocampal CA1 region. We found that NMDA receptor but not AMPA receptor-mediated current was decreased in TgDREAM mice. Moreover, synaptic plasticity, such as long-term depression (LTD) but not long-term potentiation (LTP), was impaired in TgDREAM mice. Biochemical experiments found that DREAM interacts with PSD-95 and may inhibit NMDA receptor function through this interaction. Contextual fear memory was significantly impaired in TgDREAM mice. By contrast, sensory responses to noxious stimuli were not affected. Our results demonstrate that DREAM plays a novel role in postsynaptic modulation of the NMDA receptor, and contributes to synaptic plasticity and behavioral memory. PMID:20205763

  9. Site-specific silencing of regulatory elements as a mechanism of X inactivation.

    PubMed

    Calabrese, J Mauro; Sun, Wei; Song, Lingyun; Mugford, Joshua W; Williams, Lucy; Yee, Della; Starmer, Joshua; Mieczkowski, Piotr; Crawford, Gregory E; Magnuson, Terry

    2012-11-21

    The inactive X chromosome's (Xi) physical territory is microscopically devoid of transcriptional hallmarks and enriched in silencing-associated modifications. How these microscopic signatures relate to specific Xi sequences is unknown. Therefore, we profiled Xi gene expression and chromatin states at high resolution via allele-specific sequencing in mouse trophoblast stem cells. Most notably, X-inactivated transcription start sites harbored distinct epigenetic signatures relative to surrounding Xi DNA. These sites displayed H3-lysine27-trimethylation enrichment and DNaseI hypersensitivity, similar to autosomal Polycomb targets, yet excluded Pol II and other transcriptional hallmarks, similar to nontranscribed genes. CTCF bound X-inactivated and escaping genes, irrespective of measured chromatin boundaries. Escape from X inactivation occurred within, and X inactivation was maintained exterior to, the area encompassed by Xist in cells subject to imprinted and random X inactivation. The data support a model whereby inactivation of specific regulatory elements, rather than a simple chromosome-wide separation from transcription machinery, governs gene silencing over the Xi. PMID:23178118

  10. Site-specific silencing of regulatory elements as a mechanism of X-inactivation

    PubMed Central

    Calabrese, J. Mauro; Sun, Wei; Song, Lingyun; Mugford, Joshua W.; Williams, Lucy; Yee, Della; Starmer, Joshua; Mieczkowski, Piotr; Crawford, Gregory E.; Magnuson, Terry

    2012-01-01

    The inactive X chromosome’s (Xi) physical territory is microscopically devoid of transcriptional hallmarks and enriched in silencing-associated modifications. How these microscopic signatures relate to specific Xi sequence is unknown. Therefore, we profiled Xi gene expression and chromatin states at high resolution via allele-specific sequencing in mouse trophoblast stem cells. Most notably, X-inactivated transcription start sites harbored distinct epigenetic signatures relative to surrounding Xi DNA. These sites displayed H3-lysine27-trimethylation enrichment and DNaseI hypersensitivity, similar to autosomal Polycomb targets, yet excluded Pol II and other transcriptional hallmarks, similar to non-transcribed genes. CTCF bound X-inactivated and escaping genes, irrespective of measured chromatin boundaries. Escape from X-inactivation occurred within, and X-inactivation was maintained exterior to, the area encompassed by Xist in cells subject to imprinted and random X-inactivation. The data support a model whereby inactivation of specific regulatory elements, rather than a simple chromosome-wide separation from transcription machinery, governs gene silencing over the Xi. PMID:23178118

  11. Detecting conserved regulatory elements with the model genome of the Japanese puffer fish, Fugu rubripes.

    PubMed Central

    Aparicio, S; Morrison, A; Gould, A; Gilthorpe, J; Chaudhuri, C; Rigby, P; Krumlauf, R; Brenner, S

    1995-01-01

    Comparative vertebrate genome sequencing offers a powerful method for detecting conserved regulatory sequences. We propose that the compact genome of the teleost Fugu rubripes is well suited for this purpose. The evolutionary distance of teleosts from other vertebrates offers the maximum stringency for such evolutionary comparisons. To illustrate the comparative genome approach for F. rubripes, we use sequence comparisons between mouse and Fugu Hoxb-4 noncoding regions to identify conserved sequence blocks. We have used two approaches to test the function of these conserved blocks. In the first, homologous sequences were deleted from a mouse enhancer, resulting in a tissue-specific loss of activity when assayed in transgenic mice. In the second approach, Fugu DNA sequences showing homology to mouse sequences were tested for enhancer activity in transgenic mice. This strategy identified a neural element that mediates a subset of Hoxb-4 expression that is conserved between mammals and teleosts. The comparison of noncoding vertebrate sequences with those of Fugu, coupled to a transgenic bioassay, represents a general approach suitable for many genome projects. Images Fig. 2 Fig. 3 Fig. 4 PMID:7878040

  12. Regulatory elements mediating transcription from the Drosophila melanogaster actin 5C proximal promoter.

    PubMed Central

    Chung, Y T; Keller, E B

    1990-01-01

    The major cytoskeletal actin gene of Drosophila melanogaster, the actin 5C gene, has two promoters, the proximal one of which controls constitutive synthesis of actin in all growing tissues. To locate regulatory elements required for constitutive activity of the proximal promoter, mutants of this promoter were fused to the bacterial chloramphenicol acetyltransferase gene and assayed for transient expression activity in cultured Drosophila embryonic Schneider line 2 cells. An essential regulatory element has been located 313 base pairs upstream from the cap site. Deletion of this element lowered expression to one-third of the wild-type level. The element has the sequence AAGTTGTAGTTG, as shown by protein-binding footprinting with the reagent methidiumpropyl-EDTA-Fe(II). This element is probably not a general one, since it was not detected in a search of the published 5'-flanking sequences of 27 Drosophila genes. In addition to this regulatory element, there are five GAGA elements in the actin 5C proximal promoter, some or all of which are essential for the promoter activity as shown by an in vivo competition assay. Although this promoter has no classical TATA element, there is an essential promoter region about 35 base pairs upstream from the cap site that could be a TATA surrogate. The promoter also shows sequences homologous to the alcohol dehydrogenase factor 1-binding site and to the core of the vertebrate serum response element, but mutations of these sites did not affect promoter activity in transient expression assays. Images PMID:2104658

  13. Chimeric murine interferon regulatory factor-2 (IRF-2) binds to IRF-E (IRF binding element), VREβ (virus response element) but not to VREα1.

    PubMed

    Prakash, Krishna; Kumar, Pardeep; Mukherjee, Somnath; Rath, P C

    2014-12-01

    Interferon regulatory factor-2 (IRF-2) is a multifunctional transcription factor having gene activation, repression and synergistic effect in conjunction with IRF-1. IRF-2 is also involved in type I IFN signalling by repressing INFβ gene. So far, the molecular mechanism of its DNA binding activity remains elusive. We have carried out molecular sub-cloning, expression and electrophoretically mobility shift assay study of chimeric murine IRF-2. Here, we report expression of chimeric murine IRF-2 as GST-IRF-2 fusion protein in Escherichia coli/BL21 cells and demonstrated DNA binding activity by gel retardation technique using radio (32) P-labelled IRF-E motif (GAAAGT)4 , virus response element (VRE) of human INFβ and IFNα1 gene. We observed five different masses DNA/GST-IRF-2 complexes (1-5) with IRF-E motif, three different masses DNA/GST-IRF-2 complexes (1-3) with VREß , but we could not observe any complex of DNA/GST-IRF-2 with VREα1 . The specific binding on IRF-E motif was confirmed by carrying out 100-X fold cold competition with (32) P-labelled IRF-E motif. In contrast to specific binding on VREß , we used negative control where we observed no binding complex, but we observed complexes with clones IPTG-induced extract. As far as binding on VREα1 is concerned, we could not observe any complex in negative control as well as in IPTG-inducible clones extract. Chimeric IRF-2 binds with IRF-E motif and VREβ but not with VREα1. This study is first of its kind and paves the way to understand the differential DNA binding and molecular mechanism of DNA binding activity of the IRF-2 molecule, which is crucial for its function(s). PMID:25251598

  14. The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements.

    PubMed

    Schoenfelder, Stefan; Furlan-Magaril, Mayra; Mifsud, Borbala; Tavares-Cadete, Filipe; Sugar, Robert; Javierre, Biola-Maria; Nagano, Takashi; Katsman, Yulia; Sakthidevi, Moorthy; Wingett, Steven W; Dimitrova, Emilia; Dimond, Andrew; Edelman, Lucas B; Elderkin, Sarah; Tabbada, Kristina; Darbo, Elodie; Andrews, Simon; Herman, Bram; Higgs, Andy; LeProust, Emily; Osborne, Cameron S; Mitchell, Jennifer A; Luscombe, Nicholas M; Fraser, Peter

    2015-04-01

    The mammalian genome harbors up to one million regulatory elements often located at great distances from their target genes. Long-range elements control genes through physical contact with promoters and can be recognized by the presence of specific histone modifications and transcription factor binding. Linking regulatory elements to specific promoters genome-wide is currently impeded by the limited resolution of high-throughput chromatin interaction assays. Here we apply a sequence capture approach to enrich Hi-C libraries for >22,000 annotated mouse promoters to identify statistically significant, long-range interactions at restriction fragment resolution, assigning long-range interacting elements to their target genes genome-wide in embryonic stem cells and fetal liver cells. The distal sites contacting active genes are enriched in active histone modifications and transcription factor occupancy, whereas inactive genes contact distal sites with repressive histone marks, demonstrating the regulatory potential of the distal elements identified. Furthermore, we find that coregulated genes cluster nonrandomly in spatial interaction networks correlated with their biological function and expression level. Interestingly, we find the strongest gene clustering in ES cells between transcription factor genes that control key developmental processes in embryogenesis. The results provide the first genome-wide catalog linking gene promoters to their long-range interacting elements and highlight the complex spatial regulatory circuitry controlling mammalian gene expression. PMID:25752748

  15. The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements

    PubMed Central

    Schoenfelder, Stefan; Furlan-Magaril, Mayra; Mifsud, Borbala; Tavares-Cadete, Filipe; Sugar, Robert; Javierre, Biola-Maria; Nagano, Takashi; Katsman, Yulia; Sakthidevi, Moorthy; Wingett, Steven W.; Dimitrova, Emilia; Dimond, Andrew; Edelman, Lucas B.; Elderkin, Sarah; Tabbada, Kristina; Darbo, Elodie; Andrews, Simon; Herman, Bram; Higgs, Andy; LeProust, Emily; Osborne, Cameron S.; Mitchell, Jennifer A.

    2015-01-01

    The mammalian genome harbors up to one million regulatory elements often located at great distances from their target genes. Long-range elements control genes through physical contact with promoters and can be recognized by the presence of specific histone modifications and transcription factor binding. Linking regulatory elements to specific promoters genome-wide is currently impeded by the limited resolution of high-throughput chromatin interaction assays. Here we apply a sequence capture approach to enrich Hi-C libraries for >22,000 annotated mouse promoters to identify statistically significant, long-range interactions at restriction fragment resolution, assigning long-range interacting elements to their target genes genome-wide in embryonic stem cells and fetal liver cells. The distal sites contacting active genes are enriched in active histone modifications and transcription factor occupancy, whereas inactive genes contact distal sites with repressive histone marks, demonstrating the regulatory potential of the distal elements identified. Furthermore, we find that coregulated genes cluster nonrandomly in spatial interaction networks correlated with their biological function and expression level. Interestingly, we find the strongest gene clustering in ES cells between transcription factor genes that control key developmental processes in embryogenesis. The results provide the first genome-wide catalog linking gene promoters to their long-range interacting elements and highlight the complex spatial regulatory circuitry controlling mammalian gene expression. PMID:25752748

  16. Quantitative Analysis of Cis-Regulatory Element Activity Using Synthetic Promoters in Transgenic Plants.

    PubMed

    Benn, Geoffrey; Dehesh, Katayoon

    2016-01-01

    Synthetic promoters, introduced stably or transiently into plants, are an invaluable tool for the identification of functional regulatory elements and the corresponding transcription factor(s) that regulate the amplitude, spatial distribution, and temporal patterns of gene expression. Here, we present a protocol describing the steps required to identify and characterize putative cis-regulatory elements. These steps include application of computational tools to identify putative elements, construction of a synthetic promoter upstream of luciferase, identification of transcription factors that regulate the element, testing the functionality of the element introduced transiently and/or stably into the species of interest followed by high-throughput luciferase screening assays, and subsequent data processing and statistical analysis. PMID:27557758

  17. Defining functional DNA elements in the human genome

    PubMed Central

    Kellis, Manolis; Wold, Barbara; Snyder, Michael P.; Bernstein, Bradley E.; Kundaje, Anshul; Marinov, Georgi K.; Ward, Lucas D.; Birney, Ewan; Crawford, Gregory E.; Dekker, Job; Dunham, Ian; Elnitski, Laura L.; Farnham, Peggy J.; Feingold, Elise A.; Gerstein, Mark; Giddings, Morgan C.; Gilbert, David M.; Gingeras, Thomas R.; Green, Eric D.; Guigo, Roderic; Hubbard, Tim; Kent, Jim; Lieb, Jason D.; Myers, Richard M.; Pazin, Michael J.; Ren, Bing; Stamatoyannopoulos, John A.; Weng, Zhiping; White, Kevin P.; Hardison, Ross C.

    2014-01-01

    With the completion of the human genome sequence, attention turned to identifying and annotating its functional DNA elements. As a complement to genetic and comparative genomics approaches, the Encyclopedia of DNA Elements Project was launched to contribute maps of RNA transcripts, transcriptional regulator binding sites, and chromatin states in many cell types. The resulting genome-wide data reveal sites of biochemical activity with high positional resolution and cell type specificity that facilitate studies of gene regulation and interpretation of noncoding variants associated with human disease. However, the biochemically active regions cover a much larger fraction of the genome than do evolutionarily conserved regions, raising the question of whether nonconserved but biochemically active regions are truly functional. Here, we review the strengths and limitations of biochemical, evolutionary, and genetic approaches for defining functional DNA segments, potential sources for the observed differences in estimated genomic coverage, and the biological implications of these discrepancies. We also analyze the relationship between signal intensity, genomic coverage, and evolutionary conservation. Our results reinforce the principle that each approach provides complementary information and that we need to use combinations of all three to elucidate genome function in human biology and disease. PMID:24753594

  18. LDRD Report FY 03: Structure and Function of Regulatory DNA: A Next Major Challenge in Genomics

    SciTech Connect

    Stubbs, L

    2003-02-18

    leverage their long-standing investment and expertise in mapping, sequencing, and genetics of human chromosome 19 (ch19). The starting point of this effort was the LLNL and Joint Genome Institute (JGI) genomics teams' success in sequencing mouse DNA related to ch19. This effort was the first comparative sequencing project to be conducted on such a large scale; a manuscript describing this work was published in Science shortly after this LDRD began (Dehal et al., Science 293: 104-111, 2001). Like protein-coding genes themselves, DNA elements that control gene expression are protected from structural change over evolutionary time, since their function is dependent on sequence integrity. By contrast, DNA that serves no function (''junk DNA'') can accumulate mutations that change sequence content without biological consequence. As a result, the ''junk DNA'' of human and mouse are not at all alike in sequence; however, genes and regulatory elements (REs) of human, mouse, and other animals--as far removed from human and fish and birds--are very similar in sequence and structure.

  19. Characterization of "cis"-regulatory elements ("c"RE) associated with mammary gland function

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Bos taurus genome assembly has propelled dairy science into a new era; still, most of the information encoded in the genome has not yet been decoded. The human Encyclopedia of DNA Elements (ENCODE) project has spearheaded the identification and annotation of functional genomic elements in the hu...

  20. Examining cooperative binding of Sox2 on DC5 regulatory element upon complex formation with Pax6 through excess electron transfer assay.

    PubMed

    Saha, Abhijit; Kizaki, Seiichiro; De, Debojyoti; Endo, Masayuki; Kim, Kyeong Kyu; Sugiyama, Hiroshi

    2016-08-19

    Functional cooperativity among transcription factors on regulatory genetic elements is pivotal for milestone decision-making in various cellular processes including mammalian development. However, their molecular interaction during the cooperative binding cannot be precisely understood due to lack of efficient tools for the analyses of protein-DNA interaction in the transcription complex. Here, we demonstrate that photoinduced excess electron transfer assay can be used for analysing cooperativity of proteins in transcription complex using cooperative binding of Pax6 to Sox2 on the regulatory DNA element (DC5 enhancer) as an example. In this assay, (Br)U-labelled DC5 was introduced for the efficient detection of transferred electrons from Sox2 and Pax6 to the DNA, and guanine base in the complementary strand was replaced with hypoxanthine (I) to block intra-strand electron transfer at the Sox2-binding site. By examining DNA cleavage occurred as a result of the electron transfer process, from tryptophan residues of Sox2 and Pax6 to DNA after irradiation at 280 nm, we not only confirmed their binding to DNA but also observed their increased occupancy on DC5 with respect to that of Sox2 and Pax6 alone as a result of their cooperative interaction. PMID:27229137

  1. A compilation of composite regulatory elements affecting gene transcription in vertebrates.

    PubMed Central

    Kel, O V; Romaschenko, A G; Kel, A E; Wingender, E; Kolchanov, N A

    1995-01-01

    Over the past years, evidence has been accumulating for a fundamental role of protein-protein interactions between transcription factors in gene-specific transcription regulation. Many of these interactions run within composite elements containing binding sites for several factors. We have selected 101 composite regulatory elements identified experimentally in the regulatory regions of 64 genes of vertebrates and of their viruses and briefly described them in a compilation. Of these, 82 composite elements are of the synergistic type and 19 of the antagonistic type. Within the synergistic type composite elements, transcription factors bind to the corresponding sites simultaneously, thus cooperatively activating transcription. The factors, binding to their target sites within antagonistic type composite elements, produce opposing effects on transcription. The nucleotide sequence and localization in the genes, the names and brief description of transcription factors, are provided for each composite element, including a representation of experimental data on its functioning. Most of the composite elements (3/4) fall between -250 bp and the transcription start site. The distance between the binding sites within the composite elements described varies from complete overlapping to 80 bp. The compilation of composite elements is presented in the database COMPEL which is electronically accessible by anonymous ftp via internet. PMID:7479071

  2. Gene regulation and DNA C-value paradox: a model based on diffusion of regulatory molecules.

    PubMed

    Kupiec, J J

    1989-01-01

    The general idea of the model is that regulatory molecules can move stochastically from site to site along DNA and that according to their chromosomal position, genes should have a more or less high probability to be activated (or repressed) during differentiation. In this model the role of non coding DNA is to maintain genes in a relative position that determines what is usually called the "differentiation programme". PMID:2538709

  3. Dynamics and function of distal regulatory elements during neurogenesis and neuroplasticity.

    PubMed

    Thakurela, Sudhir; Sahu, Sanjeeb Kumar; Garding, Angela; Tiwari, Vijay K

    2015-09-01

    Gene regulation in mammals involves a complex interplay between promoters and distal regulatory elements that function in concert to drive precise spatiotemporal gene expression programs. However, the dynamics of the distal gene regulatory landscape and its function in the transcriptional reprogramming that underlies neurogenesis and neuronal activity remain largely unknown. Here, we performed a combinatorial analysis of genome-wide data sets for chromatin accessibility (FAIRE-seq) and the enhancer mark H3K27ac, revealing the highly dynamic nature of distal gene regulation during neurogenesis, which gets progressively restricted to distinct genomic regions as neurons acquire a post-mitotic, terminally differentiated state. We further find that the distal accessible and active regions serve as target sites for distinct transcription factors that function in a stage-specific manner to contribute to the transcriptional program underlying neuronal commitment and maturation. Mature neurons respond to a sustained activity of NMDA receptors by epigenetic reprogramming at a large number of distal regulatory regions as well as dramatic reorganization of super-enhancers. Such massive remodeling of the distal regulatory landscape in turn results in a transcriptome that confers a transient loss of neuronal identity and gain of cellular plasticity. Furthermore, NMDA receptor activity also induces many novel prosurvival genes that function in neuroprotective pathways. Taken together, these findings reveal the dynamics of the distal regulatory landscape during neurogenesis and uncover novel regulatory elements that function in concert with epigenetic mechanisms and transcription factors to generate the transcriptome underlying neuronal development and activity. PMID:26170447

  4. Dynamics and function of distal regulatory elements during neurogenesis and neuroplasticity

    PubMed Central

    Thakurela, Sudhir; Sahu, Sanjeeb Kumar; Garding, Angela; Tiwari, Vijay K.

    2015-01-01

    Gene regulation in mammals involves a complex interplay between promoters and distal regulatory elements that function in concert to drive precise spatiotemporal gene expression programs. However, the dynamics of the distal gene regulatory landscape and its function in the transcriptional reprogramming that underlies neurogenesis and neuronal activity remain largely unknown. Here, we performed a combinatorial analysis of genome-wide data sets for chromatin accessibility (FAIRE-seq) and the enhancer mark H3K27ac, revealing the highly dynamic nature of distal gene regulation during neurogenesis, which gets progressively restricted to distinct genomic regions as neurons acquire a post-mitotic, terminally differentiated state. We further find that the distal accessible and active regions serve as target sites for distinct transcription factors that function in a stage-specific manner to contribute to the transcriptional program underlying neuronal commitment and maturation. Mature neurons respond to a sustained activity of NMDA receptors by epigenetic reprogramming at a large number of distal regulatory regions as well as dramatic reorganization of super-enhancers. Such massive remodeling of the distal regulatory landscape in turn results in a transcriptome that confers a transient loss of neuronal identity and gain of cellular plasticity. Furthermore, NMDA receptor activity also induces many novel prosurvival genes that function in neuroprotective pathways. Taken together, these findings reveal the dynamics of the distal regulatory landscape during neurogenesis and uncover novel regulatory elements that function in concert with epigenetic mechanisms and transcription factors to generate the transcriptome underlying neuronal development and activity. PMID:26170447

  5. Multiple cis Regulatory Elements Control RANTES Promoter Activity in Alveolar Epithelial Cells Infected with Respiratory Syncytial Virus

    PubMed Central

    Casola, Antonella; Garofalo, Roberto P.; Haeberle, Helene; Elliott, Todd F.; Lin, Rongtuan; Jamaluddin, Mohammad; Brasier, Allan R.

    2001-01-01

    Respiratory syncytial virus (RSV) produces intense pulmonary inflammation, in part through its ability to induce chemokine synthesis in infected airway epithelial cells. RANTES (regulated upon activation, normally T-cell expressed and presumably secreted) is a CC chemokine which recruits and activates monocytes, lymphocytes, and eosinophils, all cell types present in the lung inflammatory infiltrate induced by RSV infection. In this study, we analyzed the mechanism of RSV-induced RANTES promoter activation in human type II alveolar epithelial cells (A549 cells). Promoter deletion and mutagenesis experiments indicate that RSV requires the presence of five different cis regulatory elements, located in the promoter fragment spanning from −220 to +55 nucleotides, corresponding to NF-κB, C/EBP, Jun/CREB/ATF, and interferon regulatory factor (IRF) binding sites. Although site mutations of the NF-κB, C/EBP, and CREB/AP-1 like sites reduce RSV-induced RANTES gene transcription to 50% or less, only mutations affecting IRF binding completely abolish RANTES inducibility. Supershift and microaffinity isolation assays were used to identify the different transcription factor family members whose DNA binding activity was RSV inducible. Expression of dominant negative mutants of these transcription factors further established their central role in virus-induced RANTES promoter activation. Our finding that the presence of multiple cis regulatory elements is required for full activation of the RANTES promoter in RSV-infected alveolar epithelial cells supports the enhanceosome model for RANTES gene transcription, which is absolutely dependent on binding of IRF transcription factors. The identification of regulatory mechanisms of RANTES gene expression is fundamental for rational design of inhibitors of RSV-induced lung inflammation. PMID:11413310

  6. DNA binding and transcription activation by chicken interferon regulatory factor-3 (chIRF-3)

    PubMed Central

    Grant, Caroline E.; May, Donna L.; Deeley, Roger G.

    2000-01-01

    Interferon regulatory factors (IRFs) are a family of transcription factors involved in the cellular response to interferons and viral infection. Previously we isolated an IRF from a chicken embryonic liver cDNA library. Using a PCR-based binding site selection assay, we have characterised the binding specificity of chIRF-3. The optimal binding site (OBS) fits within the consensus interferon-stimulated response element (ISRE) but the specificity of chIRF-3 binding allows less variation in nucleotides outside the core IRF-binding sequence. A comparison of IRF-1 and chIRF-3 binding to ISREs in electrophoretic mobility shift assays confirmed that the binding specificity of chIRF-3 was clearly distinguishable from IRF-1. The selection assay also showed that chIRF-3 is capable of binding an inverted repeat of two half OBSs separated by 10–13 nt. ChIRF-3 appears to bind both the OBS and inverted repeat sites as a dimer with the protein–protein interaction requiring a domain between amino acids 117 and 311. In transfection experiments expression of chIRF-3 strongly activated a promoter containing the OBS. The activation domain was mapped to between amino acids 138 and 221 and a domain inhibitory to activation was also mapped to the C-terminal portion of chIRF-3. PMID:11095692

  7. Sterol regulatory element-binding proteins are transcriptional regulators of the thyroglobulin gene in thyroid cells.

    PubMed

    Wen, Gaiping; Eder, Klaus; Ringseis, Robert

    2016-08-01

    The genes encoding sodium/iodide symporter (NIS) and thyroid peroxidase (TPO), both of which are essential for thyroid hormone (TH) synthesis, were shown to be regulated by sterol regulatory element-binding proteins (SREBP)-1c and -2. In the present study we tested the hypothesis that transcription of a further gene essential for TH synthesis, the thyroglobulin (TG) gene, is under the control of SREBP. To test this hypothesis, we studied the influence of inhibition of SREBP maturation and SREBP knockdown on TG expression in FRTL-5 thyrocytes and explored transcriptional regulation of the TG promoter by reporter gene experiments in FRTL-5 and HepG2 cells, gel shift assays and chromatin immunoprecipitation. Inhibition of SREBP maturation by 25-hydroxycholesterol and siRNA-mediated knockdown of either SREBP-1c or SREBP-2 decreased mRNA and protein levels of TG in FRTL-5 thyrocytes. Reporter gene assays with wild-type and mutated TG promoter reporter truncation constructs revealed that the rat TG promoter is transcriptionally activated by nSREBP-1c and nSREBP-2. DNA-binding assays and chromatin immunoprecipitation assays showed that both nSREBP-1c and nSREBP-2 bind to a SREBP binding motif with characteristics of an E-box SRE at position -63 in the rat TG promoter. In connection with recent findings that NIS and TPO are regulated by SREBP in thyrocytes the present findings support the view that SREBP are regulators of essential steps of TH synthesis in the thyroid gland such as iodide uptake, iodide oxidation and iodination of tyrosyl residues of TG. This moreover suggests that SREBP may be molecular targets for pharmacological modulation of TH synthesis. PMID:27321819

  8. Sites of Predicted Stress-Induced DNA Duplex Destabilization Occur Preferentially at Regulatory Loci

    NASA Astrophysics Data System (ADS)

    Benham, Craig J.

    1993-04-01

    This paper describes a computational method to predict the sites on a DNA molecule where imposed superhelical stresses destabilize the duplex. Several DNA sequences are analyzed in this way, including the pBR322 and ColE1 plasmids, bacteriophage f1, and the polyoma and bovine papilloma virus genomes. Superhelical destabilization in these molecules is predicted to occur at small numbers of discrete sites, most of which are within regulatory regions. The most destabilized sites include the terminator and promoter regions of specific plasmid operons, the LexA binding sites of genes under SOS control, the intergenic control region of bacteriophage f1, and the polyadenylylation sites in eukaryotic viruses. These results demonstrate the existence of close correspondences between sites of predicted superhelical duplex destabilization and specific types of regulatory regions. The use of these correspondences to supplement string-matching techniques in the search for regulatory loci is discussed.

  9. Regulatory Elements of the Floral Homeotic Gene AGAMOUS Identified by Phylogenetic Footprinting and ShadowingW⃞

    PubMed Central

    Hong, Ray L.; Hamaguchi, Lynn; Busch, Maximilian A.; Weigel, Detlef

    2003-01-01

    In Arabidopsis thaliana, cis-regulatory sequences of the floral homeotic gene AGAMOUS (AG) are located in the second intron. This 3-kb intron contains binding sites for two direct activators of AG, LEAFY (LFY) and WUSCHEL (WUS), along with other putative regulatory elements. We have used phylogenetic footprinting and the related technique of phylogenetic shadowing to identify putative cis-regulatory elements in this intron. Among 29 Brassicaceae species, several other motifs, but not the LFY and WUS binding sites identified previously, are largely invariant. Using reporter gene analyses, we tested six of these motifs and found that they are all functionally important for the activity of AG regulatory sequences in A. thaliana. Although there is little obvious sequence similarity outside the Brassicaceae, the intron from cucumber AG has at least partial activity in A. thaliana. Our studies underscore the value of the comparative approach as a tool that complements gene-by-gene promoter dissection but also demonstrate that sequence-based studies alone are insufficient for a complete identification of cis-regulatory sites. PMID:12782724

  10. Regulatory elements of the floral homeotic gene AGAMOUS identified by phylogenetic footprinting and shadowing.

    SciTech Connect

    Hong, R. L., Hamaguchi, L., Busch, M. A., and Weigel, D.

    2003-06-01

    OAK-B135 In Arabidopsis thaliana, cis-regulatory sequences of the floral homeotic gene AGAMOUS (AG) are located in the second intron. This 3 kb intron contains binding sites for two direct activators of AG, LEAFY (LFY) and WUSCHEL (WUS), along with other putative regulatory elements. We have used phylogenetic footprinting and the related technique of phylogenetic shadowing to identify putative cis-regulatory elements in this intron. Among 29 Brassicaceae, several other motifs, but not the LFY and WUS binding sites previously identified, are largely invariant. Using reporter gene analyses, we tested six of these motifs and found that they are all functionally important for activity of AG regulatory sequences in A. thaliana. Although there is little obvious sequence similarity outside the Brassicaceae, the intron from cucumber AG has at least partial activity in A. thaliana. Our studies underscore the value of the comparative approach as a tool that complements gene-by-gene promoter dissection, but also highlight that sequence-based studies alone are insufficient for a complete identification of cis-regulatory sites.

  11. Transcription factor interactions: Selectors of positive or negative regulation from a single DNA element

    SciTech Connect

    Diamond, M.I.; Miner, J.N.; Yoshinaga, S.K.; Yamamoto, K.R. )

    1990-09-14

    The mechanism by which a single factor evokes opposite regulatory effects from a specific DNA sequence is not well understood. In this study, a 25-base pair element that resides upstream of the mouse proliferin gene was examined; it conferred on linked promoters either positive or negative glucocorticoid regulation, depending upon physiological context. This sequence, denoted a composite glucocorticoid response element (GRE), was bound selective in vitro both by the glucocorticoid receptor and by c-Jun and c-Fos, components of the phorbol ester-activated AP-1 transcription factor. Indeed, c-Jun and c-Fos served as selectors of hormone responsiveness: the composite GRE was inactive in the absence of c-Jun, whereas it conferred a positive glucocorticoid effect in the presence of c-Jun, and a negative glucocorticoid effect in the presence of c-Jun and relatively high levels of c-Fos. The receptor also interacted selectively with c-Jun in vitro. A general model for composite GRE action is proposed that invokes both DNA binding and protein-protein interactions by receptor and nonreceptor factors.

  12. Disease-associated variants in different categories of disease located in distinct regulatory elements

    PubMed Central

    2015-01-01

    Background The invention of high throughput sequencing technologies has led to the discoveries of hundreds of thousands of genetic variants associated with thousands of human diseases. Many of these genetic variants are located outside the protein coding regions, and as such, it is challenging to interpret the function of these genetic variants by traditional genetic approaches. Recent genome-wide functional genomics studies, such as FANTOM5 and ENCODE have uncovered a large number of regulatory elements across hundreds of different tissues or cell lines in the human genome. These findings provide an opportunity to study the interaction between regulatory elements and disease-associated genetic variants. Identifying these diseased-related regulatory elements will shed light on understanding the mechanisms of how these variants regulate gene expression and ultimately result in disease formation and progression. Results In this study, we curated and categorized 27,558 Mendelian disease variants, 20,964 complex disease variants, 5,809 cancer predisposing germline variants, and 43,364 recurrent cancer somatic mutations. Compared against nine different types of regulatory regions from FANTOM5 and ENCODE projects, we found that different types of disease variants show distinctive propensity for particular regulatory elements. Mendelian disease variants and recurrent cancer somatic mutations are 22-fold and 10- fold significantly enriched in promoter regions respectively (q<0.001), compared with allele-frequency-matched genomic background. Separate from these two categories, cancer predisposing germline variants are 27-fold enriched in histone modification regions (q<0.001), 10-fold enriched in chromatin physical interaction regions (q<0.001), and 6-fold enriched in transcription promoters (q<0.001). Furthermore, Mendelian disease variants and recurrent cancer somatic mutations share very similar distribution across types of functional effects. We further found that

  13. CCAAT displacement protein (CDP/cut) binds a negative regulatory element in the human tryptophan hydroxylase gene.

    PubMed

    Teerawatanasuk, N; Skalnik, D G; Carr, L G

    1999-01-01

    Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesis of serotonin, a neurotransmitter that has been implicated in many psychiatric illnesses. The mechanism of transcriptional regulation of the human TPH gene is largely unknown. We have identified a negative regulatory element located between nucleotides -310 and -220 in the human TPH (hTPH) gene. Electromobility shift analyses performed with the -310/-220 hTPH probe and nuclear extract from P815-HTR (a TPH-expressing cell line) revealed two slow migrating protein-DNA complexes, designated I and II. CCAAT displacement protein (CDP/Cut) is involved in complex I formation as shown in electromobility shift analysis, using consensus oligonucleotide competitor and antibody. Mutations in the CDP/Cut binding site not only disrupted the CDP-DNA complex but also disrupted the second complex, suggesting that the core binding sequences of the two proteins are overlapping. The functional importance of these protein-DNA interactions was assessed by transiently transfecting wild-type and mutant pTPH/luciferase reporter constructs into P815-HTR cells. Mutations in the core CDP/Cut site resulted in an approximately fourfold increase in relative luciferase activities. Because CDP/Cut has been shown to repress transcription of many target genes, we speculate that disruption of the CDP/Cut binding was responsible, at least in part, for the activation of hTPH gene. PMID:9886051

  14. Organisation of regulatory elements in two closely spaced Drosophila genes with common expression characteristics.

    PubMed

    Gigliotti, S; Balz, V; Malva, C; Schäfer, M A

    1997-11-01

    Sperm tail proteins that are components of a specific structure formed late during spermatid elongation have been found to be encoded by the Mst(3)CGP gene family. These genes have been demonstrated to be regulated both at the transcriptional as well as at the translational level. We report here on the dissection of the regulatory regions for two members of the gene family, Mst84Da and Mst84Db. While high level transcription and negative translational control of Mst84Da is mediated by a short gene segment of 205 nt (-152/+53), Mst84Db expression is controlled by a number of distinct regulatory elements with different effects that all reside within the gene itself. We identify a transcriptional control element between +154 and +216, a translational repression element around +216 to +275 and an RNA stability element within the 3'UTR. Irrespective of the final common expression characteristics, correct regulation for any individual member of the gene family seems to be achieved by very different means. This confirms earlier observations that did not detect any other sequence elements in common apart from the TCE (translational control element). PMID:9431808

  15. CRISPR-Cas9 Genome Editing of a Single Regulatory Element Nearly Abolishes Target Gene Expression in Mice

    PubMed Central

    Han, Yu; Slivano, Orazio J.; Christie, Christine K.; Cheng, Albert W.; Miano, Joseph M.

    2014-01-01

    Objective To ascertain the importance of a single regulatory element in the control of Cnn1 expression using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) genome editing. Approach and Results The CRISPR/Cas9 system was used to produce 3/18 founder mice carrying point mutations in an intronic CArG box of the smooth muscle cell (SMC)-restricted Cnn1 gene. Each founder was bred for germ line transmission of the mutant CArG box and littermate interbreeding to generate homozygous mutant (Cnn1ΔCArG/ΔCArG) mice. Quantitative RT-PCR, Western blotting, and confocal immunofluorescence microscopy showed dramatic reductions in Cnn1 mRNA and CNN1 protein expression in Cnn1ΔCArG/ΔCArG mice with no change in other SMC-restricted genes and little evidence of off-target edits elsewhere in the genome. In vivo chromatin immunoprecipitation assay revealed a sharp decrease in binding of SRF to the mutant CArG box. Loss of CNN1 expression was coincident with an increase in Ki-67 positive cells in the normal vessel wall. Conclusion CRISPR/Cas9 genome editing of a single CArG box nearly abolishes Cnn1 expression in vivo and evokes increases in SMC DNA synthesis. This facile genome editing system paves the way for a new generation of studies designed to test the importance of individual regulatory elements in living animals, including regulatory variants in conserved sequence blocks linked to human disease. PMID:25538209

  16. Shuffling of cis-regulatory elements is a pervasive feature of the vertebrate lineage

    PubMed Central

    Sanges, Remo; Kalmar, Eva; Claudiani, Pamela; D'Amato, Maria; Muller, Ferenc; Stupka, Elia

    2006-01-01

    Background All vertebrates share a remarkable degree of similarity in their development as well as in the basic functions of their cells. Despite this, attempts at unearthing genome-wide regulatory elements conserved throughout the vertebrate lineage using BLAST-like approaches have thus far detected noncoding conservation in only a few hundred genes, mostly associated with regulation of transcription and development. Results We used a unique combination of tools to obtain regional global-local alignments of orthologous loci. This approach takes into account shuffling of regulatory regions that are likely to occur over evolutionary distances greater than those separating mammalian genomes. This approach revealed one order of magnitude more vertebrate conserved elements than was previously reported in over 2,000 genes, including a high number of genes found in the membrane and extracellular regions. Our analysis revealed that 72% of the elements identified have undergone shuffling. We tested the ability of the elements identified to enhance transcription in zebrafish embryos and compared their activity with a set of control fragments. We found that more than 80% of the elements tested were able to enhance transcription significantly, prevalently in a tissue-restricted manner corresponding to the expression domain of the neighboring gene. Conclusion Our work elucidates the importance of shuffling in the detection of cis-regulatory elements. It also elucidates how similarities across the vertebrate lineage, which go well beyond development, can be explained not only within the realm of coding genes but also in that of the sequences that ultimately govern their expression. PMID:16859531

  17. Identification of transcriptional regulatory elements for Ntng1 and Ntng2 genes in mice

    PubMed Central

    2014-01-01

    Background Higher brain function is supported by the precise temporal and spatial regulation of thousands of genes. The mechanisms that underlie transcriptional regulation in the brain, however, remain unclear. The Ntng1 and Ntng2 genes, encoding axonal membrane adhesion proteins netrin-G1 and netrin-G2, respectively, are paralogs that have evolved in vertebrates and are expressed in distinct neuronal subsets in a complementary manner. The characteristic expression patterns of these genes provide a part of the foundation of the cortical layer structure in mammals. Results We used gene-targeting techniques, bacterial artificial chromosome (BAC)-aided transgenesis techniques, and in vivo enhancer assays to examine transcriptional mechanisms in vivo to gain insight into how the characteristic expression patterns of these genes are acquired. Analysis of the gene expression patterns in the presence or absence of netrin-G1 and netrin-G2 functional proteins allowed us to exclude the possibility that a feedback or feedforward mechanism mediates their characteristic expression patterns. Findings from the BAC deletion series revealed that widely distributed combinations of cis-regulatory elements determine the differential gene expression patterns of these genes and that major cis-regulatory elements are located in the 85–45 kb upstream region of Ntng2 and in the 75–60 kb upstream region and intronic region of Ntng1. In vivo enhancer assays using 2-kb evolutionarily conserved regions detected enhancer activity in the distal upstream regions of both genes. Conclusions The complementary expression patterns of Ntng1 and Ntng2 are determined by transcriptional cis-regulatory elements widely scattered in these loci. The cis-regulatory elements characterized in this study will facilitate the development of novel genetic tools for functionally dissecting neural circuits to better understand vertebrate brain function. PMID:24642214

  18. Recurrent Modification of a Conserved Cis-Regulatory Element Underlies Fruit Fly Pigmentation Diversity

    PubMed Central

    Rogers, William A.; Salomone, Joseph R.; Tacy, David J.; Camino, Eric M.; Davis, Kristen A.; Rebeiz, Mark; Williams, Thomas M.

    2013-01-01

    The development of morphological traits occurs through the collective action of networks of genes connected at the level of gene expression. As any node in a network may be a target of evolutionary change, the recurrent targeting of the same node would indicate that the path of evolution is biased for the relevant trait and network. Although examples of parallel evolution have implicated recurrent modification of the same gene and cis-regulatory element (CRE), little is known about the mutational and molecular paths of parallel CRE evolution. In Drosophila melanogaster fruit flies, the Bric-à-brac (Bab) transcription factors control the development of a suite of sexually dimorphic traits on the posterior abdomen. Female-specific Bab expression is regulated by the dimorphic element, a CRE that possesses direct inputs from body plan (ABD-B) and sex-determination (DSX) transcription factors. Here, we find that the recurrent evolutionary modification of this CRE underlies both intraspecific and interspecific variation in female pigmentation in the melanogaster species group. By reconstructing the sequence and regulatory activity of the ancestral Drosophila melanogaster dimorphic element, we demonstrate that a handful of mutations were sufficient to create independent CRE alleles with differing activities. Moreover, intraspecific and interspecific dimorphic element evolution proceeded with little to no alterations to the known body plan and sex-determination regulatory linkages. Collectively, our findings represent an example where the paths of evolution appear biased to a specific CRE, and drastic changes in function were accompanied by deep conservation of key regulatory linkages. PMID:24009528

  19. Repressive BMP2 gene regulatory elements near the BMP2 promoter

    SciTech Connect

    Jiang, Shan; Chandler, Ronald L.; Fritz, David T.; Mortlock, Douglas P.; Rogers, Melissa B.

    2010-02-05

    The level of bone morphogenetic protein 2 (BMP2) profoundly influences essential cell behaviors such as proliferation, differentiation, apoptosis, and migration. The spatial and temporal pattern of BMP2 synthesis, particular in diverse embryonic cells, is highly varied and dynamic. We have identified GC-rich sequences within the BMP2 promoter region that strongly repress gene expression. These elements block the activity of a highly conserved, osteoblast enhancer in response to FGF2 treatment. Both positive and negative gene regulatory elements control BMP2 synthesis. Detecting and mapping the repressive motifs is essential because they impede the identification of developmentally regulated enhancers necessary for normal BMP2 patterns and concentration.

  20. Genome-wide analysis reveals regulatory role of G4 DNA in gene transcription

    PubMed Central

    Du, Zhuo; Zhao, Yiqiang; Li, Ning

    2008-01-01

    G-quadruplex or G4 DNA, a four-stranded DNA structure formed in G-rich sequences, has been hypothesized to be a structural motif involved in gene regulation. In this study, we examined the regulatory role of potential G4 DNA motifs (PG4Ms) located in the putative transcriptional regulatory region (TRR, –500 to +500) of genes across the human genome. We found that PG4Ms in the 500-bp region downstream of the annotated transcription start site (TSS; PG4MD500) are associated with gene expression. Generally, PG4MD500-positive genes are expressed at higher levels than PG4MD500-negative genes, and an increased number of PG4MD500 provides a cumulative effect. This observation was validated by controlling for attributes, including gene family, function, and promoter similarity. We also observed an asymmetric pattern of PG4MD500 distribution between strands, whereby the frequency of PG4MD500 in the coding strand is generally higher than that in the template strand. Further analysis showed that the presence of PG4MD500 and its strand asymmetry are associated with significant enrichment of RNAP II at the putative TRR. On the basis of these results, we propose a model of G4 DNA-mediated stimulation of transcription with the hypothesis that PG4MD500 contributes to gene transcription by maintaining the DNA in an open conformation, while the asymmetric distribution of PG4MD500 considerably reduces the probability of blocking the progression of the RNA polymerase complex on the template strand. Our findings provide a comprehensive view of the regulatory function of G4 DNA in gene transcription. PMID:18096746

  1. Detection and characterization of regulatory elements using probabilistic conditional random field and hidden Markov models.

    PubMed

    Wang, Hongyan; Zhou, Xiaobo

    2013-04-01

    By altering the electrostatic charge of histones or providing binding sites to protein recognition molecules, Chromatin marks have been proposed to regulate gene expression, a property that has motivated researchers to link these marks to cis-regulatory elements. With the help of next generation sequencing technologies, we can now correlate one specific chromatin mark with regulatory elements (e.g. enhancers or promoters) and also build tools, such as hidden Markov models, to gain insight into mark combinations. However, hidden Markov models have limitation for their character of generative models and assume that a current observation depends only on a current hidden state in the chain. Here, we employed two graphical probabilistic models, namely the linear conditional random field model and multivariate hidden Markov model, to mark gene regions with different states based on recurrent and spatially coherent character of these eight marks. Both models revealed chromatin states that may correspond to enhancers and promoters, transcribed regions, transcriptional elongation, and low-signal regions. We also found that the linear conditional random field model was more effective than the hidden Markov model in recognizing regulatory elements, such as promoter-, enhancer-, and transcriptional elongation-associated regions, which gives us a better choice. PMID:23237214

  2. A novel positive regulatory element for exfoliative toxin A gene expression in Staphylococcus aureus.

    PubMed

    Sakurai, Susumu; Suzuki, Hitoshi; Hata, Toshiaki; Yoshizawa, Yukio; Nakayama, Ritsuko; Machida, Katsuhiko; Masuda, Shogo; Tsukiyama, Takashi

    2004-04-01

    A 1.4 kb positive regulatory element (ETA(exp)) that controls staphylococcal exfoliative toxin A (sETA) transcription was cloned from Staphylococcus aureus. ETA(exp) is located upstream of the cloned 5.8 kb eta gene (etaJ1) obtained from the chomosomal DNA of S. aureus ZM, the standard ETA-producing strain. The cETA prepared from an Escherichia coli transformant into which the recombinant plasmid petaJ1 (5.8 kb eta/pUC9) had been introduced was expressed at high levels in the culture supernatant and the ammonium-sulfate-precipitated culture supernatant fraction as shown by immunoblotting and the single radial immunodiffusion test. However, cETA produced by the recombinant plasmid petaJ3 containing the 1.7 kb eta sequence (etaJ3) with a 1.45 kb ETA(exp)-deficient eta fragment (1.7 kb eta/pUC9) obtained from the 5.8 kb eta sequence by subcloning was not detected in either the culture supernatant or the ammonium-sulfate-precipitated culture supernatant fraction (167-fold concentrate of the culture supernatant) by immunoblotting or the single radial immunodiffusion test. A large amount of cETA was produced by the 1.7 kb eta sequence when it was linked to ETA(exp) amplified by PCR (1.7 kb eta-ETA(exp)/pUC9), regardless of the orientation of ETA(exp) insertion. Northern blot hybridization showed lower levels of the transcripts of the 1.7 kb eta sequence than of the 5.8 kb eta sequence. The rsETA prepared from an S. aureus transformant into which the recombinant plasmid 3.4 kb eta-ETA(exp)/pYT3 (pYT3-etaJ6) had been introduced was expressed at high levels in the culture supernatant fraction as shown by the latex agglutination test. However, the agglutination titre in the culture supernatant fraction of rsETA produced by the recombinant plasmid (1.7 kb eta/pYT3) containing the 1.7 kb eta sequence carrying the 1.4 kb ETA(exp)-deficient eta fragment (pYT3-etaJ3) was 2500-4000 times lower than that of pYT3-etaJ6. PMID:15073304

  3. A Structural Basis for the Regulatory Inactivation of DnaA

    PubMed Central

    Xu, Qingping; McMullan, Daniel; Abdubek, Polat; Astakhova, Tamara; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Clayton, Thomas; Das, Debanu; Deller, Marc C.; Duan, Lian; Elsliger, Marc-Andre; Feuerhelm, Julie; Hale, Joanna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Johnson, Hope A.; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Kumar, Abhinav; Marciano, David; Miller, Mitchell D.; Morse, Andrew T.; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Oommachen, Silvya; Paulsen, Jessica; Puckett, Christina; Reyes, Ron; Rife, Christopher L.; Sefcovic, Natasha; Trame, Christine; van den Bedem, Henry; Weekes, Dana; Hodgson, Keith O.; Wooley, John; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2009-01-01

    Summary Regulatory inactivation of DnaA is dependent on Hda, a protein homologous to the AAA+ ATPase region of the replication initiator DnaA. When bound to the sliding clamp loaded onto duplex DNA, Hda can stimulate the transformation of active DnaA-ATP into inactive DnaA-ADP. The crystal structure of Hda from Shewanella amazonensis SB2B at 1.75 Å resolution reveals that Hda resembles typical AAA+ ATPases. The arrangement of the two subdomains in Hda (residues 1-174, 175-241) differs dramatically from that of DnaA. A CDP molecule anchors the Hda domains in a conformation which promotes dimer formation. The Hda dimer adopts a novel oligomeric assembly for AAA+ proteins in which the arginine finger, crucial for ATP hydrolysis, is fully exposed and available to hydrolyze DnaA-ATP through a typical AAA+ type mechanism. The sliding clamp binding motifs at the N-terminus of each Hda monomer are partially buried and combine to form an antiparallel β-sheet at the dimer interface. The inaccessibility of the clamp binding motifs in the CDP bound structure of Hda suggests that conformational changes are required for Hda to form a functional complex with the clamp. Thus, the CDP-bound Hda dimer likely represents an inactive form of Hda. PMID:19000695

  4. Mapping of Variable DNA Methylation Across Multiple Cell Types Defines a Dynamic Regulatory Landscape of the Human Genome

    PubMed Central

    Gu, Junchen; Stevens, Michael; Xing, Xiaoyun; Li, Daofeng; Zhang, Bo; Payton, Jacqueline E.; Oltz, Eugene M.; Jarvis, James N.; Jiang, Kaiyu; Cicero, Theodore; Costello, Joseph F.; Wang, Ting

    2016-01-01

    DNA methylation is an important epigenetic modification involved in many biological processes and diseases. Many studies have mapped DNA methylation changes associated with embryogenesis, cell differentiation, and cancer at a genome-wide scale. Our understanding of genome-wide DNA methylation changes in a developmental or disease-related context has been steadily growing. However, the investigation of which CpGs are variably methylated in different normal cell or tissue types is still limited. Here, we present an in-depth analysis of 54 single-CpG-resolution DNA methylomes of normal human cell types by integrating high-throughput sequencing-based methylation data. We found that the ratio of methylated to unmethylated CpGs is relatively constant regardless of cell type. However, which CpGs made up the unmethylated complement was cell-type specific. We categorized the 26,000,000 human autosomal CpGs based on their methylation levels across multiple cell types to identify variably methylated CpGs and found that 22.6% exhibited variable DNA methylation. These variably methylated CpGs formed 660,000 variably methylated regions (VMRs), encompassing 11% of the genome. By integrating a multitude of genomic data, we found that VMRs enrich for histone modifications indicative of enhancers, suggesting their role as regulatory elements marking cell type specificity. VMRs enriched for transcription factor binding sites in a tissue-dependent manner. Importantly, they enriched for GWAS variants, suggesting that VMRs could potentially be implicated in disease and complex traits. Taken together, our results highlight the link between CpG methylation variation, genetic variation, and disease risk for many human cell types. PMID:26888867

  5. Mapping of Variable DNA Methylation Across Multiple Cell Types Defines a Dynamic Regulatory Landscape of the Human Genome.

    PubMed

    Gu, Junchen; Stevens, Michael; Xing, Xiaoyun; Li, Daofeng; Zhang, Bo; Payton, Jacqueline E; Oltz, Eugene M; Jarvis, James N; Jiang, Kaiyu; Cicero, Theodore; Costello, Joseph F; Wang, Ting

    2016-01-01

    DNA methylation is an important epigenetic modification involved in many biological processes and diseases. Many studies have mapped DNA methylation changes associated with embryogenesis, cell differentiation, and cancer at a genome-wide scale. Our understanding of genome-wide DNA methylation changes in a developmental or disease-related context has been steadily growing. However, the investigation of which CpGs are variably methylated in different normal cell or tissue types is still limited. Here, we present an in-depth analysis of 54 single-CpG-resolution DNA methylomes of normal human cell types by integrating high-throughput sequencing-based methylation data. We found that the ratio of methylated to unmethylated CpGs is relatively constant regardless of cell type. However, which CpGs made up the unmethylated complement was cell-type specific. We categorized the 26,000,000 human autosomal CpGs based on their methylation levels across multiple cell types to identify variably methylated CpGs and found that 22.6% exhibited variable DNA methylation. These variably methylated CpGs formed 660,000 variably methylated regions (VMRs), encompassing 11% of the genome. By integrating a multitude of genomic data, we found that VMRs enrich for histone modifications indicative of enhancers, suggesting their role as regulatory elements marking cell type specificity. VMRs enriched for transcription factor binding sites in a tissue-dependent manner. Importantly, they enriched for GWAS variants, suggesting that VMRs could potentially be implicated in disease and complex traits. Taken together, our results highlight the link between CpG methylation variation, genetic variation, and disease risk for many human cell types. PMID:26888867

  6. Inducer effect on the complex formation between rat liver nuclear proteins and cytochrome P450 2B gene regulatory elements.

    PubMed

    Duzhak, T G; Schwartz, E I; Gulyaeva, L F; Lyakhovich, V V

    2002-09-01

    DNA gel retardation assay has been applied to the investigation of complexes between rat liver nuclear proteins and Barbie box positive regulatory element of cytochrome P450 2B (CYP2B) genes. The intensities of B1 and B2 bands detected in the absence of an inducer increased after 30 min protein incubation with phenobarbital (PB) or triphenyldioxane (TPD), but not with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOPOB). In addition, a new complex (B3 band) was for the first time detected under induction by PB, TPD, and TCPOPOB. Increase in the incubation time up to 2 h facilitated the formation of other new complexes (B4 and B5 bands), which were detected only in the presence of TPD. The use of [3H]TPD in hybridization experiments revealed that this inducer, capable of binding to Barbie box DNA, is also present in B4 and B5 complexes. It is probable that the investigated compounds activate the same proteins at the initial induction steps, which correlates with the formation of B1, B2, and B3 complexes. The further induction step might be inducer-specific, as indicated by the formation of B4 and B5 complexes in the presence of TPD only. Thus, the present data suggest the possibility of specific gene activation signaling pathways that are dependent on a particular inducer. PMID:12387719

  7. Peak-valley-peak pattern of histone modifications delineates active regulatory elements and their directionality.

    PubMed

    Pundhir, Sachin; Bagger, Frederik O; Lauridsen, Felicia B; Rapin, Nicolas; Porse, Bo T

    2016-05-19

    Formation of nucleosome free region (NFR) accompanied by specific histone modifications at flanking nucleosomes is an important prerequisite for enhancer and promoter activity. Due to this process, active regulatory elements often exhibit a distinct shape of histone signal in the form of a peak-valley-peak (PVP) pattern. However, different features of PVP patterns and their robustness in predicting active regulatory elements have never been systematically analyzed. Here, we present PARE, a novel computational method that systematically analyzes the H3K4me1 or H3K4me3 PVP patterns to predict NFRs. We show that NFRs predicted by H3K4me1 and me3 patterns are associated with active enhancers and promoters, respectively. Furthermore, asymmetry in the height of peaks flanking the central valley can predict the directionality of stable transcription at promoters. Using PARE on ChIP-seq histone modifications from four ENCODE cell lines and four hematopoietic differentiation stages, we identified several enhancers whose regulatory activity is stage specific and correlates positively with the expression of proximal genes in a particular stage. In conclusion, our results demonstrate that PVP patterns delineate both the histone modification landscape and the transcriptional activities governed by active enhancers and promoters, and therefore can be used for their prediction. PARE is freely available at http://servers.binf.ku.dk/pare. PMID:27095194

  8. An ant colony optimization based algorithm for identifying gene regulatory elements.

    PubMed

    Liu, Wei; Chen, Hanwu; Chen, Ling

    2013-08-01

    It is one of the most important tasks in bioinformatics to identify the regulatory elements in gene sequences. Most of the existing algorithms for identifying regulatory elements are inclined to converge into a local optimum, and have high time complexity. Ant Colony Optimization (ACO) is a meta-heuristic method based on swarm intelligence and is derived from a model inspired by the collective foraging behavior of real ants. Taking advantage of the ACO in traits such as self-organization and robustness, this paper designs and implements an ACO based algorithm named ACRI (ant-colony-regulatory-identification) for identifying all possible binding sites of transcription factor from the upstream of co-expressed genes. To accelerate the ants' searching process, a strategy of local optimization is presented to adjust the ants' start positions on the searched sequences. By exploiting the powerful optimization ability of ACO, the algorithm ACRI can not only improve precision of the results, but also achieve a very high speed. Experimental results on real world datasets show that ACRI can outperform other traditional algorithms in the respects of speed and quality of solutions. PMID:23746735

  9. [Regulatory elements in the skin epithelium of Saccoglossus mereschkowskii (Enteropneusta, Hemichordata): electron microscopic and immunocytochemical study].

    PubMed

    Stoliarova, M V; Val'kovich, E I

    2013-01-01

    The aim of this investigation was to demonstrate the regulatory elements in the skin epithelium of Enteropneusta which are supposed to be related to the chordate ancestors. Using electron microscopy, it was found that in the skin epithelium of a representative of enteropneusts Saccoglossus mereschkowskii, the basal parts of some epitheliocytes took part in formation of a nerve layer. These cells were considered as receptor ciliated cells. The granular epithelial cells were shown to release secretion according to both exocrine and endocrine mechanism; these cells were characterized as endocrine-like regulatory cells. Fine granular cells possibly represent special receptor-endocrine-like cell type. The immunocytochemical detection of FMRFamid neuropeptide localization in histological sections confirmed the electron microscopic data on the presence of receptor and endocrine-like cells in the epithelium. It is suggested that the skin epithelium of Enteropneusta contains a peculiar neuro-endocrine regulatory system that is represented by receptor cells, receptor-endocrine-like cells of an open type and nerve elements of the nerve layer. PMID:24707736

  10. Peak-valley-peak pattern of histone modifications delineates active regulatory elements and their directionality

    PubMed Central

    Pundhir, Sachin; Bagger, Frederik O.; Lauridsen, Felicia B.; Rapin, Nicolas; Porse, Bo T.

    2016-01-01

    Formation of nucleosome free region (NFR) accompanied by specific histone modifications at flanking nucleosomes is an important prerequisite for enhancer and promoter activity. Due to this process, active regulatory elements often exhibit a distinct shape of histone signal in the form of a peak-valley-peak (PVP) pattern. However, different features of PVP patterns and their robustness in predicting active regulatory elements have never been systematically analyzed. Here, we present PARE, a novel computational method that systematically analyzes the H3K4me1 or H3K4me3 PVP patterns to predict NFRs. We show that NFRs predicted by H3K4me1 and me3 patterns are associated with active enhancers and promoters, respectively. Furthermore, asymmetry in the height of peaks flanking the central valley can predict the directionality of stable transcription at promoters. Using PARE on ChIP-seq histone modifications from four ENCODE cell lines and four hematopoietic differentiation stages, we identified several enhancers whose regulatory activity is stage specific and correlates positively with the expression of proximal genes in a particular stage. In conclusion, our results demonstrate that PVP patterns delineate both the histone modification landscape and the transcriptional activities governed by active enhancers and promoters, and therefore can be used for their prediction. PARE is freely available at http://servers.binf.ku.dk/pare. PMID:27095194

  11. Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

    SciTech Connect

    Matsushita, Y. Murakawa, T. Shimamura, K. Oishi, M. Ohyama, T. Kurita, N.

    2015-02-27

    The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA.

  12. Intranasal vaccination with proinsulin DNA induces regulatory CD4+ T cells that prevent experimental autoimmune diabetes.

    PubMed

    Every, Alison L; Kramer, David R; Mannering, Stuart I; Lew, Andrew M; Harrison, Leonard C

    2006-04-15

    Insulin, an autoantigen in type 1 diabetes, when administered mucosally to diabetes-prone NOD mice induces regulatory T cells (T(reg)) that protect against diabetes. Compared with protein, Ag encoded as DNA has potential advantages as a therapeutic agent. We found that intranasal vaccination of NOD mice with plasmid DNA encoding mouse proinsulin II-induced CD4+ T(reg) that suppressed diabetes development, both after adoptive cotransfer with "diabetogenic" spleen cells and after transfer into NOD mice given cyclophosphamide to accelerate diabetes onset. In contrast to prototypic CD4+ CD25+ T(reg), CD4+ T(reg) induced by proinsulin DNA were both CD25+ and CD25- and not defined by markers such as glucocorticoid-induced TNFR-related protein (GITR), CD103, or Foxp3. Intriguingly, despite induction of T(reg) and reduced islet inflammation, diabetes incidence in proinsulin DNA-treated mice was unchanged. However, diabetes was prevented when DNA vaccination was performed under the cover of CD40 ligand blockade, known to prevent priming of CTL by mucosal Ag. Thus, intranasal vaccination with proinsulin DNA has therapeutic potential to prevent diabetes, as demonstrated by induction of protective T(reg), but further modifications are required to improve its efficacy, which could be compromised by concomitant induction of pathogenic immunity. PMID:16585551

  13. Movable genetic elements: detection of changes in maize DNA at the Shrunken locus due to the intervention of Ds elements

    SciTech Connect

    Burr, B.; Burr, F.A.

    1980-05-28

    This report describes our initial attempts at the molecular characterization of a maize controlling element. We have prepared a cDNA probe and used it to detect changes at a locus where Ds elements are found. Evidence of their presence are indicated by changes in the restriction patterns, but there is as yet no information on the physical nature of the controlling elements nor on the kinds of rearrangements they cause.

  14. Movable Genetic Elements: Detection of Changes in Maize DNA at the Shrunken Locus Due to the Intervention of Ds Elements

    DOE R&D Accomplishments Database

    Burr, B.; Burr, F.A.

    1980-05-28

    This report describes our initial attempts at the molecular characterization of a maize controlling element. We have prepared a cDNA probe and used it to detect changes at a locus where Ds elements are found. Evidence of their presence are indicated by changes in the restriction patterns, but there is as yet no information on the physical nature of the controlling elements nor on the kinds of rearrangements they cause.

  15. BET bromodomain inhibition releases the Mediator complex from select cis-regulatory elements

    PubMed Central

    Bhagwat, Anand S.; Roe, Jae-Seok; Mok, Beverly A.; Hohmann, Anja F.; Shi, Junwei; Vakoc, Christopher R.

    2016-01-01

    The bromodomain and extraterminal (BET) protein BRD4 can physically interact with the Mediator complex, but the relevance of this association to the therapeutic effects of BET inhibitors in cancer is unclear. Here, we show that BET inhibition causes a rapid release of Mediator from a subset of cis-regulatory elements in the genome of acute myeloid leukemia (AML) cells. These sites of Mediator eviction were highly correlated with transcriptional suppression of neighboring genes, which are enriched for targets of the transcription factor MYB and for functions related to leukemogenesis. An shRNA screen of Mediator in AML cells identified the MED12, MED13, MED23, and MED24 subunits as performing a similar regulatory function to BRD4 in this context, including a shared role in sustaining a block in myeloid maturation. These findings suggest that the interaction between BRD4 and Mediator has functional importance for gene-specific transcriptional activation and for AML maintenance. PMID:27068464

  16. Distal cis-regulatory elements are required for tissue-specific expression of enamelin (Enam)

    PubMed Central

    Hu, Yuanyuan; Papagerakis, Petros; Ye, Ling; Feng, Jerry Q.; Simmer, James P.; Hu, Jan C-C.

    2009-01-01

    Enamel formation is orchestrated by the sequential expression of genes encoding enamel matrix proteins; however, the mechanisms sustaining the spatio–temporal order of gene transcription during amelogenesis are poorly understood. The aim of this study was to characterize the cis-regulatory sequences necessary for normal expression of enamelin (Enam). Several enamelin transcription regulatory regions, showing high sequence homology among species, were identified. DNA constructs containing 5.2 or 3.9 kb regions upstream of the enamelin translation initiation site were linked to a LacZ reporter and used to generate transgenic mice. Only the 5.2-Enam–LacZ construct was sufficient to recapitulate the endogenous pattern of enamelin tooth-specific expression. The 3.9-Enam–LacZ transgenic lines showed no expression in dental cells, but ectopic β-galactosidase activity was detected in osteoblasts. Potential transcription factor-binding sites were identified that may be important in controlling enamelin basal promoter activity and in conferring enamelin tissue-specific expression. Our study provides new insights into regulatory mechanisms governing enamelin expression. PMID:18353004

  17. The structure of the human peripherin gene (PRPH) and identification of potential regulatory elements

    SciTech Connect

    Foley, J.; Ley, C.A.; Parysek, L.M.

    1994-07-15

    The authors determined the complete nucleotide sequence of the coding region of the human peripherin gene (PRPH), as well as 742 bp 5{prime} to the cap site and 584 bp 3{prime} to the stop codon, and compared its structure and sequence to the rat and mouse genes. The overall structure of 9 exons separated by 8 introns is conserved among these three mammalian species. The nucleotide sequences of the human peripherin gene exons were 90% identical to the rat gene sequences, and the predicted human peripherin protein differed from rat peripherin at only 18 of 475 amino acid residues. Comparison of the 5{prime} flanking regions of the human peripherin gene and rodent genes revealed extensive areas of high homology. Additional conserved segments were found in introns 1 and 2. Within the 5{prime} region, potential regulatory sequences, including a nerve growth factor negative regulatory element, a Hox protein binding site, and a heat shock element, were identified in all peripherin genes. The positional conservation of each element suggests that they may be important in the tissue-specific, developmental-specific, and injury-specific expression of the peripherin gene. 24 refs., 2 figs., 1 tab.

  18. Densely ionizing radiation affects DNA methylation of selective LINE-1 elements.

    PubMed

    Prior, Sara; Miousse, Isabelle R; Nzabarushimana, Etienne; Pathak, Rupak; Skinner, Charles; Kutanzi, Kristy R; Allen, Antiño R; Raber, Jacob; Tackett, Alan J; Hauer-Jensen, Martin; Nelson, Gregory A; Koturbash, Igor

    2016-10-01

    Long Interspersed Nucleotide Element 1 (LINE-1) retrotransposons are heavily methylated and are the most abundant transposable elements in mammalian genomes. Here, we investigated the differential DNA methylation within the LINE-1 under normal conditions and in response to environmentally relevant doses of sparsely and densely ionizing radiation. We demonstrate that DNA methylation of LINE-1 elements in the lungs of C57BL6 mice is dependent on their evolutionary age, where the elder age of the element is associated with the lower extent of DNA methylation. Exposure to 5-aza-2'-deoxycytidine and methionine-deficient diet affected DNA methylation of selective LINE-1 elements in an age- and promoter type-dependent manner. Exposure to densely IR, but not sparsely IR, resulted in DNA hypermethylation of older LINE-1 elements, while the DNA methylation of evolutionary younger elements remained mostly unchanged. We also demonstrate that exposure to densely IR increased mRNA and protein levels of LINE-1 via the loss of the histone H3K9 dimethylation and an increase in the H3K4 trimethylation at the LINE-1 5'-untranslated region, independently of DNA methylation. Our findings suggest that DNA methylation is important for regulation of LINE-1 expression under normal conditions, but histone modifications may dictate the transcriptional activity of LINE-1 in response to exposure to densely IR. PMID:27419368

  19. Regulation of human PTCH1b expression by different 5' untranslated region cis-regulatory elements

    PubMed Central

    Ozretić, Petar; Bisio, Alessandra; Musani, Vesna; Trnski, Diana; Sabol, Maja; Levanat, Sonja; Inga, Alberto

    2015-01-01

    PTCH1 gene codes for a 12-pass transmembrane receptor with a negative regulatory role in the Hedgehog-Gli signaling pathway. PTCH1 germline mutations cause Gorlin syndrome, a disorder characterized by developmental abnormalities and tumor susceptibility. The autosomal dominant inheritance, and the evidence for PTCH1 haploinsufficiency, suggests that fine-tuning systems of protein patched homolog 1 (PTC1) levels exist to properly regulate the pathway. Given the role of 5' untranslated region (5'UTR) in protein expression, our aim was to thoroughly explore cis-regulatory elements in the 5'UTR of PTCH1 transcript 1b. The (CGG)n polymorphism was the main potential regulatory element studied so far but with inconsistent results and no clear association between repeat number and disease risk. Using luciferase reporter constructs in human cell lines here we show that the number of CGG repeats has no strong impact on gene expression, both at mRNA and protein levels. We observed variability in the length of 5'UTR and changes in abundance of the associated transcripts after pathway activation. We show that upstream AUG codons (uAUGs) present only in longer 5'UTRs could negatively regulate the amount of PTC1 isoform L (PTC1-L). The existence of an internal ribosome entry site (IRES) observed using different approaches and mapped in the region comprising the CGG repeats, would counteract the effect of the uAUGs and enable synthesis of PTC1-L under stressful conditions, such as during hypoxia. Higher relative translation efficiency of PTCH1b mRNA in HEK 293T cultured hypoxia was observed by polysomal profiling and Western blot analyses. All our results point to an exceptionally complex and so far unexplored role of 5'UTR PTCH1b cis-element features in the regulation of the Hedgehog-Gli signaling pathway. PMID:25826662

  20. Computational Approaches to Identify Promoters and cis-Regulatory Elements in Plant Genomes1

    PubMed Central

    Rombauts, Stephane; Florquin, Kobe; Lescot, Magali; Marchal, Kathleen; Rouzé, Pierre; Van de Peer, Yves

    2003-01-01

    The identification of promoters and their regulatory elements is one of the major challenges in bioinformatics and integrates comparative, structural, and functional genomics. Many different approaches have been developed to detect conserved motifs in a set of genes that are either coregulated or orthologous. However, although recent approaches seem promising, in general, unambiguous identification of regulatory elements is not straightforward. The delineation of promoters is even harder, due to its complex nature, and in silico promoter prediction is still in its infancy. Here, we review the different approaches that have been developed for identifying promoters and their regulatory elements. We discuss the detection of cis-acting regulatory elements using word-counting or probabilistic methods (so-called “search by signal” methods) and the delineation of promoters by considering both sequence content and structural features (“search by content” methods). As an example of search by content, we explored in greater detail the association of promoters with CpG islands. However, due to differences in sequence content, the parameters used to detect CpG islands in humans and other vertebrates cannot be used for plants. Therefore, a preliminary attempt was made to define parameters that could possibly define CpG and CpNpG islands in Arabidopsis, by exploring the compositional landscape around the transcriptional start site. To this end, a data set of more than 5,000 gene sequences was built, including the promoter region, the 5′-untranslated region, and the first introns and coding exons. Preliminary analysis shows that promoter location based on the detection of potential CpG/CpNpG islands in the Arabidopsis genome is not straightforward. Nevertheless, because the landscape of CpG/CpNpG islands differs considerably between promoters and introns on the one side and exons (whether coding or not) on the other, more sophisticated approaches can probably be

  1. Comprehensive DNA Microarray Analysis of Bacillus subtilis Two-Component Regulatory Systems

    PubMed Central

    Kobayashi, Kazuo; Ogura, Mitsuo; Yamaguchi, Hirotake; Yoshida, Ken-Ichi; Ogasawara, Naotake; Tanaka, Teruo; Fujita, Yasutaro

    2001-01-01

    It has recently been shown through DNA microarray analysis of Bacillus subtilis two-component regulatory systems (DegS-DegU, ComP-ComA, and PhoR-PhoP) that overproduction of a response regulator of the two-component systems in the background of a deficiency of its cognate sensor kinase affects the regulation of genes, including its target ones. The genome-wide effect on gene expression caused by the overproduction was revealed by DNA microarray analysis. In the present work, we newly analyzed 24 two-component systems by means of this strategy, leaving out 8 systems to which it was unlikely to be applicable. This analysis revealed various target gene candidates for these two-component systems. It is especially notable that interesting interactions appeared to take place between several two-component systems. Moreover, the probable functions of some unknown two-component systems were deduced from the list of their target gene candidates. This work is heuristic but provides valuable information for further study toward a comprehensive understanding of the B. subtilis two-component regulatory systems. The DNA microarray data obtained in this work are available at the KEGG Expression Database website (http://www.genome.ad.jp/kegg/expression). PMID:11717295

  2. Inferring regulatory element landscapes and transcription factor networks from cancer methylomes.

    PubMed

    Yao, Lijing; Shen, Hui; Laird, Peter W; Farnham, Peggy J; Berman, Benjamin P

    2015-01-01

    Recent studies indicate that DNA methylation can be used to identify transcriptional enhancers, but no systematic approach has been developed for genome-wide identification and analysis of enhancers based on DNA methylation. We describe ELMER (Enhancer Linking by Methylation/Expression Relationships), an R-based tool that uses DNA methylation to identify enhancers and correlates enhancer state with expression of nearby genes to identify transcriptional targets. Transcription factor motif analysis of enhancers is coupled with expression analysis of transcription factors to infer upstream regulators. Using ELMER, we investigated more than 2,000 tumor samples from The Cancer Genome Atlas. We identified networks regulated by known cancer drivers such as GATA3 and FOXA1 (breast cancer), SOX17 and FOXA2 (endometrial cancer), and NFE2L2, SOX2, and TP63 (squamous cell lung cancer). We also identified novel networks with prognostic associations, including RUNX1 in kidney cancer. We propose ELMER as a powerful new paradigm for understanding the cis-regulatory interface between cancer-associated transcription factors and their functional target genes. PMID:25994056

  3. Identification of cis-acting repressive sequences within the negative regulatory element of human immunodeficiency virus type 1.

    PubMed Central

    Lu, Y C; Touzjian, N; Stenzel, M; Dorfman, T; Sodroski, J G; Haseltine, W A

    1990-01-01

    The negative regulatory element of human immunodeficiency virus type 1 is a 260-nucleotide-long sequence that decreases the rate of RNA transcription initiation specified by the long terminal repeat. This region has the potential to bind several cellular transcription factors. Here it is shown that sequences which recognize the NFAT-1 and USF cellular transcription factors contribute to this negative regulatory effect. The sequences within the negative regulatory element which resemble the AP-1 site and the URS do not negatively regulate human immunodeficiency virus long terminal repeat transcription initiation. PMID:2398545

  4. Tissue-Specific Enrichment of Lymphoma Risk Loci in Regulatory Elements

    PubMed Central

    Hayes, James E.; Trynka, Gosia; Vijai, Joseph; Offit, Kenneth; Raychaudhuri, Soumya; Klein, Robert J.

    2015-01-01

    Though numerous polymorphisms have been associated with risk of developing lymphoma, how these variants function to promote tumorigenesis is poorly understood. Here, we report that lymphoma risk SNPs, especially in the non-Hodgkin’s lymphoma subtype chronic lymphocytic leukemia, are significantly enriched for co-localization with epigenetic marks of active gene regulation. These enrichments were seen in a lymphoid-specific manner for numerous ENCODE datasets, including DNase-hypersensitivity as well as multiple segmentation-defined enhancer regions. Furthermore, we identify putatively functional SNPs that are both in regulatory elements in lymphocytes and are associated with gene expression changes in blood. We developed an algorithm, UES, that uses a Monte Carlo simulation approach to calculate the enrichment of previously identified risk SNPs in various functional elements. This multiscale approach integrating multiple datasets helps disentangle the underlying biology of lymphoma, and more broadly, is generally applicable to GWAS results from other diseases as well. PMID:26422229

  5. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  6. Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation.

    PubMed

    Simandi, Zoltan; Horvath, Attila; Nagy, Peter; Nagy, Laszlo

    2016-01-01

    Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system. PMID:27403939

  7. Genomic divergence and brain evolution: How regulatory DNA influences development of the cerebral cortex.

    PubMed

    Silver, Debra L

    2016-02-01

    The cerebral cortex controls our most distinguishing higher cognitive functions. Human-specific gene expression differences are abundant in the cerebral cortex, yet we have only begun to understand how these variations impact brain function. This review discusses the current evidence linking non-coding regulatory DNA changes, including enhancers, with neocortical evolution. Functional interrogation using animal models reveals converging roles for our genome in key aspects of cortical development including progenitor cell cycle and neuronal signaling. New technologies, including iPS cells and organoids, offer potential alternatives to modeling evolutionary modifications in a relevant species context. Several diseases rooted in the cerebral cortex uniquely manifest in humans compared to other primates, thus highlighting the importance of understanding human brain differences. Future studies of regulatory loci, including those implicated in disease, will collectively help elucidate key cellular and genetic mechanisms underlying our distinguishing cognitive traits. PMID:26642006

  8. A compact, in vivo screen of all 6-mers reveals drivers of tissue-specific expression and guides synthetic regulatory element design

    PubMed Central

    2013-01-01

    Background Large-scale annotation efforts have improved our ability to coarsely predict regulatory elements throughout vertebrate genomes. However, it is unclear how complex spatiotemporal patterns of gene expression driven by these elements emerge from the activity of short, transcription factor binding sequences. Results We describe a comprehensive promoter extension assay in which the regulatory potential of all 6 base-pair (bp) sequences was tested in the context of a minimal promoter. To enable this large-scale screen, we developed algorithms that use a reverse-complement aware decomposition of the de Bruijn graph to design a library of DNA oligomers incorporating every 6-bp sequence exactly once. Our library multiplexes all 4,096 unique 6-mers into 184 double-stranded 15-bp oligomers, which is sufficiently compact for in vivo testing. We injected each multiplexed construct into zebrafish embryos and scored GFP expression in 15 tissues at two developmental time points. Twenty-seven constructs produced consistent expression patterns, with the majority doing so in only one tissue. Functional sequences are enriched near biologically relevant genes, match motifs for developmental transcription factors, and are required for enhancer activity. By concatenating tissue-specific functional sequences, we generated completely synthetic enhancers for the notochord, epidermis, spinal cord, forebrain and otic lateral line, and show that short regulatory sequences do not always function modularly. Conclusions This work introduces a unique in vivo catalog of short, functional regulatory sequences and demonstrates several important principles of regulatory element organization. Furthermore, we provide resources for designing compact, reverse-complement aware k-mer libraries. PMID:23867016

  9. Quantitative comparison of cis-regulatory element (CRE) activities in transgenic Drosophila melanogaster.

    PubMed

    Rogers, William A; Williams, Thomas M

    2011-01-01

    Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism's program for development, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE's activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites. This capability has made the quantitative measurement of gene and, relevant here, CRE activity feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at

  10. Splicing regulation: From a parts list of regulatory elements to an integrated splicing code

    PubMed Central

    Wang, Zefeng; Burge, Christopher B.

    2008-01-01

    Alternative splicing of pre-mRNAs is a major contributor to both proteomic diversity and control of gene expression levels. Splicing is tightly regulated in different tissues and developmental stages, and its disruption can lead to a wide range of human diseases. An important long-term goal in the splicing field is to determine a set of rules or “code” for splicing that will enable prediction of the splicing pattern of any primary transcript from its sequence. Outside of the core splice site motifs, the bulk of the information required for splicing is thought to be contained in exonic and intronic cis-regulatory elements that function by recruitment of sequence-specific RNA-binding protein factors that either activate or repress the use of adjacent splice sites. Here, we summarize the current state of knowledge of splicing cis-regulatory elements and their context-dependent effects on splicing, emphasizing recent global/genome-wide studies and open questions. PMID:18369186

  11. Transcription of Mammalian cis-Regulatory Elements Is Restrained by Actively Enforced Early Termination.

    PubMed

    Austenaa, Liv M I; Barozzi, Iros; Simonatto, Marta; Masella, Silvia; Della Chiara, Giulia; Ghisletti, Serena; Curina, Alessia; de Wit, Elzo; Bouwman, Britta A M; de Pretis, Stefano; Piccolo, Viviana; Termanini, Alberto; Prosperini, Elena; Pelizzola, Mattia; de Laat, Wouter; Natoli, Gioacchino

    2015-11-01

    Upon recruitment to active enhancers and promoters, RNA polymerase II (Pol II) generates short non-coding transcripts of unclear function. The mechanisms that control the length and the amount of ncRNAs generated by cis-regulatory elements are largely unknown. Here, we show that the adaptor protein WDR82 and its associated complexes actively limit such non-coding transcription. WDR82 targets the SET1 H3K4 methyltransferases and the nuclear protein phosphatase 1 (PP1) complexes to the initiating Pol II. WDR82 and PP1 also interact with components of the transcriptional termination and RNA processing machineries. Depletion of WDR82, SET1, or the PP1 subunit required for its nuclear import caused distinct but overlapping transcription termination defects at highly expressed genes and active enhancers and promoters, thus enabling the increased synthesis of unusually long ncRNAs. These data indicate that transcription initiated from cis-regulatory elements is tightly coordinated with termination mechanisms that impose the synthesis of short RNAs. PMID:26593720

  12. Identification of active transcriptional regulatory elements from GRO-seq data.

    PubMed

    Danko, Charles G; Hyland, Stephanie L; Core, Leighton J; Martins, Andre L; Waters, Colin T; Lee, Hyung Won; Cheung, Vivian G; Kraus, W Lee; Lis, John T; Siepel, Adam

    2015-05-01

    Modifications to the global run-on and sequencing (GRO-seq) protocol that enrich for 5'-capped RNAs can be used to reveal active transcriptional regulatory elements (TREs) with high accuracy. Here, we introduce discriminative regulatory-element detection from GRO-seq (dREG), a sensitive machine learning method that uses support vector regression to identify active TREs from GRO-seq data without requiring cap-based enrichment (https://github.com/Danko-Lab/dREG/). This approach allows TREs to be assayed together with gene expression levels and other transcriptional features in a single experiment. Predicted TREs are more enriched for several marks of transcriptional activation—including expression quantitative trait loci, disease-associated polymorphisms, acetylated histone 3 lysine 27 (H3K27ac) and transcription factor binding—than those identified by alternative functional assays. Using dREG, we surveyed TREs in eight human cell types and provide new insights into global patterns of TRE function. PMID:25799441

  13. Structure of Proximal and Distant Regulatory Elements in the Human Genome

    NASA Astrophysics Data System (ADS)

    Ovcharenko, Ivan

    Clustering of multiple transcription factor binding sites (TFBSs) for the same transcription factor (TF) is a common feature of cis-regulatory modules in invertebrate animals, but the occurrence of such homotypic clusters of TFBSs (HCTs) in the human genome has remained largely unknown. To explore whether HCTs are also common in human and other vertebrates, we used known binding motifs for vertebrate TFs and a hidden Markov model-based approach to detect HCTs in the human, mouse, chicken, and fugu genomes, and examined their association with cis-regulatory modules. We found that evolutionarily conserved HCTs occupy nearly 2% of the human genome, with experimental evidence for individual TFs supporting their binding to predicted HCTs. More than half of promoters of human genes contain HCTs, with a distribution around the transcription start site in agreement with the experimental data from the ENCODE project. In addition, almost half of 487 experimentally validated developmental enhancers contain them as well - a number more than 25-fold larger than expected by chance. We also found evidence of negative selection acting on TFBSs within HCTs, as the conservation of TFBSs is stronger than the conservation of sequences separating them. The important role of HCTs as components of developmental enhancers is additionally supported by a strong correlation between HCTs and the binding of the enhancer-associated co-activator protein p300. Experimental validation of HCT-containing elements in both zebrafish and mouse suggest that HCTs could be used to predict both the presence of enhancers and their tissue specificity, and are thus a feature that can be effectively used in deciphering the gene regulatory code. In conclusion, our results indicate that HCTs are a pervasive feature of human cis-regulatory modules and suggest that they play an important role in gene regulation in the human and other vertebrate genomes.

  14. Deciphering Cis-Regulatory Element Mediated Combinatorial Regulation in Rice under Blast Infected Condition.

    PubMed

    Deb, Arindam; Kundu, Sudip

    2015-01-01

    Combinations of cis-regulatory elements (CREs) present at the promoters facilitate the binding of several transcription factors (TFs), thereby altering the consequent gene expressions. Due to the eminent complexity of the regulatory mechanism, the combinatorics of CRE-mediated transcriptional regulation has been elusive. In this work, we have developed a new methodology that quantifies the co-occurrence tendencies of CREs present in a set of promoter sequences; these co-occurrence scores are filtered in three consecutive steps to test their statistical significance; and the significantly co-occurring CRE pairs are presented as networks. These networks of co-occurring CREs are further transformed to derive higher order of regulatory combinatorics. We have further applied this methodology on the differentially up-regulated gene-sets of rice tissues under fungal (Magnaporthe) infected conditions to demonstrate how it helps to understand the CRE-mediated combinatorial gene regulation. Our analysis includes a wide spectrum of biologically important results. The CRE pairs having a strong tendency to co-occur often exhibit very similar joint distribution patterns at the promoters of rice. We couple the network approach with experimental results of plant gene regulation and defense mechanisms and find evidences of auto and cross regulation among TF families, cross-talk among multiple hormone signaling pathways, similarities and dissimilarities in regulatory combinatorics between different tissues, etc. Our analyses have pointed a highly distributed nature of the combinatorial gene regulation facilitating an efficient alteration in response to fungal attack. All together, our proposed methodology could be an important approach in understanding the combinatorial gene regulation. It can be further applied to unravel the tissue and/or condition specific combinatorial gene regulation in other eukaryotic systems with the availability of annotated genomic sequences and suitable

  15. Deciphering Cis-Regulatory Element Mediated Combinatorial Regulation in Rice under Blast Infected Condition

    PubMed Central

    Deb, Arindam; Kundu, Sudip

    2015-01-01

    Combinations of cis-regulatory elements (CREs) present at the promoters facilitate the binding of several transcription factors (TFs), thereby altering the consequent gene expressions. Due to the eminent complexity of the regulatory mechanism, the combinatorics of CRE-mediated transcriptional regulation has been elusive. In this work, we have developed a new methodology that quantifies the co-occurrence tendencies of CREs present in a set of promoter sequences; these co-occurrence scores are filtered in three consecutive steps to test their statistical significance; and the significantly co-occurring CRE pairs are presented as networks. These networks of co-occurring CREs are further transformed to derive higher order of regulatory combinatorics. We have further applied this methodology on the differentially up-regulated gene-sets of rice tissues under fungal (Magnaporthe) infected conditions to demonstrate how it helps to understand the CRE-mediated combinatorial gene regulation. Our analysis includes a wide spectrum of biologically important results. The CRE pairs having a strong tendency to co-occur often exhibit very similar joint distribution patterns at the promoters of rice. We couple the network approach with experimental results of plant gene regulation and defense mechanisms and find evidences of auto and cross regulation among TF families, cross-talk among multiple hormone signaling pathways, similarities and dissimilarities in regulatory combinatorics between different tissues, etc. Our analyses have pointed a highly distributed nature of the combinatorial gene regulation facilitating an efficient alteration in response to fungal attack. All together, our proposed methodology could be an important approach in understanding the combinatorial gene regulation. It can be further applied to unravel the tissue and/or condition specific combinatorial gene regulation in other eukaryotic systems with the availability of annotated genomic sequences and suitable

  16. Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements

    SciTech Connect

    Tully, D.B.; Cidlowski, J.A. )

    1989-03-07

    Sucrose density gradient shift assays were used to study the interactions of human glucocorticoid receptors (GR) with small DNA fragments either containing or lacking glucocorticoid response element (GRE) DNA consensus sequences. When crude cytoplasmic extracts containing ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) labeled GR were incubated with unlabeled DNA under conditions of DNA excess, a GRE-containing DNA fragment obtained from the 5' long terminal repeat of mouse mammary tumor virus (MMTV LTR) formed a stable 12-16S complex with activated, but not nonactivated, ({sup 3}H)TA receptor. By contrast, if the cytosols were treated with calf thymus DNA-cellulose to deplete non-GR-DNA-binding proteins prior to heat activation, a smaller 7-10S complex was formed with the MMTV LTR DNA fragment. Activated ({sup 3}H)TA receptor from DNA-cellulose pretreated cytosols also interacted with two similarly sized fragments from pBR322 DNA. Stability of the complexes formed between GR and these three DNA fragments was strongly affected by even moderate alterations in either the salt concentration or the pH of the gradient buffer. Under all conditions tested, the complex formed with the MMTV LTR DNA fragment was more stable than the complexes formed with either of the pBR322 DNA fragments. Together these observations indicate that the formation of stable complexes between activated GR and isolated DNA fragments requires the presence of GRE consensus sequences in the DNA.

  17. Positive and negative regulatory elements mediating transcription from the Drosophila melanogaster actin 5C distal promoter.

    PubMed Central

    Chung, Y T; Keller, E B

    1990-01-01

    The major cytoskeletal actin gene of Drosophila melanogaster, the actin 5C gene, has two promoters, the distal one of which controls synthesis of actin in a tissue- and developmental stage-specific manner. This very strong promoter has widely been used for expression of heterologous genes in cultured cells. To locate functional regulatory elements in this distal promoter, mutants of the promoter were fused to the bacterial chloramphenicol acetyltransferase gene and assayed for transient expression activity in cultured Drosophila embryonic Schneider line 2 cells. The results showed that the upstream end of the promoter extends to 522 bp from the transcription start site. In addition, there are two remote activating regions about 2 kb upstream. Between -522 and -379 are two regions that exert a strong negative effect. Downstream from these negative regions are at least six positive regions and a TATA element. The strongest positive determinant of the promoter was identified at -320 as AAAATGTG by footprinting and by a replacement experiment. When the relevant region was replaced by a synthetic sequence containing this element in a random context, the transient expression activity was restored. The sequence TGTATG located at -355 was also identified as a positive element by a similar replacement approach. Apparently the very high activity of this promoter is the result of the combined activities of multiple factors. Images PMID:2123290

  18. Structural characterization and regulatory element analysis of the heart isoform of cytochrome c oxidase VIa

    NASA Technical Reports Server (NTRS)

    Wan, B.; Moreadith, R. W.; Blomqvist, C. G. (Principal Investigator)

    1995-01-01

    In order to investigate the mechanism(s) governing the striated muscle-specific expression of cytochrome c oxidase VIaH we have characterized the murine gene and analyzed its transcriptional regulatory elements in skeletal myogenic cell lines. The gene is single copy, spans 689 base pairs (bp), and is comprised of three exons. The 5'-ends of transcripts from the gene are heterogeneous, but the most abundant transcript includes a 5'-untranslated region of 30 nucleotides. When fused to the luciferase reporter gene, the 3.5-kilobase 5'-flanking region of the gene directed the expression of the heterologous protein selectively in differentiated Sol8 cells and transgenic mice, recapitulating the pattern of expression of the endogenous gene. Deletion analysis identified a 300-bp fragment sufficient to direct the myotube-specific expression of luciferase in Sol8 cells. The region lacks an apparent TATA element, and sequence motifs predicted to bind NRF-1, NRF-2, ox-box, or PPAR factors known to regulate other nuclear genes encoding mitochondrial proteins are not evident. Mutational analysis, however, identified two cis-elements necessary for the high level expression of the reporter protein: a MEF2 consensus element at -90 to -81 bp and an E-box element at -147 to -142 bp. Additional E-box motifs at closely located positions were mutated without loss of transcriptional activity. The dependence of transcriptional activation of cytochrome c oxidase VIaH on cis-elements similar to those found in contractile protein genes suggests that the striated muscle-specific expression is coregulated by mechanisms that control the lineage-specific expression of several contractile and cytosolic proteins.

  19. Regulatory T Cell DNA Methyltransferase Inhibition Accelerates Resolution of Lung Inflammation

    PubMed Central

    Singer, Benjamin D.; Mock, Jason R.; Aggarwal, Neil R.; Garibaldi, Brian T.; Sidhaye, Venkataramana K.; Florez, Marcus A.; Chau, Eric; Gibbs, Kevin W.; Mandke, Pooja; Tripathi, Ashutosh; Yegnasubramanian, Srinivasan; King, Landon S.

    2015-01-01

    Acute respiratory distress syndrome (ARDS) is a common and often fatal inflammatory lung condition without effective targeted therapies. Regulatory T cells (Tregs) resolve lung inflammation, but mechanisms that enhance Tregs to promote resolution of established damage remain unknown. DNA demethylation at the forkhead box protein 3 (Foxp3) locus and other key Treg loci typify the Treg lineage. To test how dynamic DNA demethylation affects lung injury resolution, we administered the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) to wild-type (WT) mice beginning 24 hours after intratracheal LPS-induced lung injury. Mice that received DAC exhibited accelerated resolution of their injury. Lung CD4+CD25hiFoxp3+ Tregs from DAC-treated WT mice increased in number and displayed enhanced Foxp3 expression, activation state, suppressive phenotype, and proliferative capacity. Lymphocyte-deficient recombinase activating gene-1–null mice and Treg-depleted (diphtheria toxin-treated Foxp3DTR) mice did not resolve their injury in response to DAC. Adoptive transfer of 2 × 105 DAC-treated, but not vehicle-treated, exogenous Tregs rescued Treg-deficient mice from ongoing lung inflammation. In addition, in WT mice with influenza-induced lung inflammation, DAC rescue treatment facilitated recovery of their injury and promoted an increase in lung Treg number. Thus, DNA methyltransferase inhibition, at least in part, augments Treg number and function to accelerate repair of experimental lung injury. Epigenetic pathways represent novel manipulable targets for the treatment of ARDS. PMID:25295995

  20. Identification of two regulatory elements controlling Fucosyltransferase 7 transcription in murine CD4+ T cells.

    PubMed

    Pink, Matthias; Ratsch, Boris A; Mardahl, Maibritt; Schröter, Micha F; Engelbert, Dirk; Triebus, Julia; Hamann, Alf; Syrbe, Uta

    2014-11-01

    Fucosyltransferase VII encoded by the gene Fut7 is essential in CD4(+) T cells for the generation of E- and P-selectin ligands (E- and P-lig) which facilitate recruitment of lymphocytes into inflamed tissues and into the skin. This study aimed to identify regulatory elements controlling the inducible Fut7 expression in CD4(+) T cells that occurs upon activation and differentiation of naive T cells into effector cells. Comparative analysis of the histone modification pattern in non-hematopoetic cells and CD4(+) T cell subsets revealed a differential histone modification pattern within the Fut7 locus including a conserved non-coding sequence (CNS) identified by cross-species conservation comparison suggesting that regulatory elements are confined to this region. Cloning of the CNS located about 500 bp upstream of the Fut7 locus, into a luciferase reporter vector elicited reporter activity after transfection of the αβ-WT T cell line, but not after transfection of primary murine CD4(+) Th1 cells. As quantification of different Fut7 transcripts revealed a predominance of transcripts lacking the first exons in primary Th1 cells we searched for an alternative promoter. Cloning of an intragenic region spanning a 1kb region upstream of exon 4 into an enhancer-containing vector indeed elicited promoter activity. Interestingly, also the CNS enhanced activity of this intragenic minimal promoter in reporter assays in primary Th1 cells suggesting that both elements interact in primary CD4(+) T cells to induce Fut7 transcription. PMID:24915132

  1. Putative cis-Regulatory Elements Associated with Heat Shock Genes Activated During Excystation of Cryptosporidium parvum

    PubMed Central

    Lara, Ana M.; Serrano, Myrna; Sheth, Nihar; Buck, Gregory

    2010-01-01

    Background Cryptosporidiosis is a ubiquitous infectious disease, caused by the protozoan parasites Cryptosporidium hominis and C. parvum, leading to acute, persistent and chronic diarrhea worldwide. Although the complications of this disease can be serious, even fatal, in immunocompromised patients of any age, they have also been found to lead to long term effects, including growth inhibition and impaired cognitive development, in infected immunocompetent children. The Cryptosporidium life cycle alternates between a dormant stage, the oocyst, and a highly replicative phase that includes both asexual vegetative stages as well as sexual stages, implying fine genetic regulatory mechanisms. The parasite is extremely difficult to study because it cannot be cultured in vitro and animal models are equally challenging. The recent publication of the genome sequence of C. hominis and C. parvum has, however, significantly advanced our understanding of the biology and pathogenesis of this parasite. Methodology/Principal Findings Herein, our goal was to identify cis-regulatory elements associated with heat shock response in Cryptosporidium using a combination of in silico and real time RT-PCR strategies. Analysis with Gibbs-Sampling algorithms of upstream non-translated regions of twelve genes annotated as heat shock proteins in the Cryptosporidium genome identified a highly conserved over-represented sequence motif in eleven of them. RT-PCR analyses, described herein and also by others, show that these eleven genes bearing the putative element are induced concurrent with excystation of parasite oocysts via heat shock. Conclusions/Significance Our analyses suggest that occurrences of a motif identified in the upstream regions of the Cryptosporidium heat shock genes represent parts of the transcriptional apparatus and function as stress response elements that activate expression of these genes during excystation, and possibly at other stages in the life cycle of the parasite

  2. Genome-Wide Identification of Regulatory Elements and Reconstruction of Gene Regulatory Networks of the Green Alga Chlamydomonas reinhardtii under Carbon Deprivation

    PubMed Central

    Vischi Winck, Flavia; Arvidsson, Samuel; Riaño-Pachón, Diego Mauricio; Hempel, Sabrina; Koseska, Aneta; Nikoloski, Zoran; Urbina Gomez, David Alejandro; Rupprecht, Jens; Mueller-Roeber, Bernd

    2013-01-01

    The unicellular green alga Chlamydomonas reinhardtii is a long-established model organism for studies on photosynthesis and carbon metabolism-related physiology. Under conditions of air-level carbon dioxide concentration [CO2], a carbon concentrating mechanism (CCM) is induced to facilitate cellular carbon uptake. CCM increases the availability of carbon dioxide at the site of cellular carbon fixation. To improve our understanding of the transcriptional control of the CCM, we employed FAIRE-seq (formaldehyde-assisted Isolation of Regulatory Elements, followed by deep sequencing) to determine nucleosome-depleted chromatin regions of algal cells subjected to carbon deprivation. Our FAIRE data recapitulated the positions of known regulatory elements in the promoter of the periplasmic carbonic anhydrase (Cah1) gene, which is upregulated during CCM induction, and revealed new candidate regulatory elements at a genome-wide scale. In addition, time series expression patterns of 130 transcription factor (TF) and transcription regulator (TR) genes were obtained for cells cultured under photoautotrophic condition and subjected to a shift from high to low [CO2]. Groups of co-expressed genes were identified and a putative directed gene-regulatory network underlying the CCM was reconstructed from the gene expression data using the recently developed IOTA (inner composition alignment) method. Among the candidate regulatory genes, two members of the MYB-related TF family, Lcr1 (Low-CO2 response regulator 1) and Lcr2 (Low-CO2 response regulator 2), may play an important role in down-regulating the expression of a particular set of TF and TR genes in response to low [CO2]. The results obtained provide new insights into the transcriptional control of the CCM and revealed more than 60 new candidate regulatory genes. Deep sequencing of nucleosome-depleted genomic regions indicated the presence of new, previously unknown regulatory elements in the C. reinhardtii genome. Our work can

  3. Identification and characterization of DNA sequences that prevent glucocorticoid receptor binding to nearby response elements.

    PubMed

    Telorac, Jonas; Prykhozhij, Sergey V; Schöne, Stefanie; Meierhofer, David; Sauer, Sascha; Thomas-Chollier, Morgane; Meijsing, Sebastiaan H

    2016-07-27

    Out of the myriad of potential DNA binding sites of the glucocorticoid receptor (GR) found in the human genome, only a cell-type specific minority is actually bound, indicating that the presence of a recognition sequence alone is insufficient to specify where GR binds. Cooperative interactions with other transcription factors (TFs) are known to contribute to binding specificity. Here, we reasoned that sequence signals preventing GR recruitment to certain loci provide an alternative means to confer specificity. Motif analyses uncovered candidate Negative Regulatory Sequences (NRSs) that interfere with genomic GR binding. Subsequent functional analyses demonstrated that NRSs indeed prevent GR binding to nearby response elements. We show that NRS activity is conserved across species, found in most tissues and that they also interfere with the genomic binding of other TFs. Interestingly, the effects of NRSs appear not to be a simple consequence of changes in chromatin accessibility. Instead, we find that NRSs interact with proteins found at sub-nuclear structures called paraspeckles and that these proteins might mediate the repressive effects of NRSs. Together, our studies suggest that the joint influence of positive and negative sequence signals partition the genome into regions where GR can bind and those where it cannot. PMID:27016732

  4. DNA methylation of distal regulatory sites characterizes dysregulation of cancer genes

    PubMed Central

    2013-01-01

    Background Abnormal epigenetic marking is well documented in gene promoters of cancer cells, but the study of distal regulatory siteshas lagged behind.We performed a systematic analysis of DNA methylation sites connected with gene expression profilesacross normal and cancerous human genomes. Results Utilizing methylation and expression data in 58 cell types, we developed a model for methylation-expression relationships in gene promoters and extrapolated it to the genome. We mapped numerous sites at which DNA methylation was associated with expression of distal genes. These sites bind transcription factors in a methylation-dependent manner, and carry the chromatin marks of a particular class of transcriptional enhancers. In contrast to the traditional model of one enhancer site per cell type, we found that single enhancer sites may define gradients of expression levels across many different cell types. Strikingly, the identified sites were drastically altered in cancers: hypomethylated enhancer sites associated with upregulation of cancer-related genes and hypermethylated sites with downregulation. Moreover, the association between enhancer methylation and gene deregulation in cancerwas significantly stronger than the association of promoter methylationwith gene deregulation. Conclusions Methylation of distal regulatory sites is closely related to gene expression levels across the genome. Single enhancers may modulate ranges of cell-specific transcription levels, from constantlyopen promoters. In contrast to the remote relationships between promoter methylation and gene dysregulation in cancer, altered methylation of enhancer sites is closely related to gene expression profiles of transformed cells. PMID:23497655

  5. cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication.

    PubMed Central

    Hartl, M; Willnow, T; Fanning, E

    1990-01-01

    Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA. Images PMID:2159549

  6. Functional characterization of transcriptional regulatory elements in the upstream region of the yeast GLK1 gene.

    PubMed Central

    Herrero, P; Flores, L; de la Cera, T; Moreno, F

    1999-01-01

    The glucokinase gene GLK1 of the yeast Saccharomyces cerevisiae is transcriptionally regulated in response to the carbon source of the growth medium. Northern-blot analysis shows that the GLK1 gene is expressed at a basal level in the presence of glucose, de-repressed more than 6-fold under conditions of sugar limitation and more than 25-fold under conditions of ethanol induction. lacZ fusions of the GLK1 gene promoter were constructed and a deletion analysis was performed in order to identify the cis-acting regulatory elements of the promoter that controls GLK1 gene expression. First, the expression seemed to be mediated mainly by one GCR1 and three stress-responsive element (STRE) activating elements. Secondly, an ethanol repression autoregulation (ERA)/twelve-fold TA repeat (TAB) repressor element was identified within the promoter region of the GLK1 gene. A specific and differential protein binding to the STRE was observed with extracts from de-repressed and repressed cells. No differential binding to the GCR1 or ERA/TAB elements was observed with extracts from de-repressed and repressed cells, but, in both cases, the binding was competed for by an excess of the unlabelled GLK1(GCR1) and GLK1(ERA) sequence. The transcription factors Msn2 and Msn4, which bind to the GLK1 upstream region through the STRE, contribute to inductive activation. The transcription factor Gcr1, which binds through the GCR1 element, contributes to constitutive activation. In order to achieve the severe glucose repression of GLK1, constitutive repressor factors acting through the ERA/TAB element must counteract constitutive activation generated by Gcr1 binding to the GCR1 element. Full expression of the GLK1 gene is produced by inductive activation of three STRE when Msn2 and Msn4 proteins are translocated to the nucleus by covalent modification. The combinatorial effect of the entire region leads to the regulated transcription of GLK1, i.e., silent in media with glucose and other

  7. Palindromic repetitive DNA elements with coding potential in Methanocaldococcus jannaschii.

    PubMed

    Suyama, Mikita; Lathe, Warren C; Bork, Peer

    2005-10-10

    We have identified 141 novel palindromic repetitive elements in the genome of euryarchaeon Methanocaldococcus jannaschii. The total length of these elements is 14.3kb, which corresponds to 0.9% of the total genomic sequence and 6.3% of all extragenic regions. The elements can be divided into three groups (MJRE1-3) based on the sequence similarity. The low sequence identity within each of the groups suggests rather old origin of these elements in M. jannaschii. Three MJRE2 elements were located within the protein coding regions without disrupting the coding potential of the host genes, indicating that insertion of repeats might be a widespread mechanism to enhance sequence diversity in coding regions. PMID:16182294

  8. A transcriptional regulatory element in the coding sequence of the human Bcl-2 gene

    PubMed Central

    Lang, Georgina; Gombert, Wendy M; Gould, Hannah J

    2005-01-01

    We investigated the protein-binding sites in a DNAse I hypersensitive site associated with bcl-2 gene expression in human B cells. We mapped this hypersensitive site to the coding sequence of exon 2 of the bcl-2 gene in the bcl-2-expressing REH B-cell line. Electrophoretic mobility shift assays (EMSAs) with extracts from REH cells revealed three previously unrecognized B-Myb-binding sites in this sequence. The protein was identified as B-Myb by using a specific antibody and EMSAs. Accordingly, the levels of B-Myb and bcl-2 proteins, and of Myb EMSA activity, were correlated over a wide range of cell lines, representing different stages of B-cell development. Transfection of REH cells with antisense B-myb down-regulated EMSA activity and the level of bcl-2, and led to the apoptosis of REH cells. Transfection of the bcl-2-non-expressing RPMI 8226 cell line with a B-Myb expression vector induced B-Myb EMSA activity and the expression of bcl-2. Reporter assays indicated that the HSS8 sequence containing the three B-Myb sites may act as an enhancer when it is linked to the bcl-2 gene promoter. Interaction of B-Myb with HSS8 may enhance bcl-2 gene expression by co-operating with positive regulatory elements (e.g. previously identified B-Myb response elements) or silencing negative response elements in the bcl-2 gene promoter. PMID:15606792

  9. The Drosophila P-element KP repressor protein dimerizes and interacts with multiple sites on P-element DNA.

    PubMed

    Lee, C C; Mul, Y M; Rio, D C

    1996-10-01

    Drosophila P elements are mobile DNA elements that encode an 87-kDa transposase enzyme and transpositional repressor proteins. One of these repressor proteins is the 207-amino-acid KP protein which is encoded by a naturally occurring P element with an internal deletion. To study the molecular mechanisms by which KP represses transposition, the protein was expressed, purified, and characterized. We show that the KP protein binds to multiple sites on the ends of P-element DNA, unlike the full-length transposase protein. These sites include the high-affinity transposase binding site, an 11-bp transpositional enhancer, and, at the highest concentrations tested, the terminal 31-hp inverted repeats. The DNA binding domain was localized to the N-terminal 98 amino acids and contains a CCHC sequence, a potential metal binding motif. We also demonstrate that the KP repressor protein can dimerize and contains two protein-protein interaction regions and that this dimerization is essential for high-affinity DNA binding. PMID:8816474

  10. The Drosophila P-element KP repressor protein dimerizes and interacts with multiple sites on P-element DNA.

    PubMed Central

    Lee, C C; Mul, Y M; Rio, D C

    1996-01-01

    Drosophila P elements are mobile DNA elements that encode an 87-kDa transposase enzyme and transpositional repressor proteins. One of these repressor proteins is the 207-amino-acid KP protein which is encoded by a naturally occurring P element with an internal deletion. To study the molecular mechanisms by which KP represses transposition, the protein was expressed, purified, and characterized. We show that the KP protein binds to multiple sites on the ends of P-element DNA, unlike the full-length transposase protein. These sites include the high-affinity transposase binding site, an 11-bp transpositional enhancer, and, at the highest concentrations tested, the terminal 31-hp inverted repeats. The DNA binding domain was localized to the N-terminal 98 amino acids and contains a CCHC sequence, a potential metal binding motif. We also demonstrate that the KP repressor protein can dimerize and contains two protein-protein interaction regions and that this dimerization is essential for high-affinity DNA binding. PMID:8816474

  11. A comparative encyclopedia of DNA elements in the mouse genome.

    PubMed

    Yue, Feng; Cheng, Yong; Breschi, Alessandra; Vierstra, Jeff; Wu, Weisheng; Ryba, Tyrone; Sandstrom, Richard; Ma, Zhihai; Davis, Carrie; Pope, Benjamin D; Shen, Yin; Pervouchine, Dmitri D; Djebali, Sarah; Thurman, Robert E; Kaul, Rajinder; Rynes, Eric; Kirilusha, Anthony; Marinov, Georgi K; Williams, Brian A; Trout, Diane; Amrhein, Henry; Fisher-Aylor, Katherine; Antoshechkin, Igor; DeSalvo, Gilberto; See, Lei-Hoon; Fastuca, Meagan; Drenkow, Jorg; Zaleski, Chris; Dobin, Alex; Prieto, Pablo; Lagarde, Julien; Bussotti, Giovanni; Tanzer, Andrea; Denas, Olgert; Li, Kanwei; Bender, M A; Zhang, Miaohua; Byron, Rachel; Groudine, Mark T; McCleary, David; Pham, Long; Ye, Zhen; Kuan, Samantha; Edsall, Lee; Wu, Yi-Chieh; Rasmussen, Matthew D; Bansal, Mukul S; Kellis, Manolis; Keller, Cheryl A; Morrissey, Christapher S; Mishra, Tejaswini; Jain, Deepti; Dogan, Nergiz; Harris, Robert S; Cayting, Philip; Kawli, Trupti; Boyle, Alan P; Euskirchen, Ghia; Kundaje, Anshul; Lin, Shin; Lin, Yiing; Jansen, Camden; Malladi, Venkat S; Cline, Melissa S; Erickson, Drew T; Kirkup, Vanessa M; Learned, Katrina; Sloan, Cricket A; Rosenbloom, Kate R; Lacerda de Sousa, Beatriz; Beal, Kathryn; Pignatelli, Miguel; Flicek, Paul; Lian, Jin; Kahveci, Tamer; Lee, Dongwon; Kent, W James; Ramalho Santos, Miguel; Herrero, Javier; Notredame, Cedric; Johnson, Audra; Vong, Shinny; Lee, Kristen; Bates, Daniel; Neri, Fidencio; Diegel, Morgan; Canfield, Theresa; Sabo, Peter J; Wilken, Matthew S; Reh, Thomas A; Giste, Erika; Shafer, Anthony; Kutyavin, Tanya; Haugen, Eric; Dunn, Douglas; Reynolds, Alex P; Neph, Shane; Humbert, Richard; Hansen, R Scott; De Bruijn, Marella; Selleri, Licia; Rudensky, Alexander; Josefowicz, Steven; Samstein, Robert; Eichler, Evan E; Orkin, Stuart H; Levasseur, Dana; Papayannopoulou, Thalia; Chang, Kai-Hsin; Skoultchi, Arthur; Gosh, Srikanta; Disteche, Christine; Treuting, Piper; Wang, Yanli; Weiss, Mitchell J; Blobel, Gerd A; Cao, Xiaoyi; Zhong, Sheng; Wang, Ting; Good, Peter J; Lowdon, Rebecca F; Adams, Leslie B; Zhou, Xiao-Qiao; Pazin, Michael J; Feingold, Elise A; Wold, Barbara; Taylor, James; Mortazavi, Ali; Weissman, Sherman M; Stamatoyannopoulos, John A; Snyder, Michael P; Guigo, Roderic; Gingeras, Thomas R; Gilbert, David M; Hardison, Ross C; Beer, Michael A; Ren, Bing

    2014-11-20

    The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases. PMID:25409824

  12. A Comparative Encyclopedia of DNA Elements in the Mouse Genome

    PubMed Central

    Yue, Feng; Cheng, Yong; Breschi, Alessandra; Vierstra, Jeff; Wu, Weisheng; Ryba, Tyrone; Sandstrom, Richard; Ma, Zhihai; Davis, Carrie; Pope, Benjamin D.; Shen, Yin; Pervouchine, Dmitri D.; Djebali, Sarah; Thurman, Bob; Kaul, Rajinder; Rynes, Eric; Kirilusha, Anthony; Marinov, Georgi K.; Williams, Brian A.; Trout, Diane; Amrhein, Henry; Fisher-Aylor, Katherine; Antoshechkin, Igor; DeSalvo, Gilberto; See, Lei-Hoon; Fastuca, Meagan; Drenkow, Jorg; Zaleski, Chris; Dobin, Alex; Prieto, Pablo; Lagarde, Julien; Bussotti, Giovanni; Tanzer, Andrea; Denas, Olgert; Li, Kanwei; Bender, M. A.; Zhang, Miaohua; Byron, Rachel; Groudine, Mark T.; McCleary, David; Pham, Long; Ye, Zhen; Kuan, Samantha; Edsall, Lee; Wu, Yi-Chieh; Rasmussen, Matthew D.; Bansal, Mukul S.; Keller, Cheryl A.; Morrissey, Christapher S.; Mishra, Tejaswini; Jain, Deepti; Dogan, Nergiz; Harris, Robert S.; Cayting, Philip; Kawli, Trupti; Boyle, Alan P.; Euskirchen, Ghia; Kundaje, Anshul; Lin, Shin; Lin, Yiing; Jansen, Camden; Malladi, Venkat S.; Cline, Melissa S.; Erickson, Drew T.; Kirkup, Vanessa M; Learned, Katrina; Sloan, Cricket A.; Rosenbloom, Kate R.; de Sousa, Beatriz Lacerda; Beal, Kathryn; Pignatelli, Miguel; Flicek, Paul; Lian, Jin; Kahveci, Tamer; Lee, Dongwon; Kent, W. James; Santos, Miguel Ramalho; Herrero, Javier; Notredame, Cedric; Johnson, Audra; Vong, Shinny; Lee, Kristen; Bates, Daniel; Neri, Fidencio; Diegel, Morgan; Canfield, Theresa; Sabo, Peter J.; Wilken, Matthew S.; Reh, Thomas A.; Giste, Erika; Shafer, Anthony; Kutyavin, Tanya; Haugen, Eric; Dunn, Douglas; Reynolds, Alex P.; Neph, Shane; Humbert, Richard; Hansen, R. Scott; De Bruijn, Marella; Selleri, Licia; Rudensky, Alexander; Josefowicz, Steven; Samstein, Robert; Eichler, Evan E.; Orkin, Stuart H.; Levasseur, Dana; Papayannopoulou, Thalia; Chang, Kai-Hsin; Skoultchi, Arthur; Gosh, Srikanta; Disteche, Christine; Treuting, Piper; Wang, Yanli; Weiss, Mitchell J.; Blobel, Gerd A.; Good, Peter J.; Lowdon, Rebecca F.; Adams, Leslie B.; Zhou, Xiao-Qiao; Pazin, Michael J.; Feingold, Elise A.; Wold, Barbara; Taylor, James; Kellis, Manolis; Mortazavi, Ali; Weissman, Sherman M.; Stamatoyannopoulos, John; Snyder, Michael P.; Guigo, Roderic; Gingeras, Thomas R.; Gilbert, David M.; Hardison, Ross C.; Beer, Michael A.; Ren, Bing

    2014-01-01

    Summary As the premier model organism in biomedical research, the laboratory mouse shares the majority of protein-coding genes with humans, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications, and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of other sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases. PMID:25409824

  13. Functional conservation of cis-regulatory elements of heat-shock genes over long evolutionary distances.

    PubMed

    He, Zhengying; Eichel, Kelsie; Ruvinsky, Ilya

    2011-01-01

    Transcriptional control of gene regulation is an intricate process that requires precise orchestration of a number of molecular components. Studying its evolution can serve as a useful model for understanding how complex molecular machines evolve. One way to investigate evolution of transcriptional regulation is to test the functions of cis-elements from one species in a distant relative. Previous results suggested that few, if any, tissue-specific promoters from Drosophila are faithfully expressed in C. elegans. Here we show that, in contrast, promoters of fly and human heat-shock genes are upregulated in C. elegans upon exposure to heat. Inducibility under conditions of heat shock may represent a relatively simple "on-off" response, whereas complex expression patterns require integration of multiple signals. Our results suggest that simpler aspects of regulatory logic may be retained over longer periods of evolutionary time, while more complex ones may be diverging more rapidly. PMID:21799932

  14. Crystal Structure of the Lysine Riboswitch Regulatory mRNA Element*S⃞

    PubMed Central

    Garst, Andrew D.; Héroux, Annie; Rambo, Robert P.; Batey, Robert T.

    2008-01-01

    Riboswitches are metabolite-sensitive elements found in mRNAs that control gene expression through a regulatory secondary structural switch. Along with regulation of lysine biosynthetic genes, mutations within the lysine-responsive riboswitch (L-box) play a role in the acquisition of resistance to antimicrobial lysine analogs. To understand the structural basis for lysine binding, we have determined the 2.8Å resolution crystal structure of lysine bound to the Thermotoga maritima asd lysine riboswitch ligand-binding domain. The structure reveals a complex architecture scaffolding a binding pocket completely enveloping lysine. Mutations conferring antimicrobial resistance cluster around this site as well as highly conserved long range interactions, indicating that they disrupt lysine binding or proper folding of the RNA. Comparison of the free and bound forms by x-ray crystallography, small angle x-ray scattering, and chemical probing reveals almost identical structures, indicating that lysine induces only limited and local conformational changes upon binding. PMID:18593706

  15. Crystal structure of the lysine riboswitch regulatory mRNA element.

    PubMed

    Garst, Andrew D; Héroux, Annie; Rambo, Robert P; Batey, Robert T

    2008-08-15

    Riboswitches are metabolite-sensitive elements found in mRNAs that control gene expression through a regulatory secondary structural switch. Along with regulation of lysine biosynthetic genes, mutations within the lysine-responsive riboswitch (L-box) play a role in the acquisition of resistance to antimicrobial lysine analogs. To understand the structural basis for lysine binding, we have determined the 2.8 angstroms resolution crystal structure of lysine bound to the Thermotoga maritima asd lysine riboswitch ligand-binding domain. The structure reveals a complex architecture scaffolding a binding pocket completely enveloping lysine. Mutations conferring antimicrobial resistance cluster around this site as well as highly conserved long range interactions, indicating that they disrupt lysine binding or proper folding of the RNA. Comparison of the free and bound forms by x-ray crystallography, small angle x-ray scattering, and chemical probing reveals almost identical structures, indicating that lysine induces only limited and local conformational changes upon binding. PMID:18593706

  16. Crystal Structure of the Lysine Riboswitch Regulatory mRNA Element

    SciTech Connect

    Garst, A.; Heroux, A; Rambo, R; Batey, R

    2008-01-01

    Riboswitches are metabolite-sensitive elements found in mRNAs that control gene expression through a regulatory secondary structural switch. Along with regulation of lysine biosynthetic genes, mutations within the lysine-responsive riboswitch (L-box) play a role in the acquisition of resistance to antimicrobial lysine analogs. To understand the structural basis for lysine binding, we have determined the 2.8{angstrom} resolution crystal structure of lysine bound to the Thermotoga maritima asd lysine riboswitch ligand-binding domain. The structure reveals a complex architecture scaffolding a binding pocket completely enveloping lysine. Mutations conferring antimicrobial resistance cluster around this site as well as highly conserved long range interactions, indicating that they disrupt lysine binding or proper folding of the RNA. Comparison of the free and bound forms by x-ray crystallography, small angle x-ray scattering, and chemical probing reveals almost identical structures, indicating that lysine induces only limited and local conformational changes upon binding.

  17. Long Terminal Repeats: From Parasitic Elements to Building Blocks of the Transcriptional Regulatory Repertoire.

    PubMed

    Thompson, Peter J; Macfarlan, Todd S; Lorincz, Matthew C

    2016-06-01

    The life cycle of endogenous retroviruses (ERVs), also called long terminal repeat (LTR) retrotransposons, begins with transcription by RNA polymerase II followed by reverse transcription and re-integration into the host genome. While most ERVs are relics of ancient integration events, "young" proviruses competent for retrotransposition-found in many mammals, but not humans-represent an ongoing threat to host fitness. As a consequence, several restriction pathways have evolved to suppress their activity at both transcriptional and post-transcriptional stages of the viral life cycle. Nevertheless, accumulating evidence has revealed that LTR sequences derived from distantly related ERVs have been exapted as regulatory sequences for many host genes in a wide range of cell types throughout mammalian evolution. Here, we focus on emerging themes from recent studies cataloging the diversity of ERV LTRs acting as important transcriptional regulatory elements in mammals and explore the molecular features that likely account for LTR exaptation in developmental and tissue-specific gene regulation. PMID:27259207

  18. Identification of Regulatory Mutations in SERPINC1 Affecting Vitamin D Response Elements Associated with Antithrombin Deficiency

    PubMed Central

    Toderici, Mara; de la Morena-Barrio, María Eugenia; Padilla, José; Miñano, Antonia; Antón, Ana Isabel; Iniesta, Juan Antonio; Herranz, María Teresa; Fernández, Nuria; Vicente, Vicente; Corral, Javier

    2016-01-01

    Antithrombin is a crucial anticoagulant serpin whose even moderate deficiency significantly increases the risk of thrombosis. Most cases with antithrombin deficiency carried genetic defects affecting exons or flanking regions of SERPINC1.We aimed to identify regulatory mutations inSERPINC1 through sequencing the promoter, intron 1 and 2 of this gene in 23 patients with antithrombin deficiency but without known genetic defects. Three cases with moderate antithrombin deficiency (63–78%) carried potential regulatory mutations. One located 200 bp before the initiation ATG and two in intron 1. These mutations disrupted two out of five potential vitamin D receptor elements (VDRE) identified in SERPINC1 with different software. One genetic defect, c.42-1060_-1057dupTTGA, was a new low prevalent polymorphism (MAF: 0.01) with functional consequences on plasma antithrombin levels. The relevance of the vitamin D pathway on the regulation of SERPINC1 was confirmed in a cell model. Incubation of HepG2 with paricalcitol, a vitamin D analog, increased dose-dependently the levels of SERPINC1transcripts and antithrombin released to the conditioned medium. This study shows further evidence of the transcriptional regulation of SERPINC1 by vitamin D and first describes the functional and pathological relevance of mutations affecting VDRE of this gene. Our study opens new perspectives in the search of new genetic defects involved in antithrombin deficiency and the risk of thrombosis as well as in the design of new antithrombotic treatments. PMID:27003919

  19. Functionally conserved cis-regulatory elements of COL18A1 identified through zebrafish transgenesis.

    PubMed

    Kague, Erika; Bessling, Seneca L; Lee, Josephine; Hu, Gui; Passos-Bueno, Maria Rita; Fisher, Shannon

    2010-01-15

    Type XVIII collagen is a component of basement membranes, and expressed prominently in the eye, blood vessels, liver, and the central nervous system. Homozygous mutations in COL18A1 lead to Knobloch Syndrome, characterized by ocular defects and occipital encephalocele. However, relatively little has been described on the role of type XVIII collagen in development, and nothing is known about the regulation of its tissue-specific expression pattern. We have used zebrafish transgenesis to identify and characterize cis-regulatory sequences controlling expression of the human gene. Candidate enhancers were selected from non-coding sequence associated with COL18A1 based on sequence conservation among mammals. Although these displayed no overt conservation with orthologous zebrafish sequences, four regions nonetheless acted as tissue-specific transcriptional enhancers in the zebrafish embryo, and together recapitulated the major aspects of col18a1 expression. Additional post-hoc computational analysis on positive enhancer sequences revealed alignments between mammalian and teleost sequences, which we hypothesize predict the corresponding zebrafish enhancers; for one of these, we demonstrate functional overlap with the orthologous human enhancer sequence. Our results provide important insight into the biological function and regulation of COL18A1, and point to additional sequences that may contribute to complex diseases involving COL18A1. More generally, we show that combining functional data with targeted analyses for phylogenetic conservation can reveal conserved cis-regulatory elements in the large number of cases where computational alignment alone falls short. PMID:19895802

  20. Genetic Analysis of Transvection Effects Involving Cis-Regulatory Elements of the Drosophila Ultrabithorax Gene

    PubMed Central

    Micol, J. L.; Castelli-Gair, J. E.; Garcia-Bellido, A.

    1990-01-01

    The Ultrabithorax (Ubx) gene of Drosophila melanogaster contains two functionally distinguishable regions: the protein-coding Ubx transcription unit and, upstream of it, the transcribed but non-protein-coding bxd region. Numerous recessive, partial loss-of-function mutations which appear to be regulatory mutations map within the bxd region and within the introns of the Ubx transcription unit. In addition, mutations within the Ubx unit exons are known and most of these behave as null alleles. Ubx(1) is one such allele. We have confirmed that, although the Ubx(1) allele does not produce detectable Ubx proteins (UBX), it does retain other genetic functions detectable by their effects on the expression of a paired, homologous Ubx allele, i.e., by transvection. We have extended previous analyses made by E. B. Lewis by mapping the critical elements of the Ubx gene which participate in transvection effects. Our results show that the Ubx(1) allele retains wild-type functions whose effectiveness can be reduced (1) by additional cis mutations in the bxd region or in introns of the Ubx transcription unit, as well as (2) by rearrangements disturbing pairing between homologous Ubx genes. Our results suggest that those remnant functions in Ubx(1) are able to modulate the activity of the allele located in the homologous chromosome. We discuss the normal cis regulatory role of these functions involved in trans interactions between homologous Ubx genes, as well as the implications of our results for the current models on transvection. PMID:2123161

  1. Sterol regulatory element binding protein-1 (SREBP-1)c promoter: Characterization and transcriptional regulation by mature SREBP-1 and liver X receptor α in goat mammary epithelial cells.

    PubMed

    Xu, H F; Luo, J; Wang, H P; Wang, H; Zhang, T Y; Tian, H B; Yao, D W; Loor, J J

    2016-02-01

    Sterol regulatory element binding protein-1 (SREBP-1) is a key transcription factor that regulates lipogenesis in rodent liver. Two isoforms (SREBP-1a and SREBP-1c) of SREBP-1 are transcribed by an alternative promoter on the same gene (SREBF1), and the isoforms differ only in their first exon. Although the regulatory effects of SREBP-1 on lipid and milk fat synthesis have received much attention in ruminants, SREBP-1c promoter and its regulatory mechanisms have not been characterized in the goat. In the present study, we cloned and sequenced a 2,012-bp fragment of the SREBP-1c 5'-flanking region from goat genomic DNA. A luciferase reporter assay revealed that SREBP-1c is transcriptionally activated by the liver X receptor α (LXRα) agonist T0901317, and is decreased by SREBP-1 small interfering (si)RNA. A 5' deletion analysis revealed a core promoter region located -395 to +1 bp upstream of the transcriptional start site (TSS). Site-directed mutagenesis of LXRα binding elements (LXRE1 and LXRE2) and sterol regulatory elements (SRE1 and SRE2) revealed that the full effects of T 4506585 require the presence of both LXRE and SRE. We also characterized a new SRE (SRE1) and demonstrated a direct role of SREBP-1 (auto-loop regulation) in maintaining its basal transcription activity. Results suggest that goat SREBP-1c gene is transcriptionally regulated by mature SREBP-1 (auto-loop circuit regulation) and LXRα in goat mammary epithelial cells. PMID:26709176

  2. Mesoscale Modeling Reveals Hierarchical Looping of Chromatin Fibers Near Gene Regulatory Elements.

    PubMed

    Bascom, Gavin D; Sanbonmatsu, Karissa Y; Schlick, Tamar

    2016-08-25

    While it is well-recognized that chromatin loops play an important role in gene regulation, structural details regarding higher order chromatin loops are only emerging. Here we present a systematic study of restrained chromatin loops ranging from 25 to 427 nucleosomes (fibers of 5-80 Kb DNA in length), mimicking gene elements studied by 3C contact data. We find that hierarchical looping represents a stable configuration that can effectively bring distant regions of the GATA-4 gene together, satisfying connections reported by 3C experiments. Additionally, we find that restrained chromatin fibers larger than 100 nucleosomes (∼20Kb) form closed plectonemes, whereas fibers shorter than 100 nucleosomes form simple hairpin loops. By studying the dependence of loop structures on internal parameters, we show that loop features are sensitive to linker histone concentration, loop length, divalent ions, and DNA linker length. Specifically, increasing loop length, linker histone concentration, and divalent ion concentration are associated with increased persistence length (or decreased bending), while varying DNA linker length in a manner similar to experimentally observed "nucleosome free regions" (found near transcription start sites) disrupts intertwining and leads to loop opening and increased persistence length in linker histone depleted (-LH) fibers. Chromatin fiber structure sensitivity to these parameters, all of which vary throughout the cell cycle, tissue type, and species, suggests that caution is warranted when using uniform polymer models to fit chromatin conformation capture genome-wide data. Furthermore, the folding geometry we observe near the transcription initiation site of the GATA-4 gene suggests that hierarchical looping provides a structural mechanism for gene inhibition, and offers tunable parameters for design of gene regulation elements. PMID:27218881

  3. Unusual properties of regulatory DNA from the Drosophila engrailed gene: three "pairing-sensitive" sites within a 1.6-kb region.

    PubMed

    Kassis, J A

    1994-03-01

    We have previously shown that a 2-kb fragment of engrailed DNA can suppress expression of a linked marker gene, white, in the P element vector CaSpeR. This suppression is dependent on the presence of two copies of engrailed DNA-containing P elements (P[en]) in proximity in the Drosophila genome (either in cis or in trans). In this study, the 2-kb fragment was dissected and found to contain three fragments of DNA which could mediate white suppression [called "pairing-sensitive sites" (PS)]. A PS site was also identified in regulatory DNA from the Drosophila escargot gene. The eye colors of six different P[en] insertions in the escargot gene suggest an interaction between P[en]-encoded and genome-encoded PS sites. I hypothesize that white gene expression from P[en] is repressed by the formation of a protein complex which is initiated at the engrailed PS sites and also requires interactions with flanking genomic DNA. Genes were sought which influence the function of PS sites. Mutations in some Polycomb and trithorax group genes were found to affect the eye color from some P[en] insertion sites. However, different mutations affected expression from different P[en] insertion sites and no one mutation was found to affect expression from all P[en] insertion sites examined. These results suggest that white expression from P[en] is not directly regulated by members of the Polycomb and trithorax group genes, but in some cases can be influenced by them. I propose that engrailed PS sites normally act to promote interactions between distantly located engrailed regulatory sites and the engrailed promoter. PMID:8005412

  4. Identification and characterization of regulatory elements in the promoter of ACVR1, the gene mutated in Fibrodysplasia Ossificans Progressiva

    PubMed Central

    2013-01-01

    Background The ACVR1 gene encodes a type I receptor for bone morphogenetic proteins (BMPs). Mutations in the ACVR1 gene are associated with Fibrodysplasia Ossificans Progressiva (FOP), a rare and extremely disabling disorder characterized by congenital malformation of the great toes and progressive heterotopic endochondral ossification in muscles and other non-skeletal tissues. Several aspects of FOP pathophysiology are still poorly understood, including mechanisms regulating ACVR1 expression. This work aimed to identify regulatory elements that control ACVR1 gene transcription. Methods and results We first characterized the structure and composition of human ACVR1 gene transcripts by identifying the transcription start site, and then characterized a 2.9 kb upstream region. This region showed strong activating activity when tested by reporter gene assays in transfected cells. We identified specific elements within the 2.9 kb region that are important for transcription factor binding using deletion constructs, co-transfection experiments with plasmids expressing selected transcription factors, site-directed mutagenesis of consensus binding-site sequences, and by protein/DNA binding assays. We also characterized a GC-rich minimal promoter region containing binding sites for the Sp1 transcription factor. Conclusions Our results showed that several transcription factors such as Egr-1, Egr-2, ZBTB7A/LRF, and Hey1, regulate the ACVR1 promoter by binding to the -762/-308 region, which is essential to confer maximal transcriptional activity. The Sp1 transcription factor acts at the most proximal promoter segment upstream of the transcription start site. We observed significant differences in different cell types suggesting tissue specificity of transcriptional regulation. These findings provide novel insights into the molecular mechanisms that regulate expression of the ACVR1 gene and that could be targets of new strategies for future therapeutic treatments. PMID:24047559

  5. Short palindromic repetitive DNA elements in enterobacteria: a survey.

    PubMed

    Bachellier, S; Clément, J M; Hofnung, M

    1999-01-01

    We present a survey of short palindromic repetitive elements in enterobacteria. Seven families are presented. Five were already known (RSA, IRU, 29-bp repeats, BIMEs and boxC), and their properties are updated; in particular, a new composite element is shown to include the formerly identified boxC repeats. Two repetitions, YPAL1 and YPAL2, found primarily in Yersinia, are described here for the first time. PMID:10673002

  6. DNA Methylation Analysis of HTR2A Regulatory Region in Leukocytes of Autistic Subjects.

    PubMed

    Hranilovic, Dubravka; Blazevic, Sofia; Stefulj, Jasminka; Zill, Peter

    2016-02-01

    Disturbed brain and peripheral serotonin homeostasis is often found in subjects with autism spectrum disorder (ASD). The role of the serotonin receptor 2A (HTR2A) in the regulation of central and peripheral serotonin homeostasis, as well as its altered expression in autistic subjects, have implicated the HTR2A gene as a major candidate for the serotonin disturbance seen in autism. Several studies, yielding so far inconclusive results, have attempted to associate autism with a functional SNP -1438 G/A (rs6311) in the HTR2A promoter region, while possible contribution of epigenetic mechanisms, such as DNA methylation, to HTR2A dysregulation in autism has not yet been investigated. In this study, we compared the mean DNA methylation within the regulatory region of the HTR2A gene between autistic and control subjects. DNA methylation was analysed in peripheral blood leukocytes using bisulfite conversion and sequencing of the HTR2A region containing rs6311 polymorphism. Autistic subjects of rs6311 AG genotype displayed higher mean methylation levels within the analysed region than the corresponding controls (P < 0.05), while there was no statistically significant difference for AA and GG carriers. Our study provides preliminary evidence for increased HTR2A promoter methylation in leukocytes of a portion of adult autistic subjects, indicating that epigenetic mechanisms might contribute to HTR2A dysregulation observed in individuals with ASD. PMID:26149086

  7. Isolation of Alcohol Dehydrogenase cDNA and Basal Regulatory Region from Metroxylon sagu

    PubMed Central

    Wee, Ching Ching; Roslan, Hairul Azman

    2012-01-01

    Alcohol dehydrogenase (Adh) is a versatile enzyme involved in many biochemical pathways in plants such as in germination and stress tolerance. Sago palm is plant with much importance to the state of Sarawak as one of the most important crops that bring revenue with the advantage of being able to withstand various biotic and abiotic stresses such as heat, pathogens, and water logging. Here we report the isolation of sago palm Adh cDNA and its putative promoter region via the use of rapid amplification of cDNA ends (RACE) and genomic walking. The isolated cDNA was characterized and determined to be 1464 bp long encoding for 380 amino acids. BLAST analysis showed that the Adh is similar to the Adh1 group with 91% and 85% homology with Elaeis guineensis and Washingtonia robusta, respectively. The putative basal msAdh1 regulatory region was further determined to contain promoter signals of TATA and AGGA boxes and predicted amino acids analyses showed several Adh-specific motifs such as the two zinc-binding domains that bind to the adenosine ribose of the coenzyme and binding to alcohol substrate. A phylogenetic tree was also constructed using the predicted amino acid showed clear separation of Adh from bacteria and clustered within the plant Adh group.

  8. Isolation of Alcohol Dehydrogenase cDNA and Basal Regulatory Region from Metroxylon sagu.

    PubMed

    Wee, Ching Ching; Roslan, Hairul Azman

    2012-01-01

    Alcohol dehydrogenase (Adh) is a versatile enzyme involved in many biochemical pathways in plants such as in germination and stress tolerance. Sago palm is plant with much importance to the state of Sarawak as one of the most important crops that bring revenue with the advantage of being able to withstand various biotic and abiotic stresses such as heat, pathogens, and water logging. Here we report the isolation of sago palm Adh cDNA and its putative promoter region via the use of rapid amplification of cDNA ends (RACE) and genomic walking. The isolated cDNA was characterized and determined to be 1464 bp long encoding for 380 amino acids. BLAST analysis showed that the Adh is similar to the Adh1 group with 91% and 85% homology with Elaeis guineensis and Washingtonia robusta, respectively. The putative basal msAdh1 regulatory region was further determined to contain promoter signals of TATA and AGGA boxes and predicted amino acids analyses showed several Adh-specific motifs such as the two zinc-binding domains that bind to the adenosine ribose of the coenzyme and binding to alcohol substrate. A phylogenetic tree was also constructed using the predicted amino acid showed clear separation of Adh from bacteria and clustered within the plant Adh group. PMID:27335670

  9. Structure of a Thyroid Hormone Receptor DNA-Binding Domain Homodimer Bound to an Inverted Palindrome DNA Response Element

    SciTech Connect

    Chen, Yi; Young, Matthew A.

    2010-10-22

    Thyroid hormone receptor (TR), as a member of the nuclear hormone receptor family, can recognize and bind different classes of DNA response element targets as either a monomer, a homooligomer, or a heterooligomer. We report here the first crystal structure of a homodimer TR DNA-binding domain (DBD) in complex with an inverted repeat class of thyroid response element (TRE). The structure shows a nearly symmetric structure of the TR DBD assembled on the F2 TRE where the base recognition contacts in the homodimer DNA complex are conserved relative to the previously published structure of a TR-9-cis-retinoic acid receptor heterodimer DNA complex. The new structure also reveals that the T-box region of the DBD can function as a structural hinge that enables a large degree of flexibility in the position of the C-terminal extension helix that connects the DBD to the ligand-binding domain. Although the isolated TR DBDs exist as monomers in solution, we have measured highly cooperative binding of the two TR DBD subunits onto the inverted repeat DNA sequence. This suggests that elements of the DBD can influence the specific TR oligomerization at target genes, and it is not just interactions between the ligand-binding domains that are responsible for TR oligomerization at target genes. Mutational analysis shows that intersubunit contacts at the DBD C terminus account for some, but not all, of the cooperative homodimer TR binding to the inverted repeat class TRE.

  10. Capture of flanking DNA by a P element in Drosophila melanogaster: Creation of a transposable element

    SciTech Connect

    Tsubota, Stuart, I.; Huong Dangvu )

    1991-02-01

    A 6.1-kilobase nsertion into the rudimentary (r) gene was cloned and partially sequenced. The insertion consists of a 703-base-pair (bp) P element next to a 5.4-kilobase single-copy sequence. The normal positon of the single-copy sequence is near the tip of the X chromosome. Upon insertion into the r gene, this chimeric element generated an 8-bp target-site duplication, characteristic of P elements. At the non-P-element end of the insertion, the first 8 bp are identical to the first 8 bp of the inverted terminal repeats of the P element. Thus, this element has inverted terminal repeats of 8 bp. This large element can excise from the r gene under conditions of hybrid dysgenesis, which indicates that it behaves like a normal P element. These data support the conclusion that a normally stable single-copy sequence has now become unstable and duplicated within the genome.

  11. A Transposable Element within the Non-canonical Telomerase RNA of Arabidopsis thaliana Modulates Telomerase in Response to DNA Damage

    PubMed Central

    Xu, Hengyi; Nelson, Andrew D. L.; Shippen, Dorothy E.

    2015-01-01

    Long noncoding RNAs (lncRNAs) have emerged as critical factors in many biological processes, but little is known about how their regulatory functions evolved. One of the best-studied lncRNAs is TER, the essential RNA template for telomerase reverse transcriptase. We previously showed that Arabidopsis thaliana harbors three TER isoforms: TER1, TER2 and TER2S. TER1 serves as a canonical telomere template, while TER2 is a novel negative regulator of telomerase activity, induced in response to double-strand breaks (DSBs). TER2 contains a 529 nt intervening sequence that is removed along with 36 nt at the RNA 3’ terminus to generate TER2S, an RNA of unknown function. Here we investigate how A. thaliana TER2 acquired its regulatory function. Using data from the 1,001 Arabidopsis genomes project, we report that the intervening sequence within TER2 is derived from a transposable element termed DSB responsive element (DRE). DRE is found in the TER2 loci of most but not all A. thaliana accessions. By analyzing accessions with (TER2) and without DRE (TER2Δ) we demonstrate that this element is responsible for many of the unique properties of TER2, including its enhanced binding to TERT and telomerase inhibitory function. We show that DRE destabilizes TER2, and further that TER2 induction by DNA damage reflects increased RNA stability and not increased transcription. DRE-mediated changes in TER2 stability thus provide a rapid and sensitive switch to fine-tune telomerase enzyme activity. Altogether, our data shows that invasion of the TER2 locus by a small transposon converted this lncRNA into a DNA damage sensor that modulates telomerase enzyme activity in response to genome assault. PMID:26075395

  12. Conformational diversity of single-stranded DNA from bacterial repetitive extragenic palindromes: Implications for the DNA recognition elements of transposases.

    PubMed

    Charnavets, Tatsiana; Nunvar, Jaroslav; Nečasová, Iva; Völker, Jens; Breslauer, Kenneth J; Schneider, Bohdan

    2015-10-01

    Repetitive extragenic palindrome (REP)-associated tyrosine transposase enzymes (RAYTs) bind REP DNA domains and catalyze their cleavage. Genomic sequence analyses identify potential noncoding REP sequences associated with RAYT-encoding genes. To probe the conformational space of potential RAYT DNA binding domains, we report here spectroscopic and calorimetric measurements that detect and partially characterize the solution conformational heterogeneity of REP oligonucleotides from six bacterial species. Our data reveal most of these REP oligonucleotides adopt multiple conformations, suggesting that RAYTs confront a landscape of potential DNA substrates in dynamic equilibrium that could be selected, enriched, and/or induced via differential binding. Thus, the transposase-bound DNA motif may not be the predominant conformation of the isolated REP domain. Intriguingly, for several REPs, the circular dichroism spectra suggest guanine tetraplexes as potential alternative or additional RAYT recognition elements, an observation consistent with these REP domains being highly nonrandom, with tetraplex-favoring 5'-G and 3'-C-rich segments. In fact, the conformational heterogeneity of REP domains detected and reported here, including the formation of noncanonical DNA secondary structures, may reflect a general feature required for recognition by RAYT transposases. Based on our biophysical data, we propose guanine tetraplexes as an additional DNA recognition element for binding by RAYT transposase enzymes. PMID:25951997

  13. Integrated methylome and transcriptome analysis reveals novel regulatory elements in pediatric acute lymphoblastic leukemia.

    PubMed

    Almamun, Md; Levinson, Benjamin T; van Swaay, Annette C; Johnson, Nathan T; McKay, Stephanie D; Arthur, Gerald L; Davis, J Wade; Taylor, Kristen H

    2015-01-01

    Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children under the age of 15. In addition to genetic aberrations, epigenetic modifications such as DNA methylation are altered in cancer and impact gene expression. To identify epigenetic alterations in ALL, genome-wide methylation profiles were generated using the methylated CpG island recovery assay followed by next-generation sequencing. More than 25,000 differentially methylated regions (DMR) were observed in ALL patients with ∼ 90% present within intronic or intergenic regions. To determine the regulatory potential of the DMR, whole-transcriptome analysis was performed and integrated with methylation data. Aberrant promoter methylation was associated with the altered expression of genes involved in transcriptional regulation, apoptosis, and proliferation. Novel enhancer-like sequences were identified within intronic and intergenic DMR. Aberrant methylation in these regions was associated with the altered expression of neighboring genes involved in cell cycle processes, lymphocyte activation and apoptosis. These genes include potential epi-driver genes, such as SYNE1, PTPRS, PAWR, HDAC9, RGCC, MCOLN2, LYN, TRAF3, FLT1, and MELK, which may provide a selective advantage to leukemic cells. In addition, the differential expression of epigenetic modifier genes, pseudogenes, and non-coding RNAs was also observed accentuating the role of erroneous epigenetic gene regulation in ALL. PMID:26308964

  14. Integrated methylome and transcriptome analysis reveals novel regulatory elements in pediatric acute lymphoblastic leukemia

    PubMed Central

    Almamun, Md; Levinson, Benjamin T; van Swaay, Annette C; Johnson, Nathan T; McKay, Stephanie D; Arthur, Gerald L; Davis, J Wade; Taylor, Kristen H

    2015-01-01

    Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children under the age of 15. In addition to genetic aberrations, epigenetic modifications such as DNA methylation are altered in cancer and impact gene expression. To identify epigenetic alterations in ALL, genome-wide methylation profiles were generated using the methylated CpG island recovery assay followed by next-generation sequencing. More than 25,000 differentially methylated regions (DMR) were observed in ALL patients with ∼90% present within intronic or intergenic regions. To determine the regulatory potential of the DMR, whole-transcriptome analysis was performed and integrated with methylation data. Aberrant promoter methylation was associated with the altered expression of genes involved in transcriptional regulation, apoptosis, and proliferation. Novel enhancer-like sequences were identified within intronic and intergenic DMR. Aberrant methylation in these regions was associated with the altered expression of neighboring genes involved in cell cycle processes, lymphocyte activation and apoptosis. These genes include potential epi-driver genes, such as SYNE1, PTPRS, PAWR, HDAC9, RGCC, MCOLN2, LYN, TRAF3, FLT1, and MELK, which may provide a selective advantage to leukemic cells. In addition, the differential expression of epigenetic modifier genes, pseudogenes, and non-coding RNAs was also observed accentuating the role of erroneous epigenetic gene regulation in ALL. PMID:26308964

  15. Highly recurring sequence elements identified in eukaryotic DNAs by computer analysis are often homologous to regulatory sequences or protein binding sites.

    PubMed Central

    Bodnar, J W; Ward, D C

    1987-01-01

    We have used computer assisted dot matrix and oligonucleotide frequency analyses to identify highly recurring sequence elements of 7-11 base pairs in eukaryotic genes and viral DNAs. Such elements are found much more frequently than expected, often with an average spacing of a few hundred base pairs. Furthermore, the most abundant repetitive elements observed in the ovalbumin locus, the beta-globin gene cluster, the metallothionein gene and the viral genomes of SV40, polyoma, Herpes simplex-1 and Mouse Mammary Tumor Virus were sequences shown previously to be protein binding sites or sequences important for regulating gene expression. These sequences were present in both exons and introns as well as promoter regions. These observations suggest that such sequences are often highly overrepresented within the specific gene segments with which they are associated. Computer analysis of other genetic units, including viral genomes and oncogenes, has identified a number of highly recurring sequence elements that could serve similar regulatory or protein-binding functions. A model for the role of such reiterated sequence elements in DNA organization and function is presented. PMID:3822840

  16. Novel DNA-binding element within the C-terminal extension of the nuclear receptor DNA-binding domain

    PubMed Central

    Jakób, Michał; Kołodziejczyk, Robert; Orłowski, Marek; Krzywda, Szymon; Kowalska, Agnieszka; Dutko-Gwóźdź, Joanna; Gwóźdź, Tomasz; Kochman, Marian; Jaskólski, Mariusz; Ożyhar, Andrzej

    2007-01-01

    The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptors superfamily, is considered as the functional receptor for ecdysteroids initiating molting and metamorphosis in insects. Here we report the 1.95 Å structure of the complex formed by the DNA-binding domains (DBDs) the EcR and the Usp, bound to the natural pseudopalindromic response element. Comparison of the structure with that obtained previously, using an idealized response element, shows how the EcRDBD, which has been previously reported to possess extraordinary flexibility, accommodates DNA-induced structural changes. Part of the C-terminal extension (CTE) of the EcRDBD folds into an α-helix whose location in the minor groove does not match any of the locations previously observed for nuclear receptors. Mutational analyses suggest that the α-helix is a component of EcR-box, a novel element indispensable for DNA-binding and located within the nuclear receptor CTE. This element seems to be a general feature of all known EcRs. PMID:17426125

  17. Putative regulatory elements within the non-coding regions of Chrysomelidae Diapause Associated Transcript-1 (DAT-1) orthologs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To develop a more comprehensive understanding of diapause within Chrysomelidae, we are employing phylogenetic foot-printing to isolate and characterize the regulatory elements associated with the diapause-associated gene, DAT-1. Leptinotarsa decemlineata (Colorado potato beetle, CPB) DAT-1 has been ...

  18. Negative regulatory element associated with potentially functional promoter and enhancer elements in the long terminal repeats of endogenous murine leukemia virus-related proviral sequences.

    PubMed Central

    Ch'ang, L Y; Yang, W K; Myer, F E; Yang, D M

    1989-01-01

    Three series of recombinant DNA clones were constructed, with the bacterial chloramphenicol acetyltransferase (CAT) gene as a quantitative indicator, to examine the activities of promoter and enhancer sequence elements in the 5' long terminal repeat (LTR) of murine leukemia virus (MuLV)-related proviral sequences isolated from the mouse genome. Transient CAT expression was determined in mouse NIH 3T3, human HT1080, and mink CCL64 cultured cells transfected with the LTR-CAT constructs. The 700-base-pair (bp) LTRs of three polytropic MuLV-related proviral clones and the 750-bp LTRs of four modified polytropic proviral clones, in complete structures either with or without the adjacent downstream sequences, all showed very little or negligible activities for CAT expression, while ecotropic MuLV LTRs were highly active. The MuLV-related LTRs were divided into three portions and examined separately. The 3' portion of the MuLV-related LTRs that contains the CCAAC and TATAA boxes was found to be a functional promoter, being about one-half to one-third as active as the corresponding portion of ecotropic MuLV LTRs. A MboI-Bg/II fragment, representing the distinct 190- to 200-bp inserted segment in the middle, was found to be a potential enhancer, especially when examined in combination with the simian virus 40 promoter in CCL64 cells. A PstI-MboI fragment of the 5' portion, which contains the protein-binding motifs of the enhancer segment as well as the upstream LTR sequences, showed moderate enhancer activities in CCL6 cells but was virtually inactive in NIH 3T3 cells and HT1080 cells; addition of this fragment to the ecotropic LTR-CAT constructs depressed CAT expression. Further analyses using chimeric LTR constructs located the presence of a strong negative regulatory element within the region containing the 5' portion of the enhancer and the immediate upstream sequences in the MuLV-related LTRs. Images PMID:2542587

  19. Surveying DNA Elements within Functional Genes of Heterocyst-Forming Cyanobacteria

    PubMed Central

    Hilton, Jason A.; Meeks, John C.; Zehr, Jonathan P.

    2016-01-01

    Some cyanobacteria are capable of differentiating a variety of cell types in response to environmental factors. For instance, in low nitrogen conditions, some cyanobacteria form heterocysts, which are specialized for N2 fixation. Many heterocyst-forming cyanobacteria have DNA elements interrupting key N2 fixation genes, elements that are excised during heterocyst differentiation. While the mechanism for the excision of the element has been well-studied, many questions remain regarding the introduction of the elements into the cyanobacterial lineage and whether they have been retained ever since or have been lost and reintroduced. To examine the evolutionary relationships and possible function of DNA sequences that interrupt genes of heterocyst-forming cyanobacteria, we identified and compared 101 interruption element sequences within genes from 38 heterocyst-forming cyanobacterial genomes. The interruption element lengths ranged from about 1 kb (the minimum able to encode the recombinase responsible for element excision), up to nearly 1 Mb. The recombinase gene sequences served as genetic markers that were common across the interruption elements and were used to track element evolution. Elements were found that interrupted 22 different orthologs, only five of which had been previously observed to be interrupted by an element. Most of the newly identified interrupted orthologs encode proteins that have been shown to have heterocyst-specific activity. However, the presence of interruption elements within genes with no known role in N2 fixation, as well as in three non-heterocyst-forming cyanobacteria, indicates that the processes that trigger the excision of elements may not be limited to heterocyst development or that the elements move randomly within genomes. This comprehensive analysis provides the framework to study the history and behavior of these unique sequences, and offers new insight regarding the frequency and persistence of interruption elements in

  20. HIRF: a novel nuclear factor that binds to the human T-cell leukemia virus type I internal regulatory element (HIRE).

    PubMed

    Ariumi, Y; Copeland, T D; Nosaka, T; Hatanaka, M

    1997-04-01

    The transcription of human T-cell leukemia virus type I (HTLV-I) provirus starts from a promoter located in the 5' long terminal repeat (LTR). We have identified a second promoter at the 3' end of the pol gene. This internal promoter expresses the Tax transactivator protein, but does not require Tax for its activity. Furthermore, we have found the novel enhancer motif AGTTCTGCCC, which are located near the initiation site. We have named the sequence HIRE (HTLV-I internal regulatory element). The HIRE binding protein is a ubiquitous protein. We purified this protein from the HTLV-I producing cell line MT-2 cells by DNA affinity chromatography. SDS-PAGE analysis revealed four major bands (70, 85, 115 and more than 200 kDa) and some minor bands on the gel. We renatured each major protein and showed the 70 and 115 kDa proteins bind to DNA, although the 115 kDa protein seemed to bind nonspecifically. We have designated these components as HIRF (HTLV-I internal regulatory factor). PMID:9209287

  1. Genome Sequencing of Autism-Affected Families Reveals Disruption of Putative Noncoding Regulatory DNA

    PubMed Central

    Turner, Tychele N.; Hormozdiari, Fereydoun; Duyzend, Michael H.; McClymont, Sarah A.; Hook, Paul W.; Iossifov, Ivan; Raja, Archana; Baker, Carl; Hoekzema, Kendra; Stessman, Holly A.; Zody, Michael C.; Nelson, Bradley J.; Huddleston, John; Sandstrom, Richard; Smith, Joshua D.; Hanna, David; Swanson, James M.; Faustman, Elaine M.; Bamshad, Michael J.; Stamatoyannopoulos, John; Nickerson, Deborah A.; McCallion, Andrew S.; Darnell, Robert; Eichler, Evan E.

    2016-01-01

    We performed whole-genome sequencing (WGS) of 208 genomes from 53 families affected by simplex autism. For the majority of these families, no copy-number variant (CNV) or candidate de novo gene-disruptive single-nucleotide variant (SNV) had been detected by microarray or whole-exome sequencing (WES). We integrated multiple CNV and SNV analyses and extensive experimental validation to identify additional candidate mutations in eight families. We report that compared to control individuals, probands showed a significant (p = 0.03) enrichment of de novo and private disruptive mutations within fetal CNS DNase I hypersensitive sites (i.e., putative regulatory regions). This effect was only observed within 50 kb of genes that have been previously associated with autism risk, including genes where dosage sensitivity has already been established by recurrent disruptive de novo protein-coding mutations (ARID1B, SCN2A, NR3C2, PRKCA, and DSCAM). In addition, we provide evidence of gene-disruptive CNVs (in DISC1, WNT7A, RBFOX1, and MBD5), as well as smaller de novo CNVs and exon-specific SNVs missed by exome sequencing in neurodevelopmental genes (e.g., CANX, SAE1, and PIK3CA). Our results suggest that the detection of smaller, often multiple CNVs affecting putative regulatory elements might help explain additional risk of simplex autism. PMID:26749308

  2. Genome Sequencing of Autism-Affected Families Reveals Disruption of Putative Noncoding Regulatory DNA.

    PubMed

    Turner, Tychele N; Hormozdiari, Fereydoun; Duyzend, Michael H; McClymont, Sarah A; Hook, Paul W; Iossifov, Ivan; Raja, Archana; Baker, Carl; Hoekzema, Kendra; Stessman, Holly A; Zody, Michael C; Nelson, Bradley J; Huddleston, John; Sandstrom, Richard; Smith, Joshua D; Hanna, David; Swanson, James M; Faustman, Elaine M; Bamshad, Michael J; Stamatoyannopoulos, John; Nickerson, Deborah A; McCallion, Andrew S; Darnell, Robert; Eichler, Evan E

    2016-01-01

    We performed whole-genome sequencing (WGS) of 208 genomes from 53 families affected by simplex autism. For the majority of these families, no copy-number variant (CNV) or candidate de novo gene-disruptive single-nucleotide variant (SNV) had been detected by microarray or whole-exome sequencing (WES). We integrated multiple CNV and SNV analyses and extensive experimental validation to identify additional candidate mutations in eight families. We report that compared to control individuals, probands showed a significant (p = 0.03) enrichment of de novo and private disruptive mutations within fetal CNS DNase I hypersensitive sites (i.e., putative regulatory regions). This effect was only observed within 50 kb of genes that have been previously associated with autism risk, including genes where dosage sensitivity has already been established by recurrent disruptive de novo protein-coding mutations (ARID1B, SCN2A, NR3C2, PRKCA, and DSCAM). In addition, we provide evidence of gene-disruptive CNVs (in DISC1, WNT7A, RBFOX1, and MBD5), as well as smaller de novo CNVs and exon-specific SNVs missed by exome sequencing in neurodevelopmental genes (e.g., CANX, SAE1, and PIK3CA). Our results suggest that the detection of smaller, often multiple CNVs affecting putative regulatory elements might help explain additional risk of simplex autism. PMID:26749308

  3. Alu-mediated deletion of SOX10 regulatory elements in Waardenburg syndrome type 4.

    PubMed

    Bondurand, Nadége; Fouquet, Virginie; Baral, Viviane; Lecerf, Laure; Loundon, Natalie; Goossens, Michel; Duriez, Benedicte; Labrune, Philippe; Pingault, Veronique

    2012-09-01

    Waardenburg syndrome type 4 (WS4) is a rare neural crest disorder defined by the combination of Waardenburg syndrome (sensorineural hearing loss and pigmentation defects) and Hirschsprung disease (intestinal aganglionosis). Three genes are known to be involved in this syndrome, that is, EDN3 (endothelin-3), EDNRB (endothelin receptor type B), and SOX10. However, 15-35% of WS4 remains unexplained at the molecular level, suggesting that other genes could be involved and/or that mutations within known genes may have escaped previous screenings. Here, we searched for deletions within recently identified SOX10 regulatory sequences and describe the first characterization of a WS4 patient presenting with a large deletion encompassing three of these enhancers. Analysis of the breakpoint region suggests a complex rearrangement involving three Alu sequences that could be mediated by a FosTes/MMBIR replication mechanism. Taken together with recent reports, our results demonstrate that the disruption of highly conserved non-coding elements located within or at a long distance from the coding sequences of key genes can result in several neurocristopathies. This opens up new routes to the molecular dissection of neural crest disorders. PMID:22378281

  4. RAR/RXR binding dynamics distinguish pluripotency from differentiation associated cis-regulatory elements

    PubMed Central

    Chatagnon, Amandine; Veber, Philippe; Morin, Valérie; Bedo, Justin; Triqueneaux, Gérard; Sémon, Marie; Laudet, Vincent; d'Alché-Buc, Florence; Benoit, Gérard

    2015-01-01

    In mouse embryonic cells, ligand-activated retinoic acid receptors (RARs) play a key role in inhibiting pluripotency-maintaining genes and activating some major actors of cell differentiation. To investigate the mechanism underlying this dual regulation, we performed joint RAR/RXR ChIP-seq and mRNA-seq time series during the first 48 h of the RA-induced Primitive Endoderm (PrE) differentiation process in F9 embryonal carcinoma (EC) cells. We show here that this dual regulation is associated with RAR/RXR genomic redistribution during the differentiation process. In-depth analysis of RAR/RXR binding sites occupancy dynamics and composition show that in undifferentiated cells, RAR/RXR interact with genomic regions characterized by binding of pluripotency-associated factors and high prevalence of the non-canonical DR0-containing RA response element. By contrast, in differentiated cells, RAR/RXR bound regions are enriched in functional Sox17 binding sites and are characterized with a higher frequency of the canonical DR5 motif. Our data offer an unprecedentedly detailed view on the action of RA in triggering pluripotent cell differentiation and demonstrate that RAR/RXR action is mediated via two different sets of regulatory regions tightly associated with cell differentiation status. PMID:25897113

  5. RAR/RXR binding dynamics distinguish pluripotency from differentiation associated cis-regulatory elements.

    PubMed

    Chatagnon, Amandine; Veber, Philippe; Morin, Valérie; Bedo, Justin; Triqueneaux, Gérard; Sémon, Marie; Laudet, Vincent; d'Alché-Buc, Florence; Benoit, Gérard

    2015-05-26

    In mouse embryonic cells, ligand-activated retinoic acid receptors (RARs) play a key role in inhibiting pluripotency-maintaining genes and activating some major actors of cell differentiation. To investigate the mechanism underlying this dual regulation, we performed joint RAR/RXR ChIP-seq and mRNA-seq time series during the first 48 h of the RA-induced Primitive Endoderm (PrE) differentiation process in F9 embryonal carcinoma (EC) cells. We show here that this dual regulation is associated with RAR/RXR genomic redistribution during the differentiation process. In-depth analysis of RAR/RXR binding sites occupancy dynamics and composition show that in undifferentiated cells, RAR/RXR interact with genomic regions characterized by binding of pluripotency-associated factors and high prevalence of the non-canonical DR0-containing RA response element. By contrast, in differentiated cells, RAR/RXR bound regions are enriched in functional Sox17 binding sites and are characterized with a higher frequency of the canonical DR5 motif. Our data offer an unprecedentedly detailed view on the action of RA in triggering pluripotent cell differentiation and demonstrate that RAR/RXR action is mediated via two different sets of regulatory regions tightly associated with cell differentiation status. PMID:25897113

  6. Highly Specific Epigenome Editing by CRISPR/Cas9 Repressors for Silencing of Distal Regulatory Elements

    PubMed Central

    Thakore, Pratiksha I.; D’Ippolito, Anthony M; Song, Lingyun; Safi, Alexias; Shivakumar, Nishkala K.; Kabadi, Ami M.; Reddy, Timothy E.; Crawford, Gregory E.; Gersbach, Charles A.

    2015-01-01

    Epigenome editing with the CRISPR/Cas9 platform is a promising technology to modulate gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms of gene regulation. Fusions of the nuclease-inactive dCas9 to the KRAB repressor (dCas9-KRAB) can silence target gene expression, but the genome-wide specificity and the extent of heterochromatin formation catalyzed by dCas9-KRAB is not known. We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates expression of multiple globin genes. Genome-wide analyses demonstrated that localization of dCas9-KRAB to HS2 specifically induced H3K9 tri-methylation (H3K9me3) at the enhancer and reduced the chromatin accessibility of both the enhancer and its promoter targets. Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in gene expression. These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome. PMID:26501517

  7. RNA structure is a key regulatory element in pathological ATM and CFTR pseudoexon inclusion events

    PubMed Central

    Buratti, Emanuele; Dhir, Ashish; Lewandowska, Marzena A.; Baralle, Francisco E.

    2007-01-01

    Genomic variations deep in the intronic regions of pre-mRNA molecules are increasingly reported to affect splicing events. However, there is no general explanation why apparently similar variations may have either no effect on splicing or cause significant splicing alterations. In this work we have examined the structural architecture of pseudoexons previously described in ATM and CFTR patients. The ATM case derives from the deletion of a repressor element and is characterized by an aberrant 5′ss selection despite the presence of better alternatives. The CFTR pseudoexon instead derives from the creation of a new 5′ss that is used while a nearby pre-existing donor-like sequence is never selected. Our results indicate that RNA structure is a major splicing regulatory factor in both cases. Furthermore, manipulation of the original RNA structures can lead to pseudoexon inclusion following the exposure of unused 5′ss already present in their wild-type intronic sequences and prevented to be recognized because of their location in RNA stem structures. Our data show that intrinsic structural features of introns must be taken into account to understand the mechanism of pseudoexon activation in genetic diseases. Our observations may help to improve diagnostics prediction programmes and eventual therapeutic targeting. PMID:17580311

  8. Understanding the molecular mechanism of transcriptional regulation of banana Sucrose phosphate synthase (SPS) gene during fruit ripening: an insight into the functions of various cis-acting regulatory elements.

    PubMed

    Choudhury, Swarup Roy; Roy, Sujit; Singh, Sanjay Kumar; Sengupta, Dibyendu N

    2010-05-01

    Recently, we have reported the characterization of promoter region of Sucrose phosphate synthase (SPS) gene in banana and investigated the role of some cis-elements/motifs, present in the promoter of SPS, in the transcriptional regulation of the gene. DNA-protein interaction studies have demonstrated the presence of specific trans-acting factors which showed specific interactions with ethylene, auxin, low temperature and light responsive elements in regulating SPS transcription. Transient expression analyses have demonstrated the functional significance of the various cis-acting regulatory elements present in banana SPS promoter in regulating SPS expression during ripening. (1) Here, we have further discussed the possible role of these regulatory sequences in the regulation of transcriptional network and comment on their function in relation to sucrose metabolism during banana fruit ripening. PMID:20139735

  9. TFIIIC Bound DNA Elements in Nuclear Organization and Insulation

    PubMed Central

    Kirkland, Jacob G.; Raab, Jesse R.

    2012-01-01

    tRNA genes (tDNAs) have been known to have barrier insulator function in budding yeast, Saccharomyces cerevisiae, for over a decade. tDNAs also play a role in genome organization by clustering at sites in the nucleus and both of these functions are dependent on the transcription factor TFIIIC. More recently TFIIIC bound sites devoid of pol III, termed Extra-TFIIIC sites (ETC) have been identified in budding yeast and these sites also function as insulators and affect genome organization. Subsequent studies in Schizosaccharomyces pombe showed that TFIIIC bound sites were insulators and also functioned as Chromosome Organization Clamps (COC); tethering the sites to the nuclear periphery. Very recently studies have moved to mammalian systems where pol III genes and their associated factors have been investigated in both mouse and human cells. Short Interspersed Nuclear Elements (SINEs) that bind TFIIIC, function as insulator elements and tDNAs can also function as both enhancer -blocking and barrier insulators in these organisms. It was also recently shown that tDNAs cluster with other tDNAs and with ETCs but not with pol II transcribed genes. Intriguingly, TFIIIC is often found near pol II transcription start sites and it remains unclear what the consequences of TFIIIC based genomic organization are and what influence pol III factors have on pol II transcribed genes and vise versa. In this review we provide a comprehensive overview of the known data on pol III factors in insulation and genome organization and identify the many open questions that require further investigation. \\ PMID:23000638

  10. In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element Specific for Bromacil

    PubMed Central

    Williams, Ryan M.; Kulick, Amanda R.; Yedlapalli, Srilakshmi; Battistella, Louisa; Hajiran, Cyrus J.; Sooter, Letha J.

    2014-01-01

    Bromacil is a widely used herbicide that is known to contaminate environmental systems. Due to the hazards it presents and inefficient detection methods, it is necessary to create a rapid and efficient sensing device. Towards this end, we have utilized a stringent in vitro selection method to identify single-stranded DNA molecular recognition elements (MRE) specific for bromacil. We have identified one MRE with high affinity (Kd = 9.6 nM) and specificity for bromacil compared to negative targets of selection and other pesticides. The selected ssDNA MRE will be useful as the sensing element in a field-deployable bromacil detection device. PMID:25400940

  11. DNA capture elements for rapid detection and identification of biological agents

    NASA Astrophysics Data System (ADS)

    Kiel, Johnathan L.; Parker, Jill E.; Holwitt, Eric A.; Vivekananda, Jeeva

    2004-08-01

    DNA capture elements (DCEs; aptamers) are artificial DNA sequences, from a random pool of sequences, selected for their specific binding to potential biological warfare agents. These sequences were selected by an affinity method using filters to which the target agent was attached and the DNA isolated and amplified by polymerase chain reaction (PCR) in an iterative, increasingly stringent, process. Reporter molecules were attached to the finished sequences. To date, we have made DCEs to Bacillus anthracis spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and Francisella tularensis. These DCEs have demonstrated specificity and sensitivity equal to or better than antibody.

  12. Profiling of conserved non-coding elements upstream of SHOX and functional characterisation of the SHOX cis-regulatory landscape

    PubMed Central

    Verdin, Hannah; Fernández-Miñán, Ana; Benito-Sanz, Sara; Janssens, Sandra; Callewaert, Bert; Waele, Kathleen De; Schepper, Jean De; François, Inge; Menten, Björn; Heath, Karen E.; Gómez-Skarmeta, José Luis; Baere, Elfride De

    2015-01-01

    Genetic defects such as copy number variations (CNVs) in non-coding regions containing conserved non-coding elements (CNEs) outside the transcription unit of their target gene, can underlie genetic disease. An example of this is the short stature homeobox (SHOX) gene, regulated by seven CNEs located downstream and upstream of SHOX, with proven enhancer capacity in chicken limbs. CNVs of the downstream CNEs have been reported in many idiopathic short stature (ISS) cases, however, only recently have a few CNVs of the upstream enhancers been identified. Here, we set out to provide insight into: (i) the cis-regulatory role of these upstream CNEs in human cells, (ii) the prevalence of upstream CNVs in ISS, and (iii) the chromatin architecture of the SHOX cis-regulatory landscape in chicken and human cells. Firstly, luciferase assays in human U2OS cells, and 4C-seq both in chicken limb buds and human U2OS cells, demonstrated cis-regulatory enhancer capacities of the upstream CNEs. Secondly, CNVs of these upstream CNEs were found in three of 501 ISS patients. Finally, our 4C-seq interaction map of the SHOX region reveals a cis-regulatory domain spanning more than 1 Mb and harbouring putative new cis-regulatory elements. PMID:26631348

  13. Ubiquitous heterogeneity and asymmetry of the chromatin environment at regulatory elements.

    PubMed

    Kundaje, Anshul; Kyriazopoulou-Panagiotopoulou, Sofia; Libbrecht, Max; Smith, Cheryl L; Raha, Debasish; Winters, Elliott E; Johnson, Steven M; Snyder, Michael; Batzoglou, Serafim; Sidow, Arend

    2012-09-01

    Gene regulation at functional elements (e.g., enhancers, promoters, insulators) is governed by an interplay of nucleosome remodeling, histone modifications, and transcription factor binding. To enhance our understanding of gene regulation, the ENCODE Consortium has generated a wealth of ChIP-seq data on DNA-binding proteins and histone modifications. We additionally generated nucleosome positioning data on two cell lines, K562 and GM12878, by MNase digestion and high-depth sequencing. Here we relate 14 chromatin signals (12 histone marks, DNase, and nucleosome positioning) to the binding sites of 119 DNA-binding proteins across a large number of cell lines. We developed a new method for unsupervised pattern discovery, the Clustered AGgregation Tool (CAGT), which accounts for the inherent heterogeneity in signal magnitude, shape, and implicit strand orientation of chromatin marks. We applied CAGT on a total of 5084 data set pairs to obtain an exhaustive catalog of high-resolution patterns of histone modifications and nucleosome positioning signals around bound transcription factors. Our analyses reveal extensive heterogeneity in how histone modifications are deposited, and how nucleosomes are positioned around binding sites. With the exception of the CTCF/cohesin complex, asymmetry of nucleosome positioning is predominant. Asymmetry of histone modifications is also widespread, for all types of chromatin marks examined, including promoter, enhancer, elongation, and repressive marks. The fine-resolution signal shapes discovered by CAGT unveiled novel correlation patterns between chromatin marks, nucleosome positioning, and sequence content. Meta-analyses of the signal profiles revealed a common vocabulary of chromatin signals shared across multiple cell lines and binding proteins. PMID:22955985

  14. Functional conservation of Pax6 regulatory elements in humans and mice demonstrated with a novel transgenic reporter mouse

    PubMed Central

    Tyas, David A; Simpson, T Ian; Carr, Catherine B; Kleinjan, Dirk A; van Heyningen, Veronica; Mason, John O; Price, David J

    2006-01-01

    Background The Pax6 transcription factor is expressed during development in the eyes and in specific CNS regions, where it is essential for normal cell proliferation and differentiation. Mice lacking one or both copies of the Pax6 gene model closely humans with loss-of-function mutations in the PAX6 locus. The sequence of the Pax6/PAX6 protein is identical in mice and humans and previous studies have shown structural conservation of the gene's regulatory regions. Results We generated a transgenic mouse expressing green fluorescent protein (GFP) and neomycin resistance under the control of the entire complement of human PAX6 regulatory elements using a modified yeast artificial chromosome (YAC). Expression of GFP was studied in embryos from 9.5 days on and was confined to cells known to express Pax6. GFP expression was sufficiently strong that expressing cells could be distinguished from non-expressing cells using flow cytometry. Conclusion This work demonstrates the functional conservation of the regulatory elements controlling Pax6/PAX6 expression in mice and humans. The transgene provides an excellent tool for studying the functions of different Pax6/PAX6 regulatory elements in controlling Pax6 expression in animals that are otherwise normal. It will allow the analysis and isolation of cells in which Pax6 is activated, irrespective of the status of the endogenous locus. PMID:16674807

  15. Radiation-induced changes in DNA methylation of repetitive elements in the mouse heart.

    PubMed

    Koturbash, Igor; Miousse, Isabelle R; Sridharan, Vijayalakshmi; Nzabarushimana, Etienne; Skinner, Charles M; Melnyk, Stepan B; Pavliv, Oleksandra; Hauer-Jensen, Martin; Nelson, Gregory A; Boerma, Marjan

    2016-05-01

    DNA methylation is a key epigenetic mechanism, needed for proper control over the expression of genetic information and silencing of repetitive elements. Exposure to ionizing radiation, aside from its strong genotoxic potential, may also affect the methylation of DNA, within the repetitive elements, in particular. In this study, we exposed C57BL/6J male mice to low absorbed mean doses of two types of space radiation-proton (0.1 Gy, 150 MeV, dose rate 0.53 ± 0.08 Gy/min), and heavy iron ions ((56)Fe) (0.5 Gy, 600 MeV/n, dose rate 0.38 ± 0.06 Gy/min). Radiation-induced changes in cardiac DNA methylation associated with repetitive elements were detected. Specifically, modest hypomethylation of retrotransposon LINE-1 was observed at day 7 after irradiation with either protons or (56)Fe. This was followed by LINE-1, and other retrotransposons, ERV2 and SINE B1, as well as major satellite DNA hypermethylation at day 90 after irradiation with (56)Fe. These changes in DNA methylation were accompanied by alterations in the expression of DNA methylation machinery and affected the one-carbon metabolism pathway. Furthermore, loss of transposable elements expression was detected in the cardiac tissue at the 90-day time-point, paralleled by substantial accumulation of mRNA transcripts, associated with major satellites. Given that the one-carbon metabolism pathway can be modulated by dietary modifications, these findings suggest a potential strategy for the mitigation and, possibly, prevention of the negative effects exerted by ionizing radiation on the cardiovascular system. Additionally, we show that the methylation status and expression of repetitive elements may serve as early biomarkers of exposure to space radiation. PMID:26963372

  16. Cutting edge: a cis-acting DNA element targets AID-mediated sequence diversification to the chicken Ig light chain gene locus.

    PubMed

    Kothapalli, Nagarama; Norton, Darrell D; Fugmann, Sebastian D

    2008-02-15

    Somatic hypermutation and gene conversion are two closely related processes that increase the diversity of the primary Ig repertoire. Both processes are initiated by the activation-induced cytidine deaminase that converts cytosine residues to uracils in a transcription-dependent manner; these lesions are subsequently fixed in the genome by direct replication and error-prone DNA repair. Two alternative mechanisms were proposed to explain why this mutagenic activity is targeted almost exclusively to Ig loci: 1) specific cis-acting DNA sequences; or 2) very high levels of Ig gene transcription. In this study we now identify a novel 3' regulatory region in the chicken Ig light chain gene containing not only a classical transcriptional enhancer but also cis-acting DNA elements essential for targeting activation-induced cytidine deaminase-mediated sequence diversification to this locus. PMID:18250404

  17. Identification and Functional Characterization of Cis-Regulatory Elements Controlling Expression of the Porcine ADRB2 Gene

    PubMed Central

    Jaeger, Alexandra; Fritschka, Stephan; Ponsuksili, Siriluck; Wimmers, Klaus; Muráni, Eduard

    2015-01-01

    The beta-2 adrenergic receptor (beta-2 AR) modulates metabolic processes in skeletal muscle, liver, and adipose tissue in response to catecholamine stimulation. We showed previously that expression of the porcine beta-2 AR gene (ADRB2) is affected by cis-regulatory polymorphisms. These are most likely responsible for the association of ADRB2 with economically relevant muscle-related traits in pigs. The present study focused on characterization of promoter elements involved in basal transcriptional regulation of the porcine ADRB2 in different cell types to aid identification of its cis-regulatory polymorphisms. Based on in silico analysis, luciferase reporter gene assays and gel shift assays were performed using COS-7, HepG2, C2C12, and 3T3-L1 cells. Deletion mapping of the 5´ flanking region (-1324 to +33) of ADRB2 revealed the region between -307 and -269 to be the minimal promoter, including regulatory elements essential for the basal transcriptional activity in all four tested cell types. Directly upstream (-400 to -323) we identified an important enhancer element required for maximal promoter activity. In silico analysis and gel shift assays revealed that this GC-rich element harbors two evolutionarily conserved binding sites of Sp1, a constitutive transcriptional activator. Significant transcriptional activation of the porcine ADRB2 promoter was demonstrated by overexpression of Sp1. Our results demonstrate, for the first time, an important role of Sp1 and of the responsive enhancer element in the regulation of ADRB2 expression. Polymorphisms located in this domain of the porcine ADRB2 promoter represent candidate causal cis-regulatory variants. PMID:26221068

  18. Characterization and identification of cis-regulatory elements in Arabidopsis based on single-nucleotide polymorphism information.

    PubMed

    Korkuc, Paula; Schippers, Jos H M; Walther, Dirk

    2014-01-01

    Identifying regulatory elements and revealing their role in gene expression regulation remains a central goal of plant genome research. We exploited the detailed genomic sequencing information of a large number of Arabidopsis (Arabidopsis thaliana) accessions to characterize known and to identify novel cis-regulatory elements in gene promoter regions of Arabidopsis by relying on conservation as the hallmark signal of functional relevance. Based on the genomic layout and the obtained density profiles of single-nucleotide polymorphisms (SNPs) in sequence regions upstream of transcription start sites, the average length of promoter regions in Arabidopsis could be established at 500 bp. Genes associated with high degrees of variability of their respective upstream regions are preferentially involved in environmental response and signaling processes, while low levels of promoter SNP density are common among housekeeping genes. Known cis-elements were found to exhibit a decreased SNP density than sequence regions not associated with known motifs. For 15 known cis-element motifs, strong positional preferences relative to the transcription start site were detected based on their promoter SNP density profiles. Five novel candidate cis-element motifs were identified as consensus motifs of 17 sequence hexamers exhibiting increased sequence conservation combined with evidence of positional preferences, annotation information, and functional relevance for inducing correlated gene expression. Our study demonstrates that the currently available resolution of SNP data offers novel ways for the identification of functional genomic elements and the characterization of gene promoter sequences. PMID:24204023

  19. The role of G-quadruplex/i-motif secondary structures as cis-acting regulatory elements

    PubMed Central

    Kendrick, Samantha; Hurley, Laurence H.

    2011-01-01

    The nature of DNA has captivated scientists for more than fifty years. The discovery of the double-helix model of DNA by Watson and Crick in 1953 not only established the primary structure of DNA, but also provided the mechanism behind DNA function. Since then, researchers have continued to further the understanding of DNA structure and its pivotal role in transcription. The demonstration of DNA secondary structure formation has allowed for the proposal that the dynamics of DNA itself can function to modulate transcription. This review presents evidence that DNA can exist in a dynamic equilibrium between duplex and secondary conformations. In addition, data demonstrating that intracellular proteins as well as small molecules can shift this equilibrium in either direction to alter gene transcription will be discussed, with a focus on the modulation of proto-oncogene expression. PMID:21796223

  20. Identification of Regulatory Elements That Control Expression of the tbpBA Operon in Neisseria gonorrhoeae

    PubMed Central

    Vélez Acevedo, Rosuany N.; Ronpirin, Chalinee; Kandler, Justin L.; Shafer, William M.

    2014-01-01

    Iron is an essential nutrient for survival and establishment of infection by Neisseria gonorrhoeae. The neisserial transferrin binding proteins (Tbps) comprise a bipartite system for iron acquisition from human transferrin. TbpA is the TonB-dependent transporter that accomplishes iron internalization. TbpB is a surface-exposed lipoprotein that makes the iron uptake process more efficient. Previous studies have shown that the genes encoding these proteins are arranged in a bicistronic operon, with the tbpB gene located upstream of tbpA and separated from it by an inverted repeat. The operon is under the control of the ferric uptake regulator (Fur); however, promoter elements necessary for regulated expression of the genes have not been experimentally defined. In this study, putative regulatory motifs were identified and confirmed by mutagenesis. Further examination of the sequence upstream of these promoter/operator motifs led to the identification of several novel repeats. We hypothesized that these repeats are involved in additional regulation of the operon. Insertional mutagenesis of regions upstream of the characterized promoter region resulted in decreased tbpB and tbpA transcript levels but increased protein levels for both TbpA and TbpB. Using RNA sequencing (RNA-Seq) technology, we determined that a long RNA was produced from the region upstream of tbpB. We localized the 5′ endpoint of this transcript to between the two upstream insertions by qualitative RT-PCR. We propose that expression of this upstream RNA leads to optimized expression of the gene products from within the tbpBA operon. PMID:24837286

  1. Sterol regulatory element-binding proteins are regulators of the NIS gene in thyroid cells.

    PubMed

    Ringseis, Robert; Rauer, Christine; Rothe, Susanne; Gessner, Denise K; Schütz, Lisa-Marie; Luci, Sebastian; Wen, Gaiping; Eder, Klaus

    2013-05-01

    The uptake of iodide into the thyroid, an essential step in thyroid hormone synthesis, is an active process mediated by the sodium-iodide symporter (NIS). Despite its strong dependence on TSH, the master regulator of the thyroid, the NIS gene was also reported to be regulated by non-TSH signaling pathways. In the present study we provide evidence that the rat NIS gene is subject to regulation by sterol regulatory element-binding proteins (SREBPs), which were initially identified as master transcriptional regulators of lipid biosynthesis and uptake. Studies in FRTL-5 thyrocytes revealed that TSH stimulates expression and maturation of SREBPs and expression of classical SREBP target genes involved in lipid biosynthesis and uptake. Almost identical effects were observed when the cAMP agonist forskolin was used instead of TSH. In TSH receptor-deficient mice, in which TSH/cAMP-dependent gene regulation is blocked, the expression of SREBP isoforms in the thyroid was markedly reduced when compared with wild-type mice. Sterol-mediated inhibition of SREBP maturation and/or RNA interference-mediated knockdown of SREBPs reduced expression of NIS and NIS-specific iodide uptake in FRTL-5 cells. Conversely, overexpression of active SREBPs caused a strong activation of the 5'-flanking region of the rat NIS gene mediated by binding to a functional SREBP binding site located in the 5'-untranslated region of the rat NIS gene. These findings show that TSH acts as a regulator of SREBP expression and maturation in thyroid epithelial cells and that SREBPs are novel transcriptional regulators of NIS. PMID:23542164

  2. Structural Requirements for Sterol Regulatory Element-binding Protein (SREBP) Cleavage in Fission Yeast*

    PubMed Central

    Cheung, Rocky; Espenshade, Peter J.

    2013-01-01

    Sterol regulatory element-binding proteins (SREBPs) are central regulators of cellular lipid synthesis and homeostasis. Mammalian SREBPs are proteolytically activated and liberated from the membrane by Golgi Site-1 and Site-2 proteases. Fission yeast SREBPs, Sre1 and Sre2, employ a different mechanism that genetically requires the Golgi Dsc E3 ligase complex for cleavage activation. Here, we established Sre2 as a model to define structural requirements for SREBP cleavage. We showed that Sre2 cleavage does not require the N-terminal basic helix-loop-helix zipper transcription factor domain, thus separating cleavage of Sre2 from its transcription factor function. From a mutagenesis screen of 94 C-terminal residues of Sre2, we isolated 15 residues required for cleavage and further identified a glycine-leucine sequence required for Sre2 cleavage. Importantly, the glycine-leucine sequence is located at a conserved distance before the first transmembrane segment of both Sre1 and Sre2 and cleavage occurs in between this sequence and the membrane. Bioinformatic analysis revealed a broad conservation of this novel glycine-leucine motif in SREBP homologs of ascomycete fungi, including the opportunistic human pathogen Aspergillus fumigatus where SREBP is required for virulence. Consistent with this, the sequence was also required for cleavage of the oxygen-responsive transcription factor Sre1 and adaptation to hypoxia, demonstrating functional conservation of this cleavage recognition motif. These cleavage mutants will aid identification of the fungal SREBP protease and facilitate functional dissection of the Dsc E3 ligase required for SREBP activation and fungal pathogenesis. PMID:23729666

  3. Activation of Sterol Regulatory Element Binding Factors by Fenofibrate and Gemfibrozil Stimulates Myelination in Zebrafish

    PubMed Central

    Ashikawa, Yoshifumi; Nishimura, Yuhei; Okabe, Shiko; Sasagawa, Shota; Murakami, Soichiro; Yuge, Mizuki; Kawaguchi, Koki; Kawase, Reiko; Tanaka, Toshio

    2016-01-01

    Oligodendrocytes are major myelin-producing cells and play essential roles in the function of a healthy nervous system. However, they are also one of the most vulnerable neural cell types in the central nervous system (CNS), and myelin abnormalities in the CNS are found in a wide variety of neurological disorders, including multiple sclerosis, adrenoleukodystrophy, and schizophrenia. There is an urgent need to identify small molecular weight compounds that can stimulate myelination. In this study, we performed comparative transcriptome analysis to identify pharmacodynamic effects common to miconazole and clobetasol, which have been shown to stimulate myelination by mouse oligodendrocyte progenitor cells (OPCs). Of the genes differentially expressed in both miconazole- and clobetasol-treated mouse OPCs compared with untreated cells, we identified differentially expressed genes (DEGs) common to both drug treatments. Gene ontology analysis revealed that these DEGs are significantly associated with the sterol biosynthetic pathway, and further bioinformatics analysis suggested that sterol regulatory element binding factors (SREBFs) might be key upstream regulators of the DEGs. In silico screening of a public database for chemicals associated with SREBF activation identified fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, as a drug that increases the expression of known SREBF targets, raising the possibility that fenofibrate may also stimulate myelination. To test this, we performed in vivo imaging of zebrafish expressing a fluorescent reporter protein under the control of the myelin basic protein (mbp) promoter. Treatment of zebrafish with fenofibrate significantly increased expression of the fluorescent reporter compared with untreated zebrafish. This increase was attenuated by co-treatment with fatostatin, a specific inhibitor of SREBFs, confirming that the fenofibrate effect was mediated via SREBFs. Furthermore, incubation of zebrafish

  4. Overview of regulatory strategies and molecular elements in metabolic engineering of bacteria.

    PubMed

    Wang, Tianwen; Ma, Xingyuan; Du, Guocheng; Chen, Jian

    2012-11-01

    From a viewpoint of biotechnology, metabolic engineering mainly aims to change the natural status of a pathway in a microorganism towards the overproduction of certain bioproducts. The biochemical nature of a pathway implies us that changed pathway is often the collective results of altered behavior of the metabolic enzymes encoded by corresponding genes. By finely modulating the expression of these genes or the properties of the enzyme, we can gain efficient control on the pathway. In this article, we reviewed the typical methods that have been applied to regulate the expression of genes in metabolic engineering. These methods are grouped according to the operation targets in a typical gene. The transcription of a gene is controlled by an indispensable promoter. By utilizing promoters with different strengths, expected levels of expression can be easily achieved, and screening a promoter library may find suitable mutant promoters that can provide tunable expression of a gene. Auto-responsive promoter (quorum sensing (QS)-based or oxygen-inducible) simplifies the induction process by driving the expression of a gene in an automated manner. Light responsive promoter enables reversible and noninvasive control on gene activity, providing a promising method in controlling gene expression with time and space resolution in metabolic engineering involving complicated genetic circuits. Through directed evolution and/or rational design, the encoding sequences of a gene can be altered, leading to the possibly most profound changes in properties of a metabolic enzyme. Introducing an engineered riboswitch in mRNA can make it a regulatory molecule at the same time; ribosomal binding site is commonly engineered to be more attractive for a ribosome through design. Terminator of a gene will affect the stability of an mRNA, and intergenic region will influence the expression of many related genes. Improving the performance of these elements are generally the main activities in

  5. The Responses of Arabidopsis Early Light-Induced Protein2 to Ultraviolet B, High Light, and Cold Stress Are Regulated by a Transcriptional Regulatory Unit Composed of Two Elements1[OPEN

    PubMed Central

    Hayami, Natsuki; Sakai, Yusaku; Kimura, Mitsuhiro; Saito, Tatsunori; Tokizawa, Mutsutomo; Iuchi, Satoshi; Kurihara, Yukio; Matsui, Minami; Nomoto, Mika; Tada, Yasuomi; Yamamoto, Yoshiharu Y.

    2015-01-01

    The Arabidopsis (Arabidopsis thaliana) Early Light-Induced Protein (ELIP) is thought to act as a photoprotectant, reducing the damaging effects of high light (HL). Expression of ELIP2 is activated by multiple environmental stresses related to photoinhibition. We have identified putative regulatory elements in an ELIP2 promoter using an octamer-based frequency comparison method, analyzed the role of these elements using synthetic promoters, and revealed a key transcriptional regulatory unit for ultraviolet B (UV-B) radiation, HL, and cold stress responses. The unit is composed of two elements, designated as Elements A (TACACACC) and B (GGCCACGCCA), and shows functionality only when paired. Our genome-wide correlation analysis between possession of these elements in the promoter region and expression profiles in response to UV-B, HL, and cold suggests that Element B receives and integrates these multiple stress signals. In vitro protein-DNA binding assays revealed that LONG HYPOCOTYL5 (HY5), a basic domain-Leucine zipper transcription factor, directly binds to Element B. In addition, mutant analysis of HY5 showed partial involvement in the UV-B and HL responses but not in the cold stress response. These results suggest that signals for UV-B, HL, and cold stress join at Element B, which recognizes the signals of multiple transcription factors, including HY5. PMID:26175515

  6. The Responses of Arabidopsis Early Light-Induced Protein2 to Ultraviolet B, High Light, and Cold Stress Are Regulated by a Transcriptional Regulatory Unit Composed of Two Elements.

    PubMed

    Hayami, Natsuki; Sakai, Yusaku; Kimura, Mitsuhiro; Saito, Tatsunori; Tokizawa, Mutsutomo; Iuchi, Satoshi; Kurihara, Yukio; Matsui, Minami; Nomoto, Mika; Tada, Yasuomi; Yamamoto, Yoshiharu Y

    2015-09-01

    The Arabidopsis (Arabidopsis thaliana) Early Light-Induced Protein (ELIP) is thought to act as a photoprotectant, reducing the damaging effects of high light (HL). Expression of ELIP2 is activated by multiple environmental stresses related to photoinhibition. We have identified putative regulatory elements in an ELIP2 promoter using an octamer-based frequency comparison method, analyzed the role of these elements using synthetic promoters, and revealed a key transcriptional regulatory unit for ultraviolet B (UV-B) radiation, HL, and cold stress responses. The unit is composed of two elements, designated as Elements A (TACACACC) and B (GGCCACGCCA), and shows functionality only when paired. Our genome-wide correlation analysis between possession of these elements in the promoter region and expression profiles in response to UV-B, HL, and cold suggests that Element B receives and integrates these multiple stress signals. In vitro protein-DNA binding assays revealed that LONG HYPOCOTYL5 (HY5), a basic domain-Leucine zipper transcription factor, directly binds to Element B. In addition, mutant analysis of HY5 showed partial involvement in the UV-B and HL responses but not in the cold stress response. These results suggest that signals for UV-B, HL, and cold stress join at Element B, which recognizes the signals of multiple transcription factors, including HY5. PMID:26175515

  7. Bladder inflammatory transcriptome in response to tachykinins: Neurokinin 1 receptor-dependent genes and transcription regulatory elements

    PubMed Central

    Saban, Ricardo; Simpson, Cindy; Vadigepalli, Rajanikanth; Memet, Sylvie; Dozmorov, Igor; Saban, Marcia R

    2007-01-01

    Background Tachykinins (TK), such as substance P, and their neurokinin receptors which are ubiquitously expressed in the human urinary tract, represent an endogenous system regulating bladder inflammatory, immune responses, and visceral hypersensitivity. Increasing evidence correlates alterations in the TK system with urinary tract diseases such as neurogenic bladders, outflow obstruction, idiopathic detrusor instability, and interstitial cystitis. However, despite promising effects in animal models, there seems to be no published clinical study showing that NK-receptor antagonists are an effective treatment of pain in general or urinary tract disorders, such as detrusor overactivity. In order to search for therapeutic targets that could block the tachykinin system, we set forth to determine the regulatory network downstream of NK1 receptor activation. First, NK1R-dependent transcripts were determined and used to query known databases for their respective transcription regulatory elements (TREs). Methods An expression analysis was performed using urinary bladders isolated from sensitized wild type (WT) and NK1R-/- mice that were stimulated with saline, LPS, or antigen to provoke inflammation. Based on cDNA array results, NK1R-dependent genes were selected. PAINT software was used to query TRANSFAC database and to retrieve upstream TREs that were confirmed by electrophoretic mobility shift assays. Results The regulatory network of TREs driving NK1R-dependent genes presented cRel in a central position driving 22% of all genes, followed by AP-1, NF-kappaB, v-Myb, CRE-BP1/c-Jun, USF, Pax-6, Efr-1, Egr-3, and AREB6. A comparison between NK1R-dependent and NK1R-independent genes revealed Nkx-2.5 as a unique discriminator. In the presence of NK1R, Nkx2-5 _01 was significantly correlated with 36 transcripts which included several candidates for mediating bladder development (FGF) and inflammation (PAR-3, IL-1R, IL-6, α-NGF, TSP2). In the absence of NK1R, the matrix Nkx2

  8. Characterization of a Disease-associated Mutation Affecting a Putative Splicing Regulatory Element in Intron 6b of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene*

    PubMed Central

    Faà, Valeria; Incani, Federica; Meloni, Alessandra; Corda, Denise; Masala, Maddalena; Baffico, A. Maria; Seia, Manuela; Cao, Antonio; Rosatelli, M. Cristina

    2009-01-01

    Cystic fibrosis (CF) is a common recessive disorder caused by >1600 mutations in the CF transmembrane conductance regulator (CFTR) gene. About 13% of CFTR mutations are classified as “splicing mutations,” but for almost 40% of these, their role in affecting the pre-mRNA splicing of the gene is not yet defined. In this work, we describe a new splicing mutation detected in three unrelated Italian CF patients. By DNA analyses and mRNA studies, we identified the c.1002–1110_1113delTAAG mutation localized in intron 6b of the CFTR gene. At the mRNA level, this mutation creates an aberrant inclusion of a sequence of 101 nucleotides between exons 6b and 7. This sequence corresponds to a portion of intron 6b and resembles a cryptic exon because it is characterized by an upstream ag and a downstream gt sequence, which are most probably recognized as 5′- and 3′-splice sites by the spliceosome. Through functional analysis of this splicing defect, we show that this mutation abolishes the interaction of the splicing regulatory protein heterogeneous nuclear ribonucleoprotein A2/B1 with an intronic splicing regulatory element and creates a new recognition motif for the SRp75 splicing factor, causing activation of the cryptic exon. Our results show that the c.1002–1110_1113delTAAG mutation creates a new intronic splicing regulatory element in intron 6b of the CFTR gene exclusively recognized by SRp75. PMID:19759008

  9. Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes

    PubMed Central

    Barber-Zucker, Shiran; Gordân, Raluca; Lukatsky, David B.

    2015-01-01

    Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF) binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs) have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq) results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large-scale analysis using

  10. Sequence analysis of Vicia faba repeated DNA, the FokI repeat element.

    PubMed Central

    Kato, A; Yakura, K; Tanifuji, S

    1984-01-01

    A type of highly repeated DNA sequences present in the genome of Vicia faba was detected by digestion its nuclear DNA with FokI endonuclease and fractionating the digests on polyacrylamide gels. Four fragments of 59, 108, 177 and 246 bp of the FokI repeated sequences were collected from the gels and their primary structures were determined by the method of Maxam and Gilbert. These repeated DNA sequences were shown to be a multiple tandem array of a 59 bp sequence element. And its nucleotide sequence was almost completely conserved among all the sequence members of each the size class and also among these classes. This sequence element consists of a duplet of an about the duplet has an incomplete dyad symmetrical structure. Images PMID:6089113

  11. Analysis of genetic elements controlling Staphylococcus aureus lrgAB expression: potential role of DNA topology in SarA regulation.

    PubMed

    Fujimoto, D F; Brunskill, E W; Bayles, K W

    2000-09-01

    Penicillin-induced killing and murein hydrolase activity in Staphylococcus aureus are dependent on a variety of regulatory elements, including the LytSR two-component regulatory system and the virulence factor regulators Agr and Sar. The LytSR effects on these processes can be explained, in part, by the recent finding that a LytSR-regulated operon, designated lrgAB, affects murein hydrolase activity and penicillin tolerance. To examine the regulation of lrgAB expression in greater detail, we performed Northern blot and promoter fusion analyses. Both methods revealed that Agr and Sar, like LytSR, positively regulate lrgAB expression. A mutation in the agr locus reduced lrgAB expression approximately sixfold, while the sar mutation reduced lrgAB expression to undetectable levels. cis-acting regulatory elements involved in lrgAB expression were identified by fusing various fragments of the lrgAB promoter region to the xylE reporter gene and integrating these constructs into the chromosome. Catechol 2,3-dioxygenase assays identified DNA sequences, including an inverted repeat and intrinsic bend sites, that contribute to maximal lrgAB expression. Confirmation of the importance of the inverted repeat was achieved by demonstrating that multiple copies of the inverted repeat reduced lrgAB promoter activity, presumably by titrating out a positive regulatory factor. The results of this study demonstrate that lrgAB expression responds to a variety of positive regulatory factors and suggest that specific DNA topology requirements are important for optimal expression. PMID:10940023

  12. Genome-wide DNA methylation patterns in LSH mutant reveals de-repression of repeat elements and redundant epigenetic silencing pathways

    PubMed Central

    Yu, Weishi; McIntosh, Carl; Lister, Ryan; Zhu, Iris; Han, Yixing; Ren, Jianke; Landsman, David; Lee, Eunice; Briones, Victorino; Terashima, Minoru; Leighty, Robert; Ecker, Joseph R.

    2014-01-01

    Cytosine methylation is critical in mammalian development and plays a role in diverse biologic processes such as genomic imprinting, X chromosome inactivation, and silencing of repeat elements. Several factors regulate DNA methylation in early embryogenesis, but their precise role in the establishment of DNA methylation at a given site remains unclear. We have generated a comprehensive methylation map in fibroblasts derived from the murine DNA methylation mutant Hells−/− (helicase, lymphoid specific, also known as LSH). It has been previously shown that HELLS can influence de novo methylation of retroviral sequences and endogenous genes. Here, we describe that HELLS controls cytosine methylation in a nuclear compartment that is in part defined by lamin B1 attachment regions. Despite widespread loss of cytosine methylation at regulatory sequences, including promoter regions of protein-coding genes and noncoding RNA genes, overall relative transcript abundance levels in the absence of HELLS are similar to those in wild-type cells. A subset of promoter regions shows increases of the histone modification H3K27me3, suggesting redundancy of epigenetic silencing mechanisms. Furthermore, HELLS modulates CG methylation at all classes of repeat elements and is critical for repression of a subset of repeat elements. Overall, we provide a detailed analysis of gene expression changes in relation to DNA methylation alterations, which contributes to our understanding of the biological role of cytosine methylation. PMID:25170028

  13. GSEL version 2, an online genome-wide query system of operon organization and regulatory sequence elements of Geobacter sulfurreducens.

    PubMed

    Qu, Yanhua; Brown, Peter; Barbe, Jose F; Puljic, Marko; Merino, Enrique; Adkins, Ronald M; Lovley, Derek R; Krushkal, Julia

    2009-10-01

    Geobacter sulfurreducens is a model organism within the delta-Proteobacterial family Geobacteraceae, members of which can participate in environmental bioremediation of metal and organic waste contaminants and in production of bioenergy. In this report, we describe a new, significantly expanded and updated, version 2 of the GSEL (Geobacter Sequence Elements) database ( http://geobacter.org/research/gsel2/ and http://geobacter.org/refs/gsel2/ ) and its accompanying online query system, which compiles information on operon organization and regulatory sequence elements in the genome of G. sulfurreducens. It incorporates a new online graphical browser, provides novel search capabilities, and includes updated operon predictions along with new information on predicted and experimentally validated genome regulatory sites. The GSEL database and online search system provides a unique and comprehensive tool cataloging information about gene regulation in G. sulfurreducens, aiding in investigation of mechanisms that regulate its ability to generate electric power, bioremediate environmental waste, and adapt to environmental changes. PMID:19792871

  14. Microevolution of cis-regulatory elements: an example from the pair-rule segmentation gene fushi tarazu in the Drosophila melanogaster subgroup.

    PubMed

    Bakkali, Mohammed

    2011-01-01

    The importance of non-coding DNAs that control transcription is ever noticeable, but the characterization and analysis of the evolution of such DNAs presents challenges not found in the analysis of coding sequences. In this study of the cis-regulatory elements of the pair rule segmentation gene fushi tarazu (ftz) I report the DNA sequences of ftz's zebra element (promoter) and a region containing the proximal enhancer from a total of 45 fly lines belonging to several populations of the species Drosophila melanogaster, D. simulans, D. sechellia, D. mauritiana, D. yakuba, D. teissieri, D. orena and D. erecta. Both elements evolve at slower rate than ftz synonymous sites, thus reflecting their functional importance. The promoter evolves more slowly than the average for ftz's coding sequence while, on average, the enhancer evolves more rapidly, suggesting more functional constraint and effective purifying selection on the former. Comparative analysis of the number and nature of base substitutions failed to detect significant evidence for positive/adaptive selection in transcription-factor-binding sites. These seem to evolve at similar rates to regions not known to bind transcription factors. Although this result reflects the evolutionary flexibility of the transcription factor binding sites, it also suggests a complex and still not completely understood nature of even the characterized cis-regulatory sequences. The latter seem to contain more functional parts than those currently identified, some of which probably transcription factor binding. This study illustrates ways in which functional assignments of sequences within cis-acting sequences can be used in the search for adaptive evolution, but also highlights difficulties in how such functional assignment and analysis can be carried out. PMID:22073317

  15. Regulatory elements of the EKLF gene that direct erythroid cell-specific expression during mammalian development.

    PubMed

    Xue, Li; Chen, Xiaoyong; Chang, Yanjie; Bieker, James J

    2004-06-01

    Erythroid Krüppel-like factor (EKLF) plays an essential role in enabling beta-globin expression during erythroid ontogeny. It is first expressed in the extraembryonic mesoderm of the yolk sac within the morphologically unique cells that give rise to the blood islands, and then later within the hepatic primordia. The BMP4/Smad pathway plays a critical role in the induction of EKLF, and transient transfection analyses demonstrate that sequences located within less than 1 kb of its transcription initiation site are sufficient for high-level erythroid-specific transcription. We have used transgenic analyses to verify that 950 bp located adjacent to the EKLF start site of transcription is sufficient to generate lacZ expression within the blood islands as well as the fetal liver during embryonic development. Of particular importance are 3 regions, 2 of which overlap endogenous erythroid-specific DNase hypersensitive sites, and 1 of which includes the proximal promoter region. The onset of transgene expression mimics that of endogenous EKLF as it begins by day 7.5 (d7.5) to d8.0. In addition, it exhibits a strict hematopoietic specificity, localized only to these cells and not to the adjacent vasculature at all stages examined. Finally, expression is heterocellular, implying that although these elements are sufficient for tissue-specific expression, they do not shield against the position effects of adjacent chromatin. These analyses demonstrate that a surprisingly small DNA segment contains all the information needed to target a linked gene to the hematopoietic compartment at both early and later stages of development, and may be a useful cassette for this purpose. PMID:14764531

  16. A bidirectional promoter reporter vector for the analysis of the p53/WDR79 dual regulatory element.

    PubMed

    Polson, Amanda; Durrett, Emily; Reisman, David

    2011-09-01

    Analysis of numerous genomes has identified a class of regulatory regions that contain a head-to-head arrangement (5' to 5') on opposite strands of DNA. Often these regulatory regions have fewer than 1000 base pairs separating their corresponding transcription start sites and have been termed as being "bidirectional". This bidirectional arrangement and the divergent gene pairs under the control of these regulatory regions appear to be a common feature within genomes. Establishing methods to study these bidirectional transcriptional promoters, and understanding how they are regulated will allow researchers to gain more insight into the roles that divergent transcription plays in the expression and maintenance of protein coding genes. Recently, the p53 tumor suppressor gene was shown to have a bidirectional gene partner, WDR79. The transcription start sites (TSSs) of human and murine p53 and WDR79 genes are separated by approximately 800 and 930bp, respectively, in a head-to-head fashion, and fit the criteria of what is designated to be a putative bidirectional regulatory region. However, further testing is needed to demonstrate that the region between these genes contains a functional bidirectional promoter. Here, we have developed a bidirectional reporter vector, termed pLucRLuc, to study the transcriptional output of each promoter. This bidirectional reporter vector will allow researchers to determine the output of transcripts mediated by the bidirectional promoters. By focusing our studies on the transcriptional regulation of p53 and its bidirectional gene partner, WDR79, we hope to elucidate key factors that can control and regulate the expression of the p53 and WDR79 genes. Here, we demonstrate that pLucRLuc is a vector capable of expressing reporter genes under the control of bidirectional promoters in multiple human and murine cell lines and that the regulatory region upstream of the p53 and WDR79 TSSs is a bidirectional promoter controlled by common

  17. Identification of a non-canonical E-box motif as a regulatory element in the proximal promoter region of the apolipoprotein E gene.

    PubMed Central

    Salero, Enrique; Giménez, Cecilio; Zafra, Francisco

    2003-01-01

    We have used the yeast one-hybrid system to identify transcription factors with binding capability to specific sequences in proximal regions of the apolipoprotein E gene ( APOE ) promoter. The sequence between -113 and -80 nt, which contains regulatory elements in various cell types, was used as a bait to screen a human brain cDNA library. Four cDNA clones that encoded portions of the human upstream-stimulatory-factor (USF) transcription factor were isolated. Electrophoretic-mobility-shift assays ('EMSAs') using nuclear extracts from various human cell lines as well as from rat brain and liver revealed the formation of two DNA-protein complexes within the sequence CACCTCGTGAC (region -101/-91 of the APOE promoter) that show similarity to the E-box element. The retarded complexes contained USF1, as deduced from competition and supershift assays. Functional experiments using different APOE promoter-luciferase reporter constructs transiently transfected into U87, HepG2 or HeLa cell lines showed that mutations that precluded the formation of complexes decreased the basal activity of the promoter by about 50%. Overexpression of USF1 in U87 glioblastoma cells led to an increased activity of the promoter that was partially mediated by the atypical E-box. The stimulatory effect of USF1 was cell-type specific, as it was not observed in hepatoma HepG2 cells. Similarly, overexpression of a USF1 dominant-negative mutant decreased the basal activity of the promoter in glioblastoma, but not in hepatoma, cells. These data indicated that USF, and probably other related transcription factors, might be involved in the basal transcriptional machinery of APOE by binding to a non-canonical E-box motif within the proximal promoter. PMID:12444925

  18. Dimerization of the human papillomavirus type 16 E2 N terminus results in DNA looping within the upstream regulatory region.

    PubMed

    Hernandez-Ramon, Elena E; Burns, Julie E; Zhang, Wenke; Walker, Hannah F; Allen, Stephanie; Antson, Alfred A; Maitland, Norman J

    2008-05-01

    Papillomavirus E2 proteins play a central role in regulating viral gene expression and replication. DNA-binding activity is associated with the C-terminal domain of E2, which forms a stable dimer, while the N-terminal domain is responsible for E2's replication and transactivation functions. The crystal structure of the latter domain revealed a second dimerization interface on E2 which may be responsible for DNA loop formation in the regulatory region of the human papillomavirus (HPV) genome. We investigated the biological significance of the N-terminal dimerization by introducing single amino acid substitutions into the dimerization interface. As expected, these substitutions did not influence the C-terminal dimerization and DNA-binding functions of E2. However, the mutations led to reduced transactivation of a synthetic E2-responsive reporter gene, while HPV DNA replication was unaffected. The effect of the mutations on DNA looping was visualized by atomic force microscopy. While wild-type E2 was able to generate DNA loops, all three mutant E2 proteins were defective in this ability. Our results suggest that N-terminal dimerization plays a role in E2-mediated transactivation, probably via DNA looping, a common mechanism for remote regulation of gene transcription. PMID:18337573

  19. DNA Elements Reducing Transcriptional Gene Silencing Revealed by a Novel Screening Strategy

    PubMed Central

    Ueno, Keiichiro; Ohashi, Yuko; Mitsuhara, Ichiro

    2013-01-01

    Transcriptional gene silencing (TGS)–a phenomenon observed in endogenous genes/transgenes in eukaryotes–is a huge hindrance to transgenic technology and occurs mainly when the genes involved share sequence homology in their promoter regions. TGS depends on chromosomal position, suggesting the existence of genomic elements that suppress TGS. However, no systematic approach to identify such DNA elements has yet been reported. Here, we developed a successful novel screening strategy to identify such elements (anti-silencing regions–ASRs), based on their ability to protect a flanked transgene from TGS. A silenced transgenic tobacco plant in which a subsequently introduced transgene undergoes obligatory promoter-homology dependent TGS in trans allowed the ability of DNA elements to prevent TGS to be used as the screening criterion. We also identified ASRs in a genomic library from a different plant species (Lotus japonicus: a perennial legume); the ASRs include portions of Ty1/copia retrotransposon-like and pararetrovirus-like sequences; the retrotransposon-like sequences also showed interspecies anti-TGS activity in a TGS-induction system in Arabidopsis. Anti-TGS elements could provide effective tools to reduce TGS and ensure proper regulation of transgene expression. Furthermore, the screening strategy described here will also facilitate the efficient identification of new classes of anti-TGS elements. PMID:23382937

  20. A new regulatory element modulates homoserine lactone-mediated autoinduction of Ti plasmid conjugal transfer.

    PubMed Central

    Hwang, I; Cook, D M; Farrand, S K

    1995-01-01

    Conjugal transfer of the Agrobacterium tumefaciens nopaline-type Ti plasmid pTiC58 is induced by agrocinopines A and B, opines secreted by crown gall tumors induced by the bacterium. This regulation functions through the transcriptional repressor, AccR. However, actual transcription of the tra genes is regulated by autoinduction through the activator TraR and the substituted homoserine lactone second messenger, Agrobacterium autoinducer (AAI). We have identified a new regulatory element that modulates the response of TraR to AAI. The gene, called traM, suppresses TraR-AAI activation of transcription of tra genes carried on recombinant clones. The suppression could be relieved by increasing the expression of TraR but not by increasing AAI levels. traM is located between traR and traAF on pTiC58 and is transcribed in the clockwise direction. The 306-bp gene encodes an 11.2-kDa protein showing no significant relatedness to other proteins in the databases. Mutations in traM in pTiC58 conferred a transfer-constitutive phenotype, and strains harboring the Ti plasmid produced easily detectable amounts of AAI. These same mutations engineered into the transfer-constitutive Ti plasmid pTiC58 delta accR conferred a hyperconjugal phenotype and very high levels of AAI production. Expression of traM required TraR, indicating that transcription of the gene is regulated by the autoinduction system. TraM had no effect on the expression of traR, demonstrating that the suppressive effect is not due to repression of the gene encoding the activator. These results suggest that TraM is not a direct transcriptional regulator. Since the suppressive effect is demonstrable only when traM is overexpressed with respect to traR, we suggest that TraM functions to sequester TraR from the very small amounts of AAI produced under conditions when the agrocinopines are not present. PMID:7814335

  1. Mavericks, a novel class of giant transposable elements widespread in eukaryotes and related to DNA viruses.

    PubMed

    Pritham, Ellen J; Putliwala, Tasneem; Feschotte, Cédric

    2007-04-01

    We previously identified a group of atypical mobile elements designated Mavericks from the nematodes Caenorhabditis elegans and C. briggsae and the zebrafish Danio rerio. Here we present the results of comprehensive database searches of the genome sequences available, which reveal that Mavericks are widespread in invertebrates and non-mammalian vertebrates but show a patchy distribution in non-animal species, being present in the fungi Glomus intraradices and Phakopsora pachyrhizi and in several single-celled eukaryotes such as the ciliate Tetrahymena thermophila, the stramenopile Phytophthora infestans and the trichomonad Trichomonas vaginalis, but not detectable in plants. This distribution, together with comparative and phylogenetic analyses of Maverick-encoded proteins, is suggestive of an ancient origin of these elements in eukaryotes followed by lineage-specific losses and/or recurrent episodes of horizontal transmission. In addition, we report that Maverick elements have amplified recently to high copy numbers in T. vaginalis where they now occupy as much as 30% of the genome. Sequence analysis confirms that most Mavericks encode a retroviral-like integrase, but lack other open reading frames typically found in retroelements. Nevertheless, the length and conservation of the target site duplication created upon Maverick insertion (5- or 6-bp) is consistent with a role of the integrase-like protein in the integration of a double-stranded DNA transposition intermediate. Mavericks also display long terminal-inverted repeats but do not contain ORFs similar to proteins encoded by DNA transposons. Instead, Mavericks encode a conserved set of 5 to 9 genes (in addition to the integrase) that are predicted to encode proteins with homology to replication and packaging proteins of some bacteriophages and diverse eukaryotic double-stranded DNA viruses, including a DNA polymerase B homolog and putative capsid proteins. Based on these and other structural similarities, we

  2. Characterization of a putative cis-regulatory element that controls transcriptional activity of the pig uroplakin II gene promoter

    SciTech Connect

    Kwon, Deug-Nam; Park, Mi-Ryung; Park, Jong-Yi; Cho, Ssang-Goo; Park, Chankyu; Oh, Jae-Wook; Song, Hyuk; Kim, Jae-Hwan; Kim, Jin-Hoi

    2011-07-01

    Highlights: {yields} The sequences of -604 to -84 bp of the pUPII promoter contained the region of a putative negative cis-regulatory element. {yields} The core promoter was located in the 5F-1. {yields} Transcription factor HNF4 can directly bind in the pUPII core promoter region, which plays a critical role in controlling promoter activity. {yields} These features of the pUPII promoter are fundamental to development of a target-specific vector. -- Abstract: Uroplakin II (UPII) is a one of the integral membrane proteins synthesized as a major differentiation product of mammalian urothelium. UPII gene expression is bladder specific and differentiation dependent, but little is known about its transcription response elements and molecular mechanism. To identify the cis-regulatory elements in the pig UPII (pUPII) gene promoter region, we constructed pUPII 5' upstream region deletion mutants and demonstrated that each of the deletion mutants participates in controlling the expression of the pUPII gene in human bladder carcinoma RT4 cells. We also identified a new core promoter region and putative negative cis-regulatory element within a minimal promoter region. In addition, we showed that hepatocyte nuclear factor 4 (HNF4) can directly bind in the pUPII core promoter (5F-1) region, which plays a critical role in controlling promoter activity. Transient cotransfection experiments showed that HNF4 positively regulates pUPII gene promoter activity. Thus, the binding element and its binding protein, HNF4 transcription factor, may be involved in the mechanism that specifically regulates pUPII gene transcription.

  3. Polymorphism of t-specific DNA elements of the proximal part of Mus mouse chromosome 17

    SciTech Connect

    Shustrova, I.V.; Tokarskaya, O.N.; Chekunova, A.I.

    1995-05-01

    To study structural organization and polymorphism of the proximal part of chromosome 17, a hybridization analysis of DNA from mice of different origin was carried out using four t-specific probes. Results of the analysis allow us to conclude that the DNA element copy number is quantitatively unstable and differs in distribution in both newly formed recombinant haplotypes and in wild-type chromosome 17. Probe Tu66 was used to study D17Leh66-element organization of Mus abbotti and Mus hortulanus mice. Three types of D17Leh66-elements were identified in the genomes of these species. The copy number of each type of DNA element varied in the genome of each of four studied species. Homologs to t-specific Tu66 and Tu119 probes were found in the genome of Rattus norvegicus rat. The data obtained are discussed with respect to the evolution of the proximal part of Mus mouse chromosome 17. 29 refs., 5 figs., 2 tabs.

  4. An Ssn6-Tup1-dependent negative regulatory element controls sporulation-specific expression of DIT1 and DIT2 in Saccharomyces cerevisiae.

    PubMed Central

    Friesen, H; Hepworth, S R; Segall, J

    1997-01-01

    Sporulation of the yeast Saccharomyces cerevisiae is a process of cellular differentiation that occurs in MATa/MAT alpha diploid cells in response to starvation. The sporulation-specific genes DIT1 and DIT2, which are required for spore wall formation, are activated midway through the sporulation program, with maximal transcript accumulation occurring at the time of prospore enclosure. In this study, we have identified a negative regulatory element, termed NREDIT, that is located between the start sites of transcription of these divergently transcribed genes. This element, which prevents expression of the DIT1 and DIT2 genes during vegetative growth, reduces expression of a CYC1-lacZ reporter gene more than 1,000-fold and acts in an orientation- and position-independent manner. We found that the ability of NREDIT to turn of expression of the reporter gene and the chromosomal DIT1 and DIT2 genes in vegetative cells requires the Ssn6-Tup1 repression complex. Interestingly, NREDIT-mediated repression of the reporter gene is maintained during sporulation. Derepression during sporulation requires complex interactions among several cis-acting elements. These are present on an approximately 350-bp DNA fragment extending from NREDIT to the TATA box and an approximately 125-bp fragment spanning the TATA box of DIT1. Additionally, a region of NREDIT which is very similar in sequence to UASSPS4, an element that activates gene expression midway through sporulation, contributes both to vegetative repression and to sporulation-specific induction of DIT1. We propose a model to explain the requirement for multiple elements in overcoming NREDIT-mediated repression during sporulation. PMID:8972192

  5. Postproliferative transcription of the rat osteocalcin gene is reflected by vitamin D-responsive developmental modifications in protein-DNA interactions at basal and enhancer promoter elements.

    PubMed Central

    Owen, T A; Bortell, R; Shalhoub, V; Heinrichs, A; Stein, J L; Stein, G S; Lian, J B

    1993-01-01

    In the osteocalcin (OC) gene promoter, both independent positive and negative regulatory elements, as well as others with contiguous [TATA/glucocorticoid-responsive elements (GRE)] or overlapping [TATA/GRE, vitamin D-responsive enhancer elements (VDRE)/AP-1, and OC box/AP-1] domains, are sites for modifications in protein-DNA interactions. In the present studies, we have examined nuclear protein extracts from fetal rat calvarial cells that undergo a developmental sequence of bone cell differentiation. Our results demonstrate modifications in protein-DNA interactions that relate to the developmental stages of the osteoblast and support developmental regulation of OC gene transcription. Basal expression of the OC gene is associated with sequence-specific protein-DNA interactions at the OC box, VDRE, and TATA/GRE box. Distinct differences are observed in proliferating osteoblasts, where the OC gene is not transcribed compared to postproliferative, differentiated osteoblasts that transcribe the OC gene. Furthermore, the protein-DNA complexes that reflect hormonal control are also developmentally regulated, mediating both the transcriptionally active and repressed states of the OC gene. For example, in proliferating osteoblasts, a vitamin D receptor-antibody-sensitive complex is formed that is different from the DNA binding complex induced by vitamin D postproliferatively when the OC gene is transcribed. Mutational analysis of the steroid hormone binding domain and the overlapping AP-1 site at the VDRE supports mutually exclusive occupancy by Fos-Jun heterodimers and vitamin D receptor. Such protein-DNA interactions at the VDRE are consistent with repression of competency for vitamin D-mediated transcriptional enhancement in proliferating osteoblasts expressing high levels of Fos and Jun. Images PMID:8381969

  6. Characterization of a novel positive transcription regulatory element that differentially regulates the alpha-2-macroglobulin gene in replicative senescence.

    PubMed

    Li, Renzhong; Ma, Liwei; Huang, Yu; Zhang, Zongyu; Tong, Tanjun

    2011-12-01

    Alpha-2-macroglobulin (α2M), a protease inhibitor, is implicated in Alzheimer's disease, atherosclerosis, and other age-related diseases. The elevated level of α2M mRNA has been described in replicative senescence and it could be used as a biomarker of the aging cells. However, the mechanism responsible for the up-regulation of its expression is still unclear. This report identified a novel transcriptional regulatory element, the α2M transcription enhancement element (ATEE), within the α2M promoter. This element differentially activates α2M expression in senescent versus young fibroblasts. Electrophoretic mobility shift assays revealed abundant complexes in senescent cell nuclear extracts compared with young cell nuclear extracts. The DNase I footprint revealed the protein-binding core sequence through which the protein binds the ATEE. Mutation within ATEE selectively abolished α2M promoter activity in senescent (but not young) cells. These results indicated the ATEE, as a positive transcription regulatory element, contributes to the up-regulation of α2M during replicative senescence. PMID:21541797

  7. Solution structure of stem-loop α of the hepatitis B virus post-transcriptional regulatory element

    PubMed Central

    Schwalbe, Martin; Ohlenschläger, Oliver; Marchanka, Aliaksandr; Ramachandran, Ramadurai; Häfner, Sabine; Heise, Tilman; Görlach, Matthias

    2008-01-01

    Chronic hepatitis B virus (HBV) infections may lead to severe diseases like liver cirrhosis or hepatocellular carcinoma (HCC). The HBV post-transcriptional regulatory element (HPRE) facilitates the nuclear export of unspliced viral mRNAs, contains a splicing regulatory element and resides in the 3′-region of all viral transcripts. The HPRE consists of three sub-elements α (nucleotides 1151–1346), β1 (nucleotides 1347–1457) and β2 (nucleotides 1458–1582), which confer together full export competence. Here, we present the NMR solution structure (pdb 2JYM) of the stem-loop α (SLα, nucleotides 1292–1321) located in the sub-element α. The SLα contains a CAGGC pentaloop highly conserved in hepatoviruses, which essentially adopts a CUNG-like tetraloop conformation. Furthermore, the SLα harbours a single bulged G residue flanked by A-helical regions. The structure is highly suggestive of serving two functions in the context of export of unspliced viral RNA: binding sterile alpha motif (SAM-) domain containing proteins and/or preventing the utilization of a 3′-splice site contained within SLα. PMID:18263618

  8. Differential contribution of cis-regulatory elements to higher order chromatin structure and expression of the CFTR locus.

    PubMed

    Yang, Rui; Kerschner, Jenny L; Gosalia, Nehal; Neems, Daniel; Gorsic, Lidija K; Safi, Alexias; Crawford, Gregory E; Kosak, Steven T; Leir, Shih-Hsing; Harris, Ann

    2016-04-20

    Higher order chromatin structure establishes domains that organize the genome and coordinate gene expression. However, the molecular mechanisms controlling transcription of individual loci within a topological domain (TAD) are not fully understood. The cystic fibrosis transmembrane conductance regulator (CFTR) gene provides a paradigm for investigating these mechanisms.CFTR occupies a TAD bordered by CTCF/cohesin binding sites within which are cell-type-selective cis-regulatory elements for the locus. We showed previously that intronic and extragenic enhancers, when occupied by specific transcription factors, are recruited to the CFTR promoter by a looping mechanism to drive gene expression. Here we use a combination of CRISPR/Cas9 editing of cis-regulatory elements and siRNA-mediated depletion of architectural proteins to determine the relative contribution of structural elements and enhancers to the higher order structure and expression of the CFTR locus. We found the boundaries of the CFTRTAD are conserved among diverse cell types and are dependent on CTCF and cohesin complex. Removal of an upstream CTCF-binding insulator alters the interaction profile, but has little effect on CFTR expression. Within the TAD, intronic enhancers recruit cell-type selective transcription factors and deletion of a pivotal enhancer element dramatically decreases CFTR expression, but has minor effect on its 3D structure. PMID:26673704

  9. Increasing the dynamic control space of mammalian transcription devices by combinatorial assembly of homologous regulatory elements from different bacterial species.

    PubMed

    Bacchus, William; Weber, Wilfried; Fussenegger, Martin

    2013-01-01

    Prokaryotic transcriptional regulatory elements are widely utilized building blocks for constructing regulatory genetic circuits adapted for mammalian cells and have found their way into a broad range of biotechnological applications. Prokaryotic transcriptional repressors, fused to eukaryotic transactivation or repression domains, compose the transcription factor, which binds and adjusts transcription from chimeric promoters containing the repressor-specific operator sequence. Escherichia coli and Chlamydia trachomatis share common features in the regulatory mechanism of the biosynthesis of l-tryptophan. The repressor protein TrpR of C. trachomatis regulates the trpRBA operon and the TrpR of E. coli regulates the trpEDCBA operon, both requiring l-tryptophan as a co-repressor. Fusion of these bacterial repressors to the VP16 transactivation domain of Herpes simplex virus creates synthetic transactivators that could bind and activate chimeric promoters, assembled by placing repressor-specific operator modules adjacent to a minimal promoter, in an l-tryptophan-adjustable manner. Combinations of different transactivator and promoter variants from the same or different bacterial species resulted in a multitude of regulatory systems where l-tryptophan regulation properties, background noise, and maximal gene expression levels were significantly diverse. Different l-tryptophan analogues showed diverse regulatory capacity depending on the promoter/transactivator combination. We believe the systems approach to rationally choose promoters, transactivators and inducer molecules, to obtain desired and predefined genetic expression dynamics and control profiles, will significantly advance the design of new regulatory circuits as well as improving already existing ones. PMID:23178502

  10. Exposure to 3,3',5-triiodothyronine affects histone and RNA polymerase II modifications, but not DNA methylation status, in the regulatory region of the Xenopus laevis thyroid hormone receptor βΑ gene.

    PubMed

    Kasai, Kentaro; Nishiyama, Norihito; Izumi, Yushi; Otsuka, Shunsuke; Ishihara, Akinori; Yamauchi, Kiyoshi

    2015-11-01

    Thyroid hormones (THs) play a critical role in amphibian metamorphosis, during which the TH receptor (TR) gene, thrb, is upregulated in a tissue-specific manner. The Xenopus laevis thrb gene has 3 TH response elements (TREs) in the 5' flanking regulatory region and 1 TRE in the exon b region, around which CpG sites are highly distributed. To clarify whether exposure to 3,3',5-triiodothyronine (T3) affects histone and RNA polymerase II (RNAPII) modifications and the level of DNA methylation in the 5' regulatory region, we conducted reverse transcription-quantitative polymerase chain reaction, bisulfite sequencing and chromatin immunoprecipitation assay using X. laevis cultured cells and premetamorphic tadpoles treated with or without 2 nM T3. Exposure to T3 increased the amount of the thrb transcript, in parallel with enhanced histone H4 acetylation and RNAPII recruitment, and probably phosphorylation of RNAPII at serine 5, in the 5' regulatory and exon b regions. However, the 5' regulatory region remained hypermethylated even with exposure to T3, and there was no significant difference in the methylation status between DNAs from T3-untreated and -treated cultured cells or tadpole tissues. Our results demonstrate that exposure to T3 induced euchromatin-associated epigenetic marks by enhancing histone acetylation and RNAPII recruitment, but not by decreasing the level of DNA methylation, in the 5' regulatory region of the X. laevis thrb gene. PMID:26417689

  11. PCR detection and DNA sequence analysis of the regulatory region of lymphotropic papovavirus in peripheral blood mononuclear cells of an immunocompromised rhesus macaque

    NASA Technical Reports Server (NTRS)

    Lednicky, John A.; Halvorson, Steven J.; Butel, Janet S.

    2002-01-01

    A lymphotropic papovavirus (LPV) archetypal regulatory region was amplified from DNA from the blood of an immunocompromised rhesus monkey. We believe this is the first nonserological evidence of LPV infection in rhesus monkeys.

  12. Potential Novel Mechanism for Axenfeld-Rieger Syndrome: Deletion of a Distant Region Containing Regulatory Elements of PITX2

    PubMed Central

    Volkmann, Bethany A.; Zinkevich, Natalya S.; Mustonen, Aki; Schilter, Kala F.; Bosenko, Dmitry V.; Reis, Linda M.; Broeckel, Ulrich; Link, Brian A.

    2011-01-01

    Purpose. Mutations in PITX2 are associated with Axenfeld-Rieger syndrome (ARS), which involves ocular, dental, and umbilical abnormalities. Identification of cis-regulatory elements of PITX2 is important to better understand the mechanisms of disease. Methods. Conserved noncoding elements surrounding PITX2/pitx2 were identified and examined through transgenic analysis in zebrafish; expression pattern was studied by in situ hybridization. Patient samples were screened for deletion/duplication of the PITX2 upstream region using arrays and probes. Results. Zebrafish pitx2 demonstrates conserved expression during ocular and craniofacial development. Thirteen conserved noncoding sequences positioned within a gene desert as far as 1.1 Mb upstream of the human PITX2 gene were identified; 11 have enhancer activities consistent with pitx2 expression. Ten elements mediated expression in the developing brain, four regions were active during eye formation, and two sequences were associated with craniofacial expression. One region, CE4, located approximately 111 kb upstream of PITX2, directed a complex pattern including expression in the developing eye and craniofacial region, the classic sites affected in ARS. Screening of ARS patients identified an approximately 7600-kb deletion that began 106 to 108 kb upstream of the PITX2 gene, leaving PITX2 intact while removing regulatory elements CE4 to CE13. Conclusions. These data suggest the presence of a complex distant regulatory matrix within the gene desert located upstream of PITX2 with an essential role in its activity and provides a possible mechanism for the previous reports of ARS in patients with balanced translocations involving the 4q25 region upstream of PITX2 and the current patient with an upstream deletion. PMID:20881290

  13. P Element Regulatory Products Enhance Zeste(1) Repression of a P[white(duplicated)] Transgene in Drosophila Melanogaster

    PubMed Central

    Coen, D.

    1990-01-01

    Drosophila P element mobilization is subject to a complex array of regulatory mechanisms. A fruitful approach to study them is the use of insertion mutations whose expression is influenced by P regulation. In the present report, it is shown that P element somatic products may influence the expression of an unrelated gene inserted in a P transposon. The P[w(d1)9.3]19DE transgene carries an in vitro modified white gene harboring a duplication of the 5' regulatory sequences. Expression of this transgene is repressed in a P background. No maternal effect is detected and repression can be relieved as soon as P chromosomes are replaced by M ones. The amplitude of repression is correlated to the P transposase activity of the individuals examined. Repression appears to be exerted by somatic products of complete autonomous P elements or of in vitro modified P elements lacking the capacity to express the fourth P exon. The P repression of P[w(d1)9.3]19DE is strongly dependent on the insertion site of this transgene. This P repression effect occurs only in the presence of the zeste(1) allele and is suppressed by Su(z)2 mutations. No qualitative differences of transcription pattern are observed between white(+) and P[w(d1)9.3]19DE in any backgrounds. P repression acts to reduce the amount of the major white transcript. This suggests that P regulatory products may act through cis-interactions at a distance of over 3 kb. PMID:1963871

  14. Changes in cis-regulatory elements of a key floral regulator are associated with divergence of inflorescence architectures.

    PubMed

    Kusters, Elske; Della Pina, Serena; Castel, Rob; Souer, Erik; Koes, Ronald

    2015-08-15

    Higher plant species diverged extensively with regard to the moment (flowering time) and position (inflorescence architecture) at which flowers are formed. This seems largely caused by variation in the expression patterns of conserved genes that specify floral meristem identity (FMI), rather than changes in the encoded proteins. Here, we report a functional comparison of the promoters of homologous FMI genes from Arabidopsis, petunia, tomato and Antirrhinum. Analysis of promoter-reporter constructs in petunia and Arabidopsis, as well as complementation experiments, showed that the divergent expression of leafy (LFY) and the petunia homolog aberrant leaf and flower (ALF) results from alterations in the upstream regulatory network rather than cis-regulatory changes. The divergent expression of unusual floral organs (UFO) from Arabidopsis, and the petunia homolog double top (DOT), however, is caused by the loss or gain of cis-regulatory promoter elements, which respond to trans-acting factors that are expressed in similar patterns in both species. Introduction of pUFO:UFO causes no obvious defects in Arabidopsis, but in petunia it causes the precocious and ectopic formation of flowers. This provides an example of how a change in a cis-regulatory region can account for a change in the plant body plan. PMID:26220938

  15. S phase-specific DNA-binding proteins interacting with the Hex and Oct motifs in type I element of the wheat histone H3 promoter.

    PubMed

    Minami, M; Meshi, T; Iwabuchi, M

    2000-01-11

    The type I element (CCACGTCANCGATCCGCG), consisting of the Hex motif (CCACGTCA) and the reverse-oriented Oct motif (GATCCGCG), is necessary and sufficient to confer the S phase-specific transcription of the wheat histone H3 (TH012) gene. The transcriptional regulation via the type I element is thought to occur through interactions between transcription factors which bind specifically to the Hex and Oct motifs. Here we report S phase-specific DNA-binding proteins interacting with the type I element in partially synchronized wheat cultured cells. Hex motif-binding proteins found here resembled HBP-1a, as reported previously in terms of DNA-binding specificity. DNA-binding activities of the HBP-1a-like proteins were modulated by phosphorylation/dephosphorylation. In the electrophoretic mobility shift assay of the wheat nuclear extract, we also found three Oct motif-specific binding proteins, named OBRF (octamer-binding regulatory factor)-1, -2 and -3. One of the HBP-1a-like proteins and OBRF-1 appeared predominantly at the S phase. Thus, it was supposed that these two factors play a crucial role in the S phase-specific regulation of wheat histone gene expression. PMID:10675046

  16. Effect of oxidative DNA damage in promoter elements on transcription factor binding.

    PubMed Central

    Ghosh, R; Mitchell, D L

    1999-01-01

    Reactive oxygen species produced by endogenous metabolic activity and exposure to a multitude of exogenous agents impact cells in a variety of ways. The DNA base damage 8-oxodeoxyguanosine (8-oxodG) is a prominent indicator of oxidative stress and has been well-characterized as a premutagenic lesion in mammalian cells and putative initiator of the carcinogenic process. Commensurate with the recent interest in epigenetic pathways of cancer causation we investigated how 8-oxodG alters the interaction between cis elements located on gene promoters and sequence-specific DNA binding proteins associated with these promoters. Consensus binding sequences for the transcription factors AP-1, NF-kappaB and Sp1 were modified site-specifically at guanine residues and electrophoretic mobility shift assays were performed to assess DNA-protein interactions. Our results indicate that whereas a single 8-oxodG was sufficient to inhibit transcription factor binding to AP-1 and Sp1 sequences it had no effect on binding to NF-kappaB, regardless of its position. We conclude from these data that minor alterations in base composition at a crucial position within some, but not all, promoter elements have the ability to disrupt transcription factor binding. The lack of inhibition by damaged NF-kappaB sequences suggests that DNA-protein contact sites may not be as determinative for stable p50 binding to this promoter as other, as yet undefined, structural parameters. PMID:10454620

  17. Nanoparticle-labeled DNA capture elements for detection and identification of biological agents

    NASA Astrophysics Data System (ADS)

    Kiel, Johnathan L.; Holwitt, Eric A.; Parker, Jill E.; Vivekananda, Jeevalatha; Franz, Veronica

    2004-12-01

    Aptamers, synthetic DNA capture elements (DCEs), can be made chemically or in genetically engineered bacteria. DNA capture elements are artificial DNA sequences, from a random pool of sequences, selected for their specific binding to potential biological warfare or terrorism agents. These sequences were selected by an affinity method using filters to which the target agent was attached and the DNA isolated and amplified by polymerase chain reaction (PCR) in an iterative, increasingly stringent, process. The probes can then be conjugated to Quantum Dots and super paramagnetic nanoparticles. The former provide intense, bleach-resistant fluorescent detection of bioagent and the latter provide a means to collect the bioagents with a magnet. The fluorescence can be detected in a flow cytometer, in a fluorescence plate reader, or with a fluorescence microscope. To date, we have made DCEs to Bacillus anthracis spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and Francisella tularensis. DCEs can easily distinguish Bacillus anthracis from its nearest relatives, Bacillus cereus and Bacillus thuringiensis. Development of a high through-put process is currently being investigated.

  18. Modular sequence elements associated with origin regions in eukaryotic chromosomal DNA.

    PubMed Central

    Dobbs, D L; Shaiu, W L; Benbow, R M

    1994-01-01

    We have postulated that chromosomal replication origin regions in eukaryotes have in common clusters of certain modular sequence elements (Benbow, Zhao, and Larson, BioEssays 14, 661-670, 1992). In this study, computer analyses of DNA sequences from six origin regions showed that each contained one or more potential initiation regions consisting of a putative DUE (DNA unwinding element) aligned with clusters of SAR (scaffold associated region), and ARS (autonomously replicating sequence) consensus sequences, and pyrimidine tracts. The replication origins analyzed were from the following loci: Tetrahymena thermophila macronuclear rDNA gene, Chinese hamster ovary dihydrofolate reductase amplicon, human c-myc proto-oncogene, chicken histone H5 gene, Drosophila melanogaster chorion gene cluster on the third chromosome, and Chinese hamster ovary rhodopsin gene. The locations of putative initiation regions identified by the computer analyses were compared with published data obtained using diverse methods to map initiation sites. For at least four loci, the potential initiation regions identified by sequence analysis aligned with previously mapped initiation events. A consensus DNA sequence, WAWTTDDWWWDHWGWHMAWTT, was found within the potential initiation regions in every case. An additional 35 kb of combined flanking sequences from the six loci were also analyzed, but no additional copies of this consensus sequence were found. Images PMID:8041609

  19. The fission yeast CENP-B protein Abp1 prevents pervasive transcription of repetitive DNA elements.

    PubMed

    Daulny, Anne; Mejía-Ramírez, Eva; Reina, Oscar; Rosado-Lugo, Jesus; Aguilar-Arnal, Lorena; Auer, Herbert; Zaratiegui, Mikel; Azorin, Fernando

    2016-10-01

    It is well established that eukaryotic genomes are pervasively transcribed producing cryptic unstable transcripts (CUTs). However, the mechanisms regulating pervasive transcription are not well understood. Here, we report that the fission yeast CENP-B homolog Abp1 plays an important role in preventing pervasive transcription. We show that loss of abp1 results in the accumulation of CUTs, which are targeted for degradation by the exosome pathway. These CUTs originate from different types of genomic features, but the highest increase corresponds to Tf2 retrotransposons and rDNA repeats, where they map along the entire elements. In the absence of abp1, increased RNAPII-Ser5P occupancy is observed throughout the Tf2 coding region and, unexpectedly, RNAPII-Ser5P is enriched at rDNA repeats. Loss of abp1 also results in Tf2 derepression and increased nucleolus size. Altogether these results suggest that Abp1 prevents pervasive RNAPII transcription of repetitive DNA elements (i.e., Tf2 and rDNA repeats) from internal cryptic sites. PMID:27345571

  20. The Contribution of Alu Elements to Mutagenic DNA Double-Strand Break Repair

    PubMed Central

    Streva, Vincent A.; DeFreece, Cecily B.; Hedges, Dale J.; Deininger, Prescott L.

    2015-01-01

    Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ) deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB) repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%), we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences). Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence divergence on both

  1. Four major sequence elements of simian virus 40 large T antigen coordinate its specific and nonspecific DNA binding.

    PubMed Central

    Simmons, D T; Loeber, G; Tegtmeyer, P

    1990-01-01

    By mutational analysis, we have identified a motif critical to the proper recognition and binding of simian virus 40 large tumor antigen (T antigen) to virus DNA sequences at the origin of DNA replication. This motif is tripartite and consists of two elements (termed A1 and B2) that are necessary for sequence-specific binding of the origin and a central element (B1) which is required for nonspecific DNA-binding activity. Certain amino acids in elements A1 (residues 152 to 155) and B2 (203 to 207) may make direct contact with the GAGGC pentanucleotide sequences in binding sites I and II on the DNA. Alternatively, these two elements could determine the proper structure of the DNA-binding domain, although for a number of reasons we favor the first possibility. In contrast, element B1 (183 to 187) is most likely important for recognizing a general structural feature of DNA. Elements A1 and B2 are nearly identical in all known papovavirus T antigens, whereas B1 is identical only in the closely related papovaviruses simian virus 40, BK virus, and JC virus. In addition to these three elements, a fourth (B3; residues 215 to 219) is necessary for the binding of T antigen to site II but not to site I. We propose that additional contact sites on T antigen are involved in the interaction with site II to initiate the replication of the viral DNA. PMID:2157865

  2. metagene Profiles Analyses Reveal Regulatory Element's Factor-Specific Recruitment Patterns.

    PubMed

    Joly Beauparlant, Charles; Lamaze, Fabien C; Deschênes, Astrid; Samb, Rawane; Lemaçon, Audrey; Belleau, Pascal; Bilodeau, Steve; Droit, Arnaud

    2016-08-01

    ChIP-Sequencing (ChIP-Seq) provides a vast amount of information regarding the localization of proteins across the genome. The aggregation of ChIP-Seq enrichment signal in a metagene plot is an approach commonly used to summarize data complexity and to obtain a high level visual representation of the general occupancy pattern of a protein. Here we present the R package metagene, the graphical interface Imetagene and the companion package similaRpeak. Together, they provide a framework to integrate, summarize and compare the ChIP-Seq enrichment signal from complex experimental designs. Those packages identify and quantify similarities or dissimilarities in patterns between large numbers of ChIP-Seq profiles. We used metagene to investigate the differential occupancy of regulatory factors at noncoding regulatory regions (promoters and enhancers) in relation to transcriptional activity in GM12878 B-lymphocytes. The relationships between occupancy patterns and transcriptional activity suggest two different mechanisms of action for transcriptional control: i) a "gradient effect" where the regulatory factor occupancy levels follow transcription and ii) a "threshold effect" where the regulatory factor occupancy levels max out prior to reaching maximal transcription. metagene, Imetagene and similaRpeak are implemented in R under the Artistic license 2.0 and are available on Bioconductor. PMID:27538250

  3. Disruption of a novel regulatory element in the erythroid-specific promoter of the human PKLR gene causes severe pyruvate kinase deficiency.

    PubMed

    van Wijk, Richard; van Solinge, Wouter W; Nerlov, Claus; Beutler, Ernest; Gelbart, Terri; Rijksen, Gert; Nielsen, Finn C

    2003-02-15

    We established the molecular basis for pyruvate kinase (PK) deficiency in a white male patient with severe nonspherocytic hemolytic anemia. The paternal allele exhibited the common PKLR cDNA sequence (c.) 1529G>A mutation, known to be associated with PK deficiency. On the maternal allele, 3 in cis mutations were identified in the erythroid-specific promoter region of the gene: one deletion of thymine -248 and 2 single nucleotide substitutions, nucleotide (nt) -324T>A and nt -83G>C. Analysis of the patient's RNA demonstrated the presence of only the 1529A allele, indicating severely reduced transcription from the allele linked to the mutated promoter region. Transfection of promoter constructs into erythroleukemic K562 cells showed that the most upstream -324T>A and -248delT mutations were nonfunctional polymorphisms. In contrast, the -83G>C mutation strongly reduced promoter activity. Site-directed mutagenesis of the promoter region revealed the presence of a putative regulatory element (PKR-RE1) whose core binding motif, CTCTG, is located between nt -87 and nt -83. Electrophoretic mobility shift assay using K562 nuclear extracts indicated binding of an as-yet-unidentified trans-acting factor. This novel element mediates the effects of factors necessary for regulation of pyruvate kinase gene expression during red cell differentiation and maturation. PMID:12393511

  4. Identification of a key regulatory element for the basal activity of the human insulin-like growth factor II gene promoter P3.

    PubMed Central

    Rietveld, L E; Holthuizen, P E; Sussenbach, J S

    1997-01-01

    Transcription of the human insulin-like growth factor II (IGF-II) gene is under the control of four promoters (P1-P4) that are differentially active during growth and development. Promoter 3 (P3) is the most active promoter during fetal development as well as in most adult tissues. P3 is also the most active promoter in tumour tissues and cell lines expressing IGF-II. Transient transfections of HeLa and Hep3B cells with truncated promoter constructs revealed that the region between -289 and -183 relative to the transcription start site supports basal promoter activity in both cell lines. Footprint experiments showed that the region between positions -192 and -172 (P3-4) is the only element bound by nuclear proteins. P3-4 is bound by five proteins, of which three proteins (proteins 3, 4 and 5) bind specifically and are expressed at the same levels in HeLa and Hep3B cells. Electrophoretic mobility shift assays and differential footprint experiments revealed the presence of two protein-binding regions within the P3-4 element. Proteins 4 and 5 bind box A (-193 to -188), whereas box B (-183 to -172) is bound by protein 3. From transcription experiments in vitro it can be concluded that Box A is essential for P3 activity. Box A is part of a region 11 dG residues long and is protected by proteins 4 and 5 that bind a contiguous set of six dG residues. DNA-binding of proteins 4 and 5 to box A requires the presence of Zn2+ ions. Thus structural and functional analysis reveals that the P3-4 element is a key regulatory element of P3 that contains two separate binding sites for proteins essential for the basal activity of IGF-II P3. PMID:9581544

  5. Developmental patterns of chromatin structure and DNA methylation responsible for epigenetic expression of a maize regulatory gene.

    PubMed Central

    Hoekenga, O A; Muszynski, M G; Cone, K C

    2000-01-01

    Epigenetic regulatory mechanisms heritably alter patterns of gene expression without changes in DNA sequence. Epigenetic states are often correlated with developmentally imposed alterations in genomic DNA methylation and local chromatin structure. Pl-Blotched is a stable epigenetic allele of the maize anthocyanin regulatory gene, purple plant1(pl). Pl-Blotched plants display a variegated pattern of pigmentation that contrasts sharply with the uniformly dark purple pigmentation of plants carrying the dominant Pl-Rhoades allele. Previously, we showed that the lower level of pigmentation in Pl-Blotched is correlated with lower pl mRNA levels and increased DNA methylation at some sites. To explore how DNA methylation, chromatin structure, and developmental stage might contribute to the expression of Pl-Blotched, we used methylation-sensitive restriction enzymes and DNaseI sensitivity assays to compare the methylation status and chromatin structure of Pl-Blotched and Pl-Rhoades at different stages in development. Both alleles exhibit developmentally sensitive changes in methylation. In Pl-Blotched, methylation of two diagnostic HpaII/MspI sites increases progressively, coincident with the juvenile-to-adult transition in growth. In seedlings, the chromatin encompassing the coding region of the gene is less sensitive to DNaseI digestion in Pl-Blotched than in Pl-Rhoades. Developmental maturation from seedling to adult is accompanied by expansion of this closed chromatin domain to include the promoter and downstream flanking sequences. We provide evidence to show that chromatin structure, rather than DNA methylation, is the primary epigenetic determinant for the phenotypic differences between Pl-Blotched and Pl-Rhoades. PMID:10924483

  6. Phosphorylation-Induced Dimerization of Interferon Regulatory Factor 7 Unmasks DNA Binding and a Bipartite Transactivation Domain

    PubMed Central

    Marié, Isabelle; Smith, Eric; Prakash, Arun; Levy, David E.

    2000-01-01

    Interferon regulatory factor 7 (IRF7) is an interferon (IFN)-inducible transcription factor required for activation of a subset of IFN-α genes that are expressed with delayed kinetics following viral infection. IRF7 is synthesized as a latent protein and is posttranslationally modified by protein phosphorylation in infected cells. Phosphorylation required a carboxyl-terminal regulatory domain that controlled the retention of the active protein exclusively in the nucleus, as well as its binding to specific DNA target sequences, multimerization, and ability to induce target gene expression. Transcriptional activation by IRF7 mapped to two distinct regions, both of which were required for full activity, while all functions were masked in latent IRF7 by an autoinhibitory domain mapping to an internal region. A conditionally active form of IRF7 was constructed by fusing IRF7 with the ligand-binding and dimerization domain of estrogen receptor (ER). Hormone-dependent dimerization of chimeric IRF7-ER stimulated DNA binding and transcriptional transactivation of endogenous target genes. These studies demonstrate the regulation of IRF7 activity by phosphorylation-dependent allosteric changes that result in dimerization and that facilitate nuclear retention, derepress transactivation, and allow specific DNA binding. PMID:11073981

  7. Mapping the DNA-binding domain and target sequences of the Streptomyces peucetius daunorubicin biosynthesis regulatory protein, DnrI.

    PubMed

    Sheldon, Paul J; Busarow, Sara B; Hutchinson, C Richard

    2002-04-01

    Streptomyces antibiotic regulatory proteins (SARPs) constitute a novel family of transcriptional activators that control the expression of several diverse anti-biotic biosynthetic gene clusters. The Streptomyces peucetius DnrI protein, one of only a handful of these proteins yet discovered, controls the biosynthesis of the polyketide antitumour antibiotics daunorubicin and doxorubicin. Recently, comparative analyses have revealed significant similarities among the predicted DNA-binding domains of the SARPs and the C-terminal DNA-binding domain of the OmpR family of regulatory proteins. Using the crystal structure of the OmpR-binding domain as a template, DnrI was mapped by truncation and site-directed mutagenesis. Several highly conserved residues within the N-terminus are crucial for DNA binding and protein function. Tandemly arranged heptameric imperfect repeat sequences are found within the -35 promoter regions of target genes. Substitutions for each nucleotide within the repeats of the dnrG-dpsABCD promoter were performed by site-directed mutagenesis. The mutant promoter fragments were found to have modified binding characteristics in gel mobility shift assays. The spacing between the repeat target sequences is also critical for successful occupation by DnrI and, therefore, competent transcriptional activation of the dnrG-dpsABCD operon. PMID:11972782

  8. Effects of trace elements and pesticides on dephosphorylation of RNA and DNA added to soils

    SciTech Connect

    Frankenberger, W.T. Jr.; Johanson, J.B.; Lund L.J.

    1986-01-01

    This study was carried out to assess the effects of 14 trace elements, 12 herbicides, and two fungicides on dephosphorylation of yeast ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) added to soils (Xerollic Calciorthids and Typic Haploxeralfs). The cumulative amount of ortho phosphate (Pi) released from nucleic acids increased linearly with time of incubation (up to 72 h), decreased with profile depth, and was highly influenced by soil pH. When trace elements were applied and compared by using 2.5 mmol kg/sup -1/ of soil, the average inhibition in dephosphorylation of RNA and DNA in two soils ranged from 17% with Co(II) to 52% with Cu(II). The most effective inhibitors of nucleic acid dephosphorylation were Ag(I), Cu(I), Cd(II), Cu(II), Mn(II), Ni(II), and Pb(II) (avg inhibition greater than or equal to 35%). Other elements that inhibited dephosphorylation of RNA and DNA added to soils included Ba(II), Co(II), Hg(II), Zn(II), Ti(IV), V(IV), and W(VI). When the pesticides were compared by using 5 mg of active ingredient kg/sup -1/ of soil, the average inhibition in nucleic acid dephosphorylation ranged from 14% with butylate to 39% with chloramben. The most effective inhibitors (> 25%) were atrazine, naptalam, chloramben, dicamba, trifluralin, and maneb. Other pesticides that inhibited RNA and DNA dephosphorylation in soils included cyanazine, 2,4-D, dinitroamine, EPTC plus R-25788, alachlor, paraquat, butylate, and captan.

  9. AthaMap web tools for database-assisted identification of combinatorial cis-regulatory elements and the display of highly conserved transcription factor binding sites in Arabidopsis thaliana.

    PubMed

    Steffens, Nils Ole; Galuschka, Claudia; Schindler, Martin; Bülow, Lorenz; Hehl, Reinhard

    2005-07-01

    The AthaMap database generates a map of cis-regulatory elements for the Arabidopsis thaliana genome. AthaMap contains more than 7.4 x 10(6) putative binding sites for 36 transcription factors (TFs) from 16 different TF families. A newly implemented functionality allows the display of subsets of higher conserved transcription factor binding sites (TFBSs). Furthermore, a web tool was developed that permits a user-defined search for co-localizing cis-regulatory elements. The user can specify individually the level of conservation for each TFBS and a spacer range between them. This web tool was employed for the identification of co-localizing sites of known interacting TFs and TFs containing two DNA-binding domains. More than 1.8 x 10(5) combinatorial elements were annotated in the AthaMap database. These elements can also be used to identify more complex co-localizing elements consisting of up to four TFBSs. The AthaMap database and the connected web tools are a valuable resource for the analysis and the prediction of gene expression regulation at http://www.athamap.de. PMID:15980498

  10. The Role of Crowding Forces in Juxtaposing β-Globin Gene Domain Remote Regulatory Elements in Mouse Erythroid Cells.

    PubMed

    Golov, Arkadiy K; Gavrilov, Alexey A; Razin, Sergey V

    2015-01-01

    The extremely high concentration of macromolecules in a eukaryotic cell nucleus indicates that the nucleoplasm is a crowded macromolecular solution in which large objects tend to gather together due to crowding forces. It has been shown experimentally that crowding forces support the integrity of various nuclear compartments. However, little is known about their role in control of chromatin dynamics in vivo. Here, we experimentally addressed the possible role of crowding forces in spatial organization of the eukaryotic genome. Using the mouse β-globin domain as a model, we demonstrated that spatial juxtaposition of the remote regulatory elements of this domain in globin-expressing cells may be lost and restored by manipulation of the level of macromolecular crowding. In addition to proving the role of crowding forces in shaping interphase chromatin, our results suggest that the folding of the chromatin fiber is a major determinant in juxtaposing remote genomic elements. PMID:26436546

  11. Genetic variation in regulatory DNA elements: the case of OCA2 transcriptional regulation.

    PubMed

    Visser, Mijke; Kayser, Manfred; Grosveld, Frank; Palstra, Robert-Jan

    2014-03-01

    Mutations within the OCA2 gene or the complete absence of the OCA2 protein leads to oculocutaneous albinism type 2. The OCA2 protein plays a central role in melanosome biogenesis, and it is a strong determinant of the eumelanin content in melanocytes. Transcript levels of the OCA2 gene are strongly correlated with pigmentation intensities. Recent studies demonstrated that the transcriptional level of OCA2 is to a large extent determined by the noncoding SNP rs12913832 located 21.5 kb upstream of the OCA2 gene promoter. In this review, we discuss current hypotheses and the available data on the mechanism of OCA2 transcriptional regulation and how this is influenced by genetic variation. Finally, we will explore how future epigenetic studies can be used to advance our insight into the functional biology that connects genetic variation to human pigmentation. PMID:24387780

  12. Identification of a positive transcription regulatory element within the coding region of the nifLA operon in Azotobacter vinelandii.

    PubMed

    Mitra, Ranjana; Das, Hirendra K; Dixit, Aparna

    2005-07-01

    Nitrogen fixation in Azotobacter vinelandii is regulated by the nifLA operon. NifA activates the transcription of nif genes, while NifL antagonizes the transcriptional activator NifA in response to fixed nitrogen and molecular oxygen levels. However, transcriptional regulation of the nifLA operon of A. vinelandii itself is not fully understood. Using the S1 nuclease assay, we mapped the transcription start site of the nifLA operon, showing it to be similar to the sigma54-dependent promoters. We also identified a positive cis-acting regulatory element (+134 to +790) of the nifLA operon within the coding region of the nifL gene of A. vinelandii. Deletion of this element results in complete loss of promoter activity. Several protein factors bind to this region, and the specific binding sites have been mapped by DNase I foot printing. Two of these sites, namely dR1 (+134 to +204) and dR2 (+745 to +765), are involved in regulating the nifLA promoter activity. The absence of NtrC-like binding sites in the upstream region of the nifLA operon in A. vinelandii makes the identification of these downstream elements a highly significant finding. The interaction of the promoter with the proteins binding to the dR2 region spanning +745 to +765 appears to be dependent on the face of the helix as introduction of 4 bases just before this region completely disrupts promoter activity. Thus, the positive regulatory element present within the BglII-BglII fragment may play, in part; an important role in nifLA regulation in A. vinelandii. PMID:16000781

  13. Mutagenesis of GATA motifs controlling the endoderm regulator elt-2 reveals distinct dominant and secondary cis-regulatory elements.

    PubMed

    Du, Lawrence; Tracy, Sharon; Rifkin, Scott A

    2016-04-01

    Cis-regulatory elements (CREs) are crucial links in developmental gene regulatory networks, but in many cases, it can be difficult to discern whether similar CREs are functionally equivalent. We found that despite similar conservation and binding capability to upstream activators, different GATA cis-regulatory motifs within the promoter of the C. elegans endoderm regulator elt-2 play distinctive roles in activating and modulating gene expression throughout development. We fused wild-type and mutant versions of the elt-2 promoter to a gfp reporter and inserted these constructs as single copies into the C. elegans genome. We then counted early embryonic gfp transcripts using single-molecule RNA FISH (smFISH) and quantified gut GFP fluorescence. We determined that a single primary dominant GATA motif located 527bp upstream of the elt-2 start codon was necessary for both embryonic activation and later maintenance of transcription, while nearby secondary GATA motifs played largely subtle roles in modulating postembryonic levels of elt-2. Mutation of the primary activating site increased low-level spatiotemporally ectopic stochastic transcription, indicating that this site acts repressively in non-endoderm cells. Our results reveal that CREs with similar GATA factor binding affinities in close proximity can play very divergent context-dependent roles in regulating the expression of a developmentally critical gene in vivo. PMID:26896592

  14. A G-string positive cis-regulatory element in the LpS1 promoter binds two distinct nuclear factors distributed non-uniformly in Lytechinus pictus embryos.

    PubMed

    Xiang, M; Lu, S Y; Musso, M; Karsenty, G; Klein, W H

    1991-12-01

    The LpS1 alpha and beta genes of Lytechinus pictus are activated at the late cleavage stage of embryogenesis, with LpS1 mRNAs accumulating only in lineages contributing to aboral ectoderm. We had shown previously that 762 bp of 5' flanking DNA from the LpS1 beta gene was sufficient for proper temporal and aboral ectoderm specific expression. In the present study, we identified a strong positive cis-regulatory element at -70 bp to -75 bp in the LpS1 beta promoter with the sequence (G)6 and a similar, more distal cis-element at -721 bp to -726 bp. The proximal 'G-string' element interacted with two nuclear factors, one specific to ectoderm and one to endoderm/mesoderm nuclear extracts, whereas the distal G-string element interacted only with the ectoderm factor. The ectoderm and endoderm/mesoderm G-string factors were distinct based on their migratory behavior in electrophoretic mobility shift assays, binding site specificities, salt optima and EDTA sensitivity. The proximal G-string element shared homology with a binding site for the mammalian transcription factor IF1, a protein that binds to negative cis-regulatory elements in the mouse alpha 1(I) and alpha 2(I) collagen gene promoters. Competition experiments using wild-type and mutant oligonucleotides indicated that the ectoderm G-string factor and IF1 have similar recognition sites. Partially purified IF1 specifically bound to an oligonucleotide containing the proximal G-string of LpS1 beta. From our results, we suggest that the ectoderm G-string factor, a member of the G-rich DNA-binding protein family, activates the LpS1 gene in aboral ectoderm cells by binding to the LpS1 promoter at the proximal G-string site. PMID:1811948

  15. Identification of Rhizobium-specific intergenic mosaic elements within an essential two-component regulatory system of Rhizobium species.

    PubMed Central

    Osterås, M; Stanley, J; Finan, T M

    1995-01-01

    Analysis of the DNA regions upstream of the phosphoenolpyruvate carboxykinase gene (pckA) in Rhizobium meliloti and Rhizobium sp. strain NGR234 identified an open reading frame which was highly homologous to the Agrobacterium tumefaciens chromosomal virulence gene product ChvI. A second gene product, 500 bp downstream of the chvI-like gene in R. meliloti, was homologous to the A. tumefaciens ChvG protein. The homology between the R. meliloti and A. tumefaciens genes was confirmed, because the R. meliloti chvI and chvG genes complemented A. tumefaciens chvI and chvG mutants for growth on complex media. We were unable to construct chvI or chvG insertion mutants of R. meliloti, whereas mutants carrying insertions outside of these genes were readily obtained. A 108-bp repeat element characterized by two large palindromes was identified in the chvI and chvG intergenic regions of both Rhizobium species. This element was duplicated in Rhizobium sp. strain NGR234. Another structurally similar element with a size of 109 bp was present in R. meliloti but not in Rhizobium sp. strain NGR234. These elements were named rhizobium-specific intergenic mosaic elements (RIMEs), because their distribution seems to be limited to members of the family Rhizobiaceae. A homology search in GenBank detected six more copies of the first element (RIME1), all in Rhizobium species, and three extra copies of the second element (RIME2), only in R. meliloti. Southern blot analysis with a probe specific to RIME1 showed the presence of several copies of the element in the genome of R. meliloti, Rhizobium sp. strain NGR234, Rhizobium leguminosarum, and Agrobacterium rhizogenes, but none was present in A. tumefaciens and Bradyrhizobium japonicum. PMID:7559334

  16. In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element against Atrazine

    PubMed Central

    Williams, Ryan M.; Crihfield, Cassandra L.; Gattu, Srikanth; Holland, Lisa A.; Sooter, Letha J.

    2014-01-01

    Widespread use of the chlorotriazine herbicide, atrazine, has led to serious environmental and human health consequences. Current methods of detecting atrazine contamination are neither rapid nor cost-effective. In this work, atrazine-specific single-stranded DNA (ssDNA) molecular recognition elements (MRE) were isolated. We utilized a stringent Systematic Evolution of Ligands by Exponential Enrichment (SELEX) methodology that placed the greatest emphasis on what the MRE should not bind to. After twelve rounds of SELEX, an atrazine-specific MRE with high affinity was obtained. The equilibrium dissociation constant (Kd) of the ssDNA sequence is 0.62 ± 0.21 nM. It also has significant selectivity for atrazine over atrazine metabolites and other pesticides found in environmentally similar locations and concentrations. Furthermore, we have detected environmentally relevant atrazine concentrations in river water using this MRE. The strong affinity and selectivity of the selected atrazine-specific ssDNA validated the stringent SELEX methodology and identified a MRE that will be useful for rapid atrazine detection in environmental samples. PMID:25196435

  17. Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces

    PubMed Central

    Bai, Chaoxian; Zhang, Yang; Zhao, Xuejin; Hu, Yiling; Xiang, Sihai; Miao, Jin; Lou, Chunbo; Zhang, Lixin

    2015-01-01

    There is a great demand for precisely quantitating the expression of genes of interest in synthetic and systems biotechnology as new and fascinating insights into the genetics of streptomycetes have come to light. Here, we developed, for the first time to our knowledge, a quantitative method based on flow cytometry and a superfolder green fluorescent protein (sfGFP) at single-cell resolution in Streptomyces. Single cells of filamentous bacteria were obtained by releasing the protoplasts from the mycelium, and the dead cells could be distinguished from the viable ones by propidium iodide (PI) staining. With this sophisticated quantitative method, some 200 native or synthetic promoters and 200 ribosomal binding sites (RBSs) were characterized in a high-throughput format. Furthermore, an insulator (RiboJ) was recruited to eliminate the interference between promoters and RBSs and improve the modularity of regulatory elements. Seven synthetic promoters with gradient strength were successfully applied in a proof-of-principle approach to activate and overproduce the cryptic lycopene in a predictable manner in Streptomyces avermitilis. Our work therefore presents a quantitative strategy and universal synthetic modular regulatory elements, which will facilitate the functional optimization of gene clusters and the drug discovery process in Streptomyces. PMID:26374838

  18. Comparative Analysis of Regulatory Elements between Escherichia coli and Klebsiella pneumoniae by Genome-Wide Transcription Start Site Profiling

    PubMed Central

    Qiu, Yu; Nagarajan, Harish; Seo, Joo-Hyun; Cho, Byung-Kwan; Tsai, Shih-Feng; Palsson, Bernhard Ø.

    2012-01-01

    Genome-wide transcription start site (TSS) profiles of the enterobacteria Escherichia coli and Klebsiella pneumoniae were experimentally determined through modified 5′ RACE followed by deep sequencing of intact primary mRNA. This identified 3,746 and 3,143 TSSs for E. coli and K. pneumoniae, respectively. Experimentally determined TSSs were then used to define promoter regions and 5′ UTRs upstream of coding genes. Comparative analysis of these regulatory elements revealed the use of multiple TSSs, identical sequence motifs of promoter and Shine-Dalgarno sequence, reflecting conserved gene expression apparatuses between the two species. In both species, over 70% of primary transcripts were expressed from operons having orthologous genes during exponential growth. However, expressed orthologous genes in E. coli and K. pneumoniae showed a strikingly different organization of upstream regulatory regions with only 20% identical promoters with TSSs in both species. Over 40% of promoters had TSSs identified in only one species, despite conserved promoter sequences existing in the other species. 662 conserved promoters having TSSs in both species resulted in the same number of comparable 5′ UTR pairs, and that regulatory element was found to be the most variant region in sequence among promoter, 5′ UTR, and ORF. In K. pneumoniae, 48 sRNAs were predicted and 36 of them were expressed during exponential growth. Among them, 34 orthologous sRNAs between two species were analyzed in depth, and the analysis showed that many sRNAs of K. pneumoniae, including pleiotropic sRNAs such as rprA, arcZ, and sgrS, may work in the same way as in E. coli. These results reveal a new dimension of comparative genomics such that a comparison of two genomes needs to be comprehensive over all levels of genome organization. PMID:22912590

  19. Molecular stripping in the NFκB / IκB / DNA genetic regulatory network

    NASA Astrophysics Data System (ADS)

    Potoyan, Davit; Wolynes, Peter

    Genetic switches based on the NFκB / IκB / DNA system are master regulators of an array of cellular responses. Recent kinetic experiments have shown that IκB can actively remove NF κB bound to its genetic sites via a process called ''molecular stripping''. This allows the NFκB / IκB / DNA switch to function under kinetic control rather than the thermodynamic control contemplated in the traditional models of gene switches. Using molecular dynamics simulations of coarse grained predictive energy landscape models for the constituent proteins by themselves and interacting with the DNA we explore the functional motions of the transcription factor NFκB and its various binary and ternary complexes with DNA and the inhibitor I κB. These studies show that the function of the NFκB / IκB / DNA genetic switch is realized via an allosteric mechanism. Molecular stripping occurs through the activation of a domain twist mode by the binding of IκB which occurs through conformational selection. Free energy calculations for DNA binding show that the binding of IκB not only results in a significant decrease of the affinity of the transcription factor for the DNA but also kinetically speeds DNA release. Projections of the

  20. Molecular stripping in the NF-κB/IκB/DNA genetic regulatory network

    PubMed Central

    Potoyan, Davit A.; Zheng, Weihua; Komives, Elizabeth A.; Wolynes, Peter G.

    2016-01-01

    Genetic switches based on the NF-κB/IκB/DNA system are master regulators of an array of cellular responses. Recent kinetic experiments have shown that IκB can actively remove NF-κB bound to its genetic sites via a process called “molecular stripping.” This allows the NF-κB/IκB/DNA switch to function under kinetic control rather than the thermodynamic control contemplated in the traditional models of gene switches. Using molecular dynamics simulations of coarse-grained predictive energy landscape models for the constituent proteins by themselves and interacting with the DNA we explore the functional motions of the transcription factor NF-κB and its various binary and ternary complexes with DNA and the inhibitor IκB. These studies show that the function of the NF-κB/IκB/DNA genetic switch is realized via an allosteric mechanism. Molecular stripping occurs through the activation of a domain twist mode by the binding of IκB that occurs through conformational selection. Free energy calculations for DNA binding show that the binding of IκB not only results in a significant decrease of the affinity of the transcription factor for the DNA but also kinetically speeds DNA release. Projections of the free energy onto various reaction coordinates reveal the structural details of the stripping pathways. PMID:26699500

  1. Iron Deprivation in Synechocystis: Inference of Pathways, Non-coding RNAs, and Regulatory Elements from Comprehensive Expression Profiling

    PubMed Central

    Hernández-Prieto, Miguel A.; Schön, Verena; Georg, Jens; Barreira, Luísa; Varela, João; Hess, Wolfgang R.; Futschik, Matthias E.

    2012-01-01

    Iron is an essential cofactor in many metabolic reactions. Mechanisms controlling iron homeostasis need to respond rapidly to changes in extracellular conditions, but they must also keep the concentration of intracellular iron under strict control to avoid the generation of damaging reactive oxygen species. Due to its role as a redox carrier in photosynthesis, the iron quota in cyanobacteria is about 10 times higher than in model enterobacteria. The molecular details of how such a high quota is regulated are obscure. Here we present experiments that shed light on the iron regulatory system in cyanobacteria. We measured time-resolved changes in gene expression after iron depletion in the cyanobacterium Synechocystis sp. PCC 6803 using a comprehensive microarray platform, monitoring both protein-coding and non-coding transcripts. In total, less than a fifth of all protein-coding genes were differentially expressed during the first 72 hr. Many of these proteins are associated with iron transport, photosynthesis, or ATP synthesis. Comparing our data with three previous studies, we identified a core set of 28 genes involved in iron stress response. Among them were genes important for assimilation of inorganic carbon, suggesting a link between the carbon and iron regulatory networks. Nine of the 28 genes have unknown functions and constitute key targets for further functional analysis. Statistical and clustering analyses identified 10 small RNAs, 62 antisense RNAs, four 5′UTRs, and seven intragenic elements as potential novel components of the iron regulatory network in Synechocystis. Hence, our genome-wide expression profiling indicates an unprecedented complexity in the iron regulatory network of cyanobacteria. PMID:23275872

  2. DNA binding and regulatory effects of transcription factors SP1 and USF at the rat amyloid precursor protein gene promoter.

    PubMed Central

    Hoffman, P W; Chernak, J M

    1995-01-01

    Two DNA elements which we have termed SAA and GAG have been shown to control expression of the rat amyloid precursor protein (APP) gene, and the region containing the SAA element has been shown to interact with nuclear proteins [Hoffman and Chernak (1994) Biochem. Biophys. Res. Commun. 201, 610-617]. In this report we study DNA sequences and proteins which influence the activity of the SAA element. An oligonucleotide containing the SAA element is specifically bound by nuclear proteins derived from rat PC12 cells, consistently forming four complexes designated C25, C30, C35 and C40 in electrophoretic mobility shift assays (EMSAs). We demonstrate that the C25, C30 and C40 complexes involve the binding of nuclear proteins to an SP1 consensus sequence located within the SAA element and that the C25 complex contains a protein antigenically related to the human SP1 protein. We establish further that the C35 complex requires a USF recognition site located within the SAA element and contains a protein antigenically related to the human upstream stimulatory factor (USF) protein. Using APP promoter/luciferase reporter gene constructs, we demonstrate that both the SP1 and the USF sites can play a role in the transcriptional activity of the SAA element. Finally, we show that complexes similar to the C25, C30 and C35 complexes are formed by rat cortex nuclear extracts and the SAA element in EMSA experiments, suggesting the relevance of our in vitro observations to the in vivo functioning of the rat APP promoter. Images PMID:7610052

  3. DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

    PubMed Central

    Rajeev, Lara; Luning, Eric G.; Mukhopadhyay, Aindrila

    2014-01-01

    In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of limited use in exploring bacterial two component regulatory systems with uncharacterized activation conditions. Such systems regulate transcription only when activated in the presence of unique signals. Since these signals are often unknown, the in vitro microarray based method described in this video article can be used to determine gene targets and binding sites for response regulators. This DNA-affinity-purified-chip method may be used for any purified regulator in any organism with a sequenced genome. The protocol involves allowing the purified tagged protein to bind to sheared genomic DNA and then affinity purifying the protein-bound DNA, followed by fluorescent labeling of the DNA and hybridization to a custom tiling array. Preceding steps that may be used to optimize the assay for specific regulators are also described. The peaks generated by the array data analysis are used to predict binding site motifs, which are then experimentally validated. The motif predictions can be further used to determine gene targets of orthologous response regulators in closely related species. We demonstrate the applicability of this method by determining the gene targets and binding site motifs and thus predicting the function for a sigma54-dependent response regulator DVU3023 in the environmental bacterium Desulfovibrio vulgaris Hildenborough. PMID:25079303

  4. A multistep bioinformatic approach detects putative regulatory elements in gene promoters

    PubMed Central

    Bortoluzzi, Stefania; Coppe, Alessandro; Bisognin, Andrea; Pizzi, Cinzia; Danieli, Gian Antonio

    2005-01-01

    Background Searching for approximate patterns in large promoter sequences frequently produces an exceedingly high numbers of results. Our aim was to exploit biological knowledge for definition of a sheltered search space and of appropriate search parameters, in order to develop a method for identification of a tractable number of sequence motifs. Results Novel software (COOP) was developed for extraction of sequence motifs, based on clustering of exact or approximate patterns according to the frequency of their overlapping occurrences. Genomic sequences of 1 Kb upstream of 91 genes differentially expressed and/or encoding proteins with relevant function in adult human retina were analyzed. Methodology and results were tested by analysing 1,000 groups of putatively unrelated sequences, randomly selected among 17,156 human gene promoters. When applied to a sample of human promoters, the method identified 279 putative motifs frequently occurring in retina promoters sequences. Most of them are localized in the proximal portion of promoters, less variable in central region than in lateral regions and similar to known regulatory sequences. COOP software and reference manual are freely available upon request to the Authors. Conclusion The approach described in this paper seems effective for identifying a tractable number of sequence motifs with putative regulatory role. PMID:15904489

  5. A novel mutation of the GATA site in the erythroid cell-specific regulatory element of the ABO gene in a blood donor with the Am B phenotype.

    PubMed

    Oda, A; Isa, K; Ogasawara, K; Kameyama, K; Okuda, K; Hirashima, M; Ishii, H; Kimura, K; Matsukura, H; Hirayama, F; Kawa, K

    2015-05-01

    The Am and Bm phenotypes are characterized by weak expression of the A or B antigens, respectively, by red blood cells with a normal expression by the saliva of secretors. Deletion of the regulatory element in the first intron of the ABO gene and disruption of the GATA motif in the element were found to be responsible. In this study, we identified a novel mutation within the GATA motif (G>C substitution at position c.28 + 5830) in the regulatory element of the A allele that might diminish transcription activity causing the generation of the Am B phenotype. PMID:25557060

  6. DANIO-CODE: Toward an Encyclopedia of DNA Elements in Zebrafish

    PubMed Central

    2016-01-01

    Abstract The zebrafish has emerged as a model organism for genomics studies. The symposium “Toward an encyclopedia of DNA elements in zebrafish” held in London in December 2014, was coorganized by Ferenc Müller and Fiona Wardle. This meeting is a follow-up of a similar previous workshop held 2 years earlier and represents a push toward the formalization of a community effort to annotate functional elements in the zebrafish genome. The meeting brought together zebrafish researchers, bioinformaticians, as well as members of established consortia, to exchange scientific findings and experience, as well as to discuss the initial steps toward the formation of a DANIO-CODE consortium. In this study, we provide the latest updates on the current progress of the consortium's efforts, opening up a broad invitation to researchers to join in and contribute to DANIO-CODE. PMID:26671609

  7. Preventing Phosphorylation of Sterol Regulatory Element-Binding Protein 1a by MAP-Kinases Protects Mice from Fatty Liver and Visceral Obesity

    PubMed Central

    Haas, Jutta; Kremer, Lorena; Jacob, Sylvia; Hartwig, Sonja; Nitzgen, Ulrike; Muller–Wieland, Dirk

    2012-01-01

    The transcription factor sterol regulatory element binding protein (SREBP)-1a plays a pivotal role in lipid metabolism. Using the SREBP-1a expressing human hepatoma cell line HepG2 we have shown previously that human SREBP-1a is phosphorylated at serine 117 by ERK-mitogen-activated protein kinases (MAPK). Using a combination of cell biology and protein chemistry approach we show that SREBP-1a is also target of other MAPK-families, i.e. c-JUN N-terminal protein kinases (JNK) or p38 stress activated MAP kinases. Serine 117 is also the major phosphorylation site in SREBP-1a for JNK. In contrast to that the major phosphorylation sites of p38 MAPK family are serine 63 and threonine 426. Functional analyses reveal that phosphorylation of SREBP-1a does not alter protein/DNA interaction. The identified phosphorylation sites are specific for both kinase families also in cellular context. To provide direct evidence that phosphorylation of SREBP-1a is a regulatory principle of biological and clinical relevance, we generated transgenic mice expressing mature transcriptionally active N-terminal domain of human SREBP–1a variant lacking all identified phosphorylaton sites designed as alb-SREBP-1aΔP and wild type SREBP-1a designed as alb-SREBP-1a liver specific under control of the albumin promoter and a liver specific enhancer. In contrast to alb-SREBP–1a mice the phosphorylation–deficient mice develop no enlarged fatty livers under normocaloric conditions. Phenotypical examination reveales a massive accumulation of adipose tissue in alb-SREBP-1a but not in the phosphorylation deficient alb-SREBP-1aΔP mice. Moreover, preventing phosphorylation of SREBP-1a protects mice also from dyslipidemia. In conclusion, phosphorylation of SREBP-1a by ERK, JNK and p38 MAPK-families resembles a biological principle and plays a significant role, in vivo. PMID:22384276

  8. Hepatitis B virus nuclear export elements: RNA stem-loop α and β, key parts of the HBV post-transcriptional regulatory element.

    PubMed

    Lim, Chun Shen; Brown, Chris M

    2016-09-01

    Many viruses contain RNA elements that modulate splicing and/or promote nuclear export of their RNAs. The RNAs of the major human pathogen, hepatitis B virus (HBV) contain a large (~600 bases) composite cis-acting 'post-transcriptional regulatory element' (PRE). This element promotes expression from these naturally intronless transcripts. Indeed, the related woodchuck hepadnavirus PRE (WPRE) is used to enhance expression in gene therapy and other expression vectors. These PRE are likely to act through a combination of mechanisms, including promotion of RNA nuclear export. Functional components of both the HBV PRE and WPRE are 2 conserved RNA cis-acting stem-loop (SL) structures, SLα and SLβ. They are within the coding regions of polymerase (P) gene, and both P and X genes, respectively. Based on previous studies using mutagenesis and/or nuclear magnetic resonance (NMR), here we propose 2 covariance models for SLα and SLβ. The model for the 30-nucleotide SLα contains a G-bulge and a CNGG(U) apical loop of which the first and the fourth loop residues form a CG pair and the fifth loop residue is bulged out, as observed in the NMR structure. The model for the 23-nucleotide SLβ contains a 7-base-pair stem and a 9-nucleotide loop. Comparison of the models with other RNA structural elements, as well as similarity searches of human transcriptome and viral genomes demonstrate that SLα and SLβ are specific to HBV transcripts. However, they are well conserved among the hepadnaviruses of non-human primates, the woodchuck and ground squirrel. PMID:27031749

  9. A single-laboratory validated method for the generation of DNA barcodes for the identification of fish for regulatory compliance.

    PubMed

    Handy, Sara M; Deeds, Jonathan R; Ivanova, Natalia V; Hebert, Paul D N; Hanner, Robert H; Ormos, Andrea; Weigt, Lee A; Moore, Michelle M; Yancy, Haile F

    2011-01-01

    The U.S. Food and Drug Administration is responsible for ensuring that the nation's food supply is safe and accurately labeled. This task is particularly challenging in the case of seafood where a large variety of species are marketed, most of this commodity is imported, and processed product is difficult to identify using traditional morphological methods. Reliable species identification is critical for both foodborne illness investigations and for prevention of deceptive practices, such as those where species are intentionally mislabeled to circumvent import restrictions or for resale as species of higher value. New methods that allow accurate and rapid species identifications are needed, but any new methods to be used for regulatory compliance must be both standardized and adequately validated. "DNA barcoding" is a process by which species discriminations are achieved through the use of short, standardized gene fragments. For animals, a fragment (655 base pairs starting near the 5' end) of the cytochrome c oxidase subunit 1 mitochondrial gene has been shown to provide reliable species level discrimination in most cases. We provide here a protocol with single-laboratory validation for the generation of DNA barcodes suitable for the identification of seafood products, specifically fish, in a manner that is suitable for FDA regulatory use. PMID:21391497

  10. Genome-wide characterization of cis-acting DNA targets reveals the transcriptional regulatory framework of opaque2 in maize.

    PubMed

    Li, Chaobin; Qiao, Zhenyi; Qi, Weiwei; Wang, Qian; Yuan, Yue; Yang, Xi; Tang, Yuanping; Mei, Bing; Lv, Yuanda; Zhao, Han; Xiao, Han; Song, Rentao

    2015-03-01

    Opaque2 (O2) is a transcription factor that plays important roles during maize endosperm development. Mutation of the O2 gene improves the nutritional value of maize seeds but also confers pleiotropic effects that result in reduced agronomic quality. To reveal the transcriptional regulatory framework of O2, we studied the transcriptome of o2 mutants using RNA sequencing (RNA-Seq) and determined O2 DNA binding targets using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq). The RNA-Seq analysis revealed 1605 differentially expressed genes (DEGs) and 383 differentially expressed long, noncoding RNAs. The DEGs cover a wide range of functions related to nutrient reservoir activity, nitrogen metabolism, stress resistance, etc. ChIP-Seq analysis detected 1686 O2 DNA binding sites distributed over 1143 genes. Overlay of the RNA-Seq and ChIP-Seq results revealed 35 O2-modulated target genes. We identified four O2 binding motifs; among them, TGACGTGG appears to be the most conserved and strongest. We confirmed that, except for the 16- and 18-kD zeins, O2 directly regulates expression of all other zeins. O2 directly regulates two transcription factors, genes linked to carbon and amino acid metabolism and abiotic stress resistance. We built a hierarchical regulatory model for O2 that provides an understanding of its pleiotropic biological effects. PMID:25691733

  11. The role of an inverted CCAAT element in transcriptional activation of the human DNA topoisomerase IIalpha gene by heat shock.

    PubMed

    Furukawa, M; Uchiumi, T; Nomoto, M; Takano, H; Morimoto, R I; Naito, S; Kuwano, M; Kohno, K

    1998-04-24

    Expression of the DNA topoisomerase IIalpha (topoIIalpha) gene is highly sensitive to various environmental stimuli including heat shock. The amount of topoIIalpha mRNA was increased 1.5-3-fold 6-24 h after exposure of T24 human urinary bladder cancer cells to heat shock stress at 43 degreesC for 1 h. The effect of heat shock on the transcriptional activity of the human topoIIalpha gene promoter was investigated by transient transfection of T24 cells with luciferase reporter plasmids containing various lengths of the promoter sequence. The transcriptional activity of the full-length promoter (nucleotides (nt) -295 to +85) and of three deletion constructs (nt -197 to +85, -154 to +85, and -74 to +85) was increased approximately 3-fold 24 h after heat shock stress. In contrast, the transcriptional activity of the minimal promoter (nt -20 to +85), which lacks the first inverted CCAAT element (ICE1), the GC box, and the heat shock element located between nt -74 and -21, was not increased by heat shock. Furthermore, the transcriptional activity of promoter constructs containing mutations in the GC box or heat shock element, but not that of a construct containing mutations in ICE1, was significantly increased by heat shock. Electrophoretic mobility shift assays revealed reduced binding of a nuclear factor to an oligonucleotide containing ICE1 when nuclear extracts were derived from cells cultured for 3-24 h after heat shock. No such change in factor binding was apparent with an oligonucleotide containing the heat shock element of the topoIIalpha gene promoter. Finally, in vivo footprint analysis of the topoIIalpha gene promoter revealed that two G residues of ICE1 that were protected in control cells became sensitive to dimethyl sulfate modification after heat shock. These results suggest that transcriptional activation of the topoIIalpha gene by heat shock requires the release of a negative regulatory factor from ICE1. PMID:9553115

  12. Expression of active iron regulatory factor from a full-length human cDNA by in vitro transcription/translation.

    PubMed Central

    Hirling, H; Emery-Goodman, A; Thompson, N; Neupert, B; Seiser, C; Kühn, L C

    1992-01-01

    Iron regulatory factor (IRF), also called iron responsive element-binding protein (IRE-BP), is a cytoplasmic RNA-binding protein which regulates post-transcriptionally transferrin receptor mRNA stability and ferritin mRNA translation. By using the polymerase chain reaction (PCR) and the sequence published by Rouault et al. (1990) a probe was derived which permitted the isolation of three human IRF cDNA clones. Hybridization to genomic DNA and mRNA, as well as sequencing data indicated a single copy gene of about 40 kb specifying a 4.0 kb mRNA that translates into a protein of 98,400 dalton. By in vitro transcription of a assembled IRF cDNA coupled to in vitro translation in a wheat germ extract, we obtained full sized IRF that bound specifically to a human ferritin IRE. In vitro translated IRF retained sensitivity to sulfhydryl oxidation by diamide and could be reactivated by beta-mercaptoethanol in the same way as native placental IRF. An IRF deletion mutant shortened by 132 amino acids at the COOH-terminus was no longer able to bind to an IRE, indicating that this region of the protein plays a role in RNA recognition. Placental IRF has previously been shown to migrate as a doublet on SDS-polyacrylamide gels. After V8 protease digestion the heterogeneity was located in a 65/70 kDa NH2-terminal doublet. The liberated 31 kDa COOH-terminal polypeptide was found to be homogeneous by amino acid sequencing supporting the conclusion of a single IRF gene. Images PMID:1738601

  13. Transcription of T cell receptor beta-chain genes is controlled by a downstream regulatory element.

    PubMed Central

    Krimpenfort, P; de Jong, R; Uematsu, Y; Dembic, Z; Ryser, S; von Boehmer, H; Steinmetz, M; Berns, A

    1988-01-01

    To characterize cis-acting elements controlling the expression of T cell receptor beta-chains we generated a number of transgenic mouse lines harboring a rearranged T cell receptor beta-chain with different extensions of 5' and 3' flanking sequences. Transcriptional analysis of transgenic mice carrying these clones showed that sequences located downstream of the polyadenylation signal of the C beta 2 region are indispensable for expression in transgenic mice. The sequences conferring enhancer activity in this fragment were further defined by transient CAT assays. Strong enhancer activity was found to reside in a 550 bp fragment located 5 kb downstream from C beta 2. The nucleotide sequence of this fragment revealed a number of oligonucleotide motifs characteristic for enhancer elements. Images PMID:3396541

  14. csrT Represents a New Class of csrA-Like Regulatory Genes Associated with Integrative Conjugative Elements of Legionella pneumophila

    PubMed Central

    Abbott, Zachary D.; Flynn, Kaitlin J.; Byrne, Brenda G.; Mukherjee, Sampriti; Kearns, Daniel B.

    2015-01-01

    ABSTRACT Bacterial evolution is accelerated by mobile genetic elements. To spread horizontally and to benefit the recipient bacteria, genes encoded on these elements must be properly regulated. Among the legionellae are multiple integrative conjugative elements (ICEs) that each encode a paralog of the broadly conserved regulator csrA. Using bioinformatic analyses, we deduced that specific csrA paralogs are coinherited with particular lineages of the type IV secretion system that mediates horizontal spread of its ICE, suggesting a conserved regulatory interaction. As a first step to investigate the contribution of csrA regulators to this class of mobile genetic elements, we analyzed here the activity of the csrA paralog encoded on Legionella pneumophila ICE-βox. Deletion of this gene, which we name csrT, had no observed effect under laboratory conditions. However, ectopic expression of csrT abrogated the protection to hydrogen peroxide and macrophage degradation that ICE-βox confers to L. pneumophila. When ectopically expressed, csrT also repressed L. pneumophila flagellin production and motility, a function similar to the core genome's canonical csrA. Moreover, csrT restored the repression of motility to csrA mutants of Bacillus subtilis, a finding consistent with the predicted function of CsrT as an mRNA binding protein. Since all known ICEs of legionellae encode coinherited csrA-type IV secretion system pairs, we postulate that CsrA superfamily proteins regulate ICE activity to increase their horizontal spread, thereby expanding L. pneumophila versatility. IMPORTANCE ICEs are mobile DNA elements whose type IV secretion machineries mediate spread among bacterial populations. All surveyed ICEs within the Legionella genus also carry paralogs of the essential life cycle regulator csrA. It is striking that the csrA loci could be classified into distinct families based on either their sequence or the subtype of the adjacent type IV secretion system locus. To

  15. Dual Masking of Specific Negative Splicing Regulatory Elements Resulted in Maximal Exon 7 Inclusion of SMN2 Gene

    PubMed Central

    Pao, Peng Wen; Wee, Keng Boon; Yee, Woon Chee; DwiPramono, Zacharias Aloysius

    2014-01-01

    Spinal muscular atrophy (SMA) is a fatal autosomal recessive disease caused by survival motor neuron (SMN) protein insufficiency due to SMN1 mutations. Boosting SMN2 expression is a potential therapy for SMA. SMN2 has identical coding sequence as SMN1 except for a silent C-to-T transition at the 6th nucleotide of exon 7, converting a splicing enhancer to a silencer motif. Consequently, most SMN2 transcripts lack exon 7. More than ten putative splicing regulatory elements (SREs) were reported to regulate exon 7 splicing. To investigate the relative strength of each negative SRE in inhibiting exon 7 inclusion, antisense oligonucleotides (AONs) were used to mask each element, and the fold increase of full-length SMN transcripts containing exon 7 were compared. The most potent negative SREs are at intron 7 (in descending order): ISS-N1, 3′ splice site of exon 8 (ex8 3′ss) and ISS+100. Dual-targeting AONs were subsequently used to mask two nonadjacent SREs simultaneously. Notably, masking of both ISS-N1 and ex8 3′ss induced the highest fold increase of full-length SMN transcripts and proteins. Therefore, efforts should be directed towards the two elements simultaneously for the development of optimal AONs for SMA therapy. PMID:24317636

  16. Chemical Elemental Distribution and Soil DNA Fingerprints Provide the Critical Evidence in Murder Case Investigation

    PubMed Central

    Concheri, Giuseppe; Bertoldi, Daniela; Polone, Elisa; Otto, Stefan; Larcher, Roberto; Squartini, Andrea

    2011-01-01

    Background The scientific contribution to the solution of crime cases, or throughout the consequent forensic trials, is a crucial aspect of the justice system. The possibility to extract meaningful information from trace amounts of samples, and to match and validate evidences with robust and unambiguous statistical tests, are the key points of such process. The present report is the authorized disclosure of an investigation, carried out by Attorney General appointment, on a murder case in northern Italy, which yielded the critical supporting evidence for the judicial trial. Methodology/Principal Findings The proportional distribution of 54 chemical elements and the bacterial community DNA fingerprints were used as signature markers to prove the similarity of two soil samples. The first soil was collected on the crime scene, along a corn field, while the second was found in trace amounts on the carpet of a car impounded from the main suspect in a distant location. The matching similarity of the two soils was proven by crossing the results of two independent techniques: a) elemental analysis via inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectrometry (ICP-OES) approaches, and b) amplified ribosomal DNA restriction analysis by gel electrophoresis (ARDRA). Conclusions Besides introducing the novel application of these methods to forensic disciplines, the highly accurate level of resolution observed, opens new possibilities also in the fields of soil typing and tracking, historical analyses, geochemical surveys and global land mapping. PMID:21674041

  17. An ARS element inhibits DNA replication through a SIR2-dependent mechanism.

    PubMed

    Crampton, Amber; Chang, FuJung; Pappas, Donald L; Frisch, Ryan L; Weinreich, Michael

    2008-04-25

    During G1 phase, a prereplicative complex (pre-RC) that determines where DNA synthesis initiates forms at origins. The Sir2p histone deacetylase inhibits pre-RC assembly at a subset of origins, suggesting that Sir2p inhibits DNA replication through a unique aspect of origin structure. Here, we identified five SIR2-sensitive origins on chromosomes III and VI. Linker scan analysis of two origins indicated that they share a common organization, including an inhibitory sequence positioned 3' to the sites of origin recognition complex (ORC) binding and pre-RC assembly. This inhibitory sequence (I(S)) required SIR2 for its activity, suggesting that SIR2 inhibits origins through this sequence. Furthermore, I(S) elements occurred within positioned nucleosomes, and Abf1p-mediated exclusion of nucleosomes from the origin abrogated the inhibition. These data suggest that Sir2p and I(S) elements inhibit origin activity by promoting an unfavorable chromatin structure for pre-RC assembly. PMID:18439895

  18. Altered Response Hierarchy and Increased T-Cell Breadth upon HIV-1 Conserved Element DNA Vaccination in Macaques

    PubMed Central

    Kulkarni, Viraj; Valentin, Antonio; Rosati, Margherita; Alicea, Candido; Singh, Ashish K.; Jalah, Rashmi; Broderick, Kate E.; Sardesai, Niranjan Y.; Le Gall, Sylvie; Mothe, Beatriz; Brander, Christian; Rolland, Morgane; Mullins, James I.; Pavlakis, George N.; Felber, Barbara K.

    2014-01-01

    HIV sequence diversity and potential decoy epitopes are hurdles in the development of an effective AIDS vaccine. A DNA vaccine candidate comprising of highly conserved p24gag elements (CE) induced robust immunity in all 10 vaccinated macaques, whereas full-length gag DNA vaccination elicited responses to these conserved elements in only 5 of 11 animals, targeting fewer CE per animal. Importantly, boosting CE-primed macaques with DNA expressing full-length p55gag increased both magnitude of CE responses and breadth of Gag immunity, demonstrating alteration of the hierarchy of epitope recognition in the presence of pre-existing CE-specific responses. Inclusion of a conserved element immunogen provides a novel and effective strategy to broaden responses against highly diverse pathogens by avoiding decoy epitopes, while focusing responses to critical viral elements for which few escape pathways exist. PMID:24465991

  19. Altered response hierarchy and increased T-cell breadth upon HIV-1 conserved element DNA vaccination in macaques.

    PubMed

    Kulkarni, Viraj; Valentin, Antonio; Rosati, Margherita; Alicea, Candido; Singh, Ashish K; Jalah, Rashmi; Broderick, Kate E; Sardesai, Niranjan Y; Le Gall, Sylvie; Mothe, Beatriz; Brander, Christian; Rolland, Morgane; Mullins, James I; Pavlakis, George N; Felber, Barbara K

    2014-01-01

    HIV sequence diversity and potential decoy epitopes are hurdles in the development of an effective AIDS vaccine. A DNA vaccine candidate comprising of highly conserved p24(gag) elements (CE) induced robust immunity in all 10 vaccinated macaques, whereas full-length gag DNA vaccination elicited responses to these conserved elements in only 5 of 11 animals, targeting fewer CE per animal. Importantly, boosting CE-primed macaques with DNA expressing full-length p55(gag) increased both magnitude of CE responses and breadth of Gag immunity, demonstrating alteration of the hierarchy of epitope recognition in the presence of pre-existing CE-specific responses. Inclusion of a conserved element immunogen provides a novel and effective strategy to broaden responses against highly diverse pathogens by avoiding decoy epitopes, while focusing responses to critical viral elements for which few escape pathways exist. PMID:24465991

  20. The sterol regulatory element binding proteins are essential for the metabolic programming of effector T cells and adaptive immunity

    PubMed Central

    Kidani, Yoko; Elsaesser, Heidi; Hock, M Benjamin; Vergnes, Laurent; Williams, Kevin J; Argus, Joseph P; Marbois, Beth N; Komisopoulou, Evangelia; Wilson, Elizabeth B; Osborne, Timothy F; Graeber, Thomas G; Reue, Karen; Brooks, David G; Bensinger, Steven J

    2013-01-01

    Newly activated CD8+ T cells reprogram their metabolism to meet the extraordinary biosynthetic demands of clonal expansion; however, the signals mediating metabolic reprogramming remain poorly defined. Herein, we demonstrate an essential role for sterol regulatory element binding proteins (SREBPs) in the acquisition of effector cell metabolism. Without SREBP signaling, CD8+ T cells are unable to blast, resulting in markedly attenuated clonal expansion during viral infection. Mechanistic studies indicate that SREBPs are essential to meet the heightened lipid requirements of membrane synthesis during blastogenesis. SREBPs are dispensable for homeostatic proliferation, indicating a context-specific requirement for SREBPs in effector responses. These studies provide insights into the molecular signals underlying metabolic reprogramming of CD8+ T cells during the transition from quiescence to activation. PMID:23563690

  1. In vivo promoter analysis on refeeding response of hepatic sterol regulatory element-binding protein-1c expression

    SciTech Connect

    Takeuchi, Yoshinori; Yahagi, Naoya; Nakagawa, Yoshimi; Matsuzaka, Takashi; Shimizu, Ritsuko; Sekiya, Motohiro; Iizuka, Yoko; Ohashi, Ken; Gotoda, Takanari; Yamamoto, Masayuki; Nagai, Ryozo; Kadowaki, Takashi; Yamada, Nobuhiro; Osuga, Jun-ichi; Shimano, Hitoshi

    2007-11-16

    Sterol regulatory element-binding protein (SREBP)-1c is the master regulator of lipogenic gene expression in liver. The mRNA abundance of SREBP-1c is markedly induced when animals are refed after starvation, although the regulatory mechanism is so far unknown. To investigate the mechanism of refeeding response of SREBP-1c gene expression in vivo, we generated a transgenic mouse model that carries 2.2 kb promoter region fused to the luciferase reporter gene. These transgenic mice exhibited refeeding responses of the reporter in liver and adipose tissues with extents essentially identical to those of endogenous SREBP-1c mRNA. The same results were obtained from experiments using adenovirus-mediated SREBP-1c-promoter-luciferase fusion gene transduction to liver. These data demonstrate that the regulation of SREBP-1c gene expression is at the transcription level, and that the 2.2 kb 5'-flanking region is sufficient for this regulation. Moreover, when these transgenic or adenovirus-infected mice were placed on insulin-depleted state by streptozotocin treatment, the reporter expression was upregulated as strongly as in control mice, demonstrating that this regulation is not dominated by serum insulin level. These mice are the first models to provide the mechanistic insight into the transcriptional regulation of SREBP-1c gene in vivo.

  2. Global identification of the genetic networks and cis-regulatory elements of the cold response in zebrafish

    PubMed Central

    Hu, Peng; Liu, Mingli; Zhang, Dong; Wang, Jinfeng; Niu, Hongbo; Liu, Yimeng; Wu, Zhichao; Han, Bingshe; Zhai, Wanying; Shen, Yu; Chen, Liangbiao

    2015-01-01

    The transcriptional programs of ectothermic teleosts are directly influenced by water temperature. However, the cis- and trans-factors governing cold responses are not well characterized. We profiled transcriptional changes in eight zebrafish tissues exposed to mildly and severely cold temperatures using RNA-Seq. A total of 1943 differentially expressed genes (DEGs) were identified, from which 34 clusters representing distinct tissue and temperature response expression patterns were derived using the k-means fuzzy clustering algorithm. The promoter regions of the clustered DEGs that demonstrated strong co-regulation were analysed for enriched cis-regulatory elements with a motif discovery program, DREME. Seventeen motifs, ten known and seven novel, were identified, which covered 23% of the DEGs. Two motifs predicted to be the binding sites for the transcription factors Bcl6 and Jun, respectively, were chosen for experimental verification, and they demonstrated the expected cold-induced and cold-repressed patterns of gene regulation. Protein interaction modeling of the network components followed by experimental validation suggested that Jun physically interacts with Bcl6 and might be a hub factor that orchestrates the cold response in zebrafish. Thus, the methodology used and the regulatory networks uncovered in this study provide a foundation for exploring the mechanisms of cold adaptation in teleosts. PMID:26227973

  3. GTP cyclohydrolase I feedback regulatory protein is a pentamer of identical subunits. Purification, cDNA cloning, and bacterial expression.

    PubMed

    Yoneyama, T; Brewer, J M; Hatakeyama, K

    1997-04-11

    GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by tetrahydrobiopterin and also mediates the stimulatory effect of phenylalanine on the enzyme activity. To characterize the molecular structure of GFRP, we have purified it from rat liver using an efficient step of affinity chromatography and isolated cDNA clones, based on partial amino acid sequences of peptides derived from purified GFRP. Comparison between the amino acid sequence deduced from the cDNA and the N-terminal amino acid sequence of purified GFRP showed that the mature form of GFRP consists of 83 amino acid residues with a calculated Mr of 9,542. The isolated GFRP cDNA was expressed in Escherichia coli as a fusion protein with six consecutive histidine residues at its N terminus. The fusion protein was affinity-purified and digested with thrombin to remove the histidine tag. The resulting recombinant GFRP showed kinetic properties similar to those of GFRP purified from rat liver. Cross-linking experiments using dimethyl suberimidate indicated that GFRP was a pentamer of 52 kDa. Sedimentation equilibrium measurements confirmed the pentameric structure of GFRP by giving an average Mr of 49,734, which is 5 times the calculated molecular weight of the recombinant GFRP polypeptide. Based on the pentameric structure of GFRP, we have proposed a model for the quaternary structure of GFRP and GTP cyclohydrolase I complexes. PMID:9092499

  4. Expression of an early gene in the flagellar regulatory hierarchy is sensitive to an interruption in DNA replication.

    PubMed Central

    Dingwall, A; Zhuang, W Y; Quon, K; Shapiro, L

    1992-01-01

    Genes involved in the biogenesis of the flagellum in Caulobacter crescentus are expressed in a temporal order and are controlled by a trans-acting regulatory hierarchy. Strains with mutations in one of these genes, flaS, cannot transcribe flagellar structural genes and divide abnormally. This gene was cloned, and it was found that its transcription is initiated early in the cell cycle. Subclones that restored motility to FlaS mutants also restored normal cell division. Although transcription of flaS was not dependent on any other known gene in the flagellar hierarchy, it was autoregulated and subject to mild negative control by other genes at the same level of the hierarchy. An additional level of control was revealed when it was found that an interruption of DNA replication caused the inhibition of flaS transcription. The flaS transcript initiation site was identified, and an apparently unique promoter sequence was found to be highly conserved among the genes at the same level of the hierarchy. The flagellar genes with this conserved 5' region all initiate transcription early in the cell cycle and are all sensitive to a disruption in DNA replication. Mutations in these genes also cause an aberrant cell division phenotype. Therefore, flagellar genes at or near the top of the hierarchy may be controlled, in part, by a unique transcription factor and may be responsive to the same DNA replication cues that mediate other cell cycle events, such as cell division. Images PMID:1372311

  5. Demystifying the secret mission of enhancers: linking distal regulatory elements to target genes

    PubMed Central

    Yao, Lijing; Berman, Benjamin P.; Farnham, Peggy J.

    2015-01-01

    Abstract Enhancers are short regulatory sequences bound by sequence-specific transcription factors and play a major role in the spatiotemporal specificity of gene expression patterns in development and disease. While it is now possible to identify enhancer regions genomewide in both cultured cells and primary tissues using epigenomic approaches, it has been more challenging to develop methods to understand the function of individual enhancers because enhancers are located far from the gene(s) that they regulate. However, it is essential to identify target genes of enhancers not only so that we can understand the role of enhancers in disease but also because this information will assist in the development of future therapeutic options. After reviewing models of enhancer function, we discuss recent methods for identifying target genes of enhancers. First, we describe chromatin structure-based approaches for directly mapping interactions between enhancers and promoters. Second, we describe the use of correlation-based approaches to link enhancer state with the activity of nearby promoters and/or gene expression. Third, we describe how to test the function of specific enhancers experimentally by perturbing enhancer–target relationships using high-throughput reporter assays and genome editing. Finally, we conclude by discussing as yet unanswered questions concerning how enhancers function, how target genes can be identified, and how to distinguish direct from indirect changes in gene expression mediated by individual enhancers. PMID:26446758

  6. Distinct Functional Constraints Partition Sequence Conservation in a cis-Regulatory Element

    PubMed Central

    Ruvinsky, Ilya

    2011-01-01

    Different functional constraints contribute to different evolutionary rates across genomes. To understand why some sequences evolve faster than others in a single cis-regulatory locus, we investigated function and evolutionary dynamics of the promoter of the Caenorhabditis elegans unc-47 gene. We found that this promoter consists of two distinct domains. The proximal promoter is conserved and is largely sufficient to direct appropriate spatial expression. The distal promoter displays little if any conservation between several closely related nematodes. Despite this divergence, sequences from all species confer robustness of expression, arguing that this function does not require substantial sequence conservation. We showed that even unrelated sequences have the ability to promote robust expression. A prominent feature shared by all of these robustness-promoting sequences is an AT-enriched nucleotide composition consistent with nucleosome depletion. Because general sequence composition can be maintained despite sequence turnover, our results explain how different functional constraints can lead to vastly disparate rates of sequence divergence within a promoter. PMID:21655084

  7. A MAPT mutation in a regulatory element upstream of exon 10 causes frontotemporal dementia.

    PubMed

    Malkani, Roneil; D'Souza, Ian; Gwinn-Hardy, Katrina; Schellenberg, Gerard D; Hardy, John; Momeni, Parastoo

    2006-05-01

    We report here the genetic analysis of a newly ascertained kindred in which frontotemporal dementia occurs in an apparent autosomal dominant fashion, and in which a novel MAPT gene mutation co-segregates with disease. Sequencing the MAPT gene in affected individuals revealed a change in intron 9. This finding supports earlier studies on the effect of a splice-accepting element in inclusion of exon 10 in the MAPT transcript. This mutation sheds light on a novel mechanism by which over-expression of 4-repeat tau leads to disease. Based on our current findings, we propose a novel mechanism by which intronic mutations can lead to frontotemporal dementia. PMID:16503405

  8. Impact of Age at Administration, Lysosomal Storage, and Transgene Regulatory Elements on AAV2/8-Mediated Rat Liver Transduction

    PubMed Central

    Cotugno, Gabriella; Annunziata, Patrizia; Barone, Maria Vittoria; Karali, Marianthi; Banfi, Sandro; Auricchio, Alberto

    2012-01-01

    Liver-directed gene transfer is being investigated for the treatment of systemic or liver-specific diseases. Recombinant vectors based on adeno-associated virus serotype 8 (AAV2/8) efficiently transduce liver cells allowing long term transgene expression after a single administration in animal models and in patients. We evaluated the impact on AAV2/8-mediated rat liver transduction of the following variables: i) age at vector administration, ii) presence of lysosomal storage in liver cells, and iii) regulatory elements included in the transgene expression cassette. We found that systemic administration of AAV2/8 to newborn rats results in vector genome dilution and reduced transduction efficacy when compared to adult injected animals, presumably due to hepatocyte proliferation. Accumulation of glycosaminoglycans in lysosomes does not impact on levels and distribution of AAV2/8-mediated liver transduction. Transgene expression occurs in hepatocytes but not in Kupffer or liver endothelial cells when the liver-specific thyroxine-binding-globulin promoter is used. However, extra-hepatic transduction is observed in the spleen and kidney of animals injected at birth. The use of target sequences for the hematopoietic-specific microRNA miR142-3p does not improve liver transduction efficacy neither reduce immune responses to the lysosomal enzyme arylsulfatase B. The inclusion of a variant of the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE-m) decreases AAV2/8-mediated liver transduction levels. As AAV2/8-mediated liver gene transfer is entering in the clinical arena, these data will provide relevant information to the design of efficient AAV2/8-based therapeutic strategies. PMID:22428010

  9. Mutation of a transcriptional motif of a distant regulatory element reduces the expression of embryonic and fetal globin genes

    PubMed Central

    Navas, Patrick A.; Swank, Richard A.; Yu, Man; Peterson, Kenneth R.; Stamatoyannopoulos, George

    2010-01-01

    High-level β-globin gene expression is dependent on the presence of the locus control region (LCR), a powerful regulatory element physically characterized by five DNase I-hypersensitive sites (HS), designated HS1–HS5. Of these, HS3 contains seven GT motifs that are essential for its activity. One of the motifs, GT6, has been shown by in vivo footprinting to display the largest difference in signal between fetal and adult globin expressing cells. We assessed the contribution of GT6 on the downstream globin gene expression by mutating this motif in a 248 kb β-globin locus yeast artificial chromosome and measuring the activity of β-globin genes in GT6m β-YAC transgenic mice. Seven transgenic lines were established, three of which contained at least one intact copy of the β-globin locus and were further investigated. The mutation of the GT6 motif reduced the expression of ε- and γ-globin genes during embryonic erythropoiesis. During definitive erythropoiesis, γ-globin gene expression was significantly reduced while β-globin gene expression was virtually indistinguishable from wild-type controls. We conclude that the GT6 motif of hypersensitive site 3 of the LCR is required for normal ε- and γ-globin gene expression during embryonic erythropoiesis and for γ-globin gene expression during definitive erythropoiesis in the fetal liver. Our results provide evidence that mutations of single transcriptional motifs of distant regulatory elements can have profound effects on gene expression. PMID:14506128

  10. Characterization of DNA sequences that mediate nuclear protein binding to the regulatory region of the Pisum sativum (pea) chlorophyl a/b binding protein gene AB80: identification of a repeated heptamer motif.

    PubMed

    Argüello, G; García-Hernández, E; Sánchez, M; Gariglio, P; Herrera-Estrella, L; Simpson, J

    1992-05-01

    Two protein factors binding to the regulatory region of the pea chlorophyl a/b binding protein gene AB80 have been identified. One of these factors is found only in green tissue but not in etiolated or root tissue. The second factor (denominated ABF-2) binds to a DNA sequence element that contains a direct heptamer repeat TCTCAAA. It was found that presence of both of the repeats is essential for binding. ABF-2 is present in both green and etiolated tissue and in roots and factors analogous to ABF-2 are present in several plant species. Computer analysis showed that the TCTCAAA motif is present in the regulatory region of several plant genes. PMID:1303797

  11. Organization of multiple regulatory elements in the control region of the adenovirus type 2-specific VARNA1 gene: fine mapping with linker-scanning mutants.

    PubMed

    Railey, J F; Wu, G J

    1988-03-01

    The adenovirus type 2-specific virus-associated RNA 1 (VARNA1) gene is transcribed by eucaryotic RNA polymerase III. Previous studies using deletion mutants for transcription have shown that the VARNA1 gene has a large control region which is composed of several regulatory elements. Twenty-five exact linker-scanning mutations in the control region, from -33 to +77, of this gene were used for definition of the number and boundaries of these elements. The effects of these mutations on transcription and competition for transcription factors in human KB cell extracts revealed five positive regulatory elements. The essential element, which coincided with the B block, was absolutely required for both transcription and formation of stable complexes. A second element, which included the A block, was also required for both transcription and formation of stable complexes. Although this element is not as essential as the B-block element, together with the B-block element it may be necessary for formation of the most basal form of transcription machinery. Therefore, these two elements are the promoter elements in this gene. In addition, one possible element in the interblock region and two elements in the 5' flanking region were also required for efficient transcription, but they were moderately required for formation of stable complexes. Transcription of these mutants and the wild-type gene using an extract of 293 cells was stimulated at least threefold over that with the KB cell extract, as expected. Similar regulatory elements of this gene were revealed, however, when the 293 cell extract was used for transcription of these mutants, suggesting that the E1A-mediated specific transcription factors act on the transcription machinery in a sequence-nonspecific manner. PMID:3367906

  12. Organization of multiple regulatory elements in the control region of the adenovirus type 2-specific VARNA1 gene: fine mapping with linker-scanning mutants.

    PubMed Central

    Railey, J F; Wu, G J

    1988-01-01

    The adenovirus type 2-specific virus-associated RNA 1 (VARNA1) gene is transcribed by eucaryotic RNA polymerase III. Previous studies using deletion mutants for transcription have shown that the VARNA1 gene has a large control region which is composed of several regulatory elements. Twenty-five exact linker-scanning mutations in the control region, from -33 to +77, of this gene were used for definition of the number and boundaries of these elements. The effects of these mutations on transcription and competition for transcription factors in human KB cell extracts revealed five positive regulatory elements. The essential element, which coincided with the B block, was absolutely required for both transcription and formation of stable complexes. A second element, which included the A block, was also required for both transcription and formation of stable complexes. Although this element is not as essential as the B-block element, together with the B-block element it may be necessary for formation of the most basal form of transcription machinery. Therefore, these two elements are the promoter elements in this gene. In addition, one possible element in the interblock region and two elements in the 5' flanking region were also required for efficient transcription, but they were moderately required for formation of stable complexes. Transcription of these mutants and the wild-type gene using an extract of 293 cells was stimulated at least threefold over that with the KB cell extract, as expected. Similar regulatory elements of this gene were revealed, however, when the 293 cell extract was used for transcription of these mutants, suggesting that the E1A-mediated specific transcription factors act on the transcription machinery in a sequence-nonspecific manner. Images PMID:3367906

  13. Organization of multiple regulatory elements in the control region of the adenovirus type 2-specific VARNA1 gene: Fine mapping with linker-scanning mutants

    SciTech Connect

    Railey, J.F.; Wu, G.J.

    1988-03-01

    The adenovirus type 2-specific virus-associated RNA 1 (VARNA1) gene is transcribed by eucaryotic RNA polymerase III. Previous studies using deletion mutants for transcription have shown that the VARNA1 gene has a large control region which is composed of several regulatory elements. Twenty-five exact linker-scanning mutations in the control region, from -33 to +77, of this gene were used for definition of the number and boundaries of these elements. The effects of these mutations on transcription and competition for transcription factors in human KB cell extracts revealed five positive regulatory elements. The essential element, which coincided with the B block, was absolutely required for both transcription and formation of stable complexes. A second element, which included the A block, was also required for both transcription and formation of stable complexes. Although this element is not as essential as the B-block element, together with the B-block element it may be necessary for formation of the most basal form of transcription machinery. Therefore, these two elements are the promoter elements in this gene. In addition, one possible element in the interblock region and two elements in the 5' flanking region were also required for efficient transcription, but they were moderately required for formation of stable complexes. Transcription of these mutants and the wild-type genes using an extract of 293 cells was stimulated at least threefold over that with the KB cell extract, as expected. Similar regulatory elements of this gene were revealed, however, when the 292 cell extract was used for transcription of these mutants, suggesting that the E1A-mediated specific transcription factors act on the transcription machinery in a sequence-nonspecific manner.

  14. Multiple Herbicide Resistance in Lolium multiflorum and Identification of Conserved Regulatory Elements of Herbicide Resistance Genes.

    PubMed

    Mahmood, Khalid; Mathiassen, Solvejg K; Kristensen, Michael; Kudsk, Per

    2016-01-01

    Herbicide resistance is a ubiquitous challenge to herbicide sustainability and a looming threat to control weeds in crops. Recently four genes were found constituently over-expressed in herbicide resistant individuals of Lolium rigidum, a close relative of Lolium multiflorum. These include two cytochrome P450s, one nitronate monooxygenase and one glycosyl-transferase. Higher expressions of these four herbicide metabolism related (HMR) genes were also observed after herbicides exposure in the gene expression databases, indicating them as reliable markers. In order to get an overview of herbicidal resistance status of L. multiflorum L, 19 field populations were collected. Among these populations, four populations were found to be resistant to acetolactate synthase (ALS) inhibitors while three exhibited resistance to acetyl-CoA carboxylase (ACCase) inhibitors in our initial screening and dose response study. The genotyping showed the presence of mutations Trp-574-Leu and Ile-2041-Asn in ALS and ACCase, respectively, and qPCR experiments revealed the enhanced expression of HMR genes in individuals of certain resistant populations. Moreover, co-expression networks and promoter analyses of HMR genes in O. sativa and A. thaliana resulted in the identification of a cis-regulatory motif and zinc finger transcription factors. The identified transcription factors were highly expressed similar to HMR genes in response to xenobiotics whereas the identified motif is known to play a vital role in coping with environmental stresses and maintaining genome stability. Overall, our findings provide an important step forward toward a better understanding of metabolism-based herbicide resistance that can be utilized to devise novel strategies of weed management. PMID:27547209

  15. Multiple Herbicide Resistance in Lolium multiflorum and Identification of Conserved Regulatory Elements of Herbicide Resistance Genes

    PubMed Central

    Mahmood, Khalid; Mathiassen, Solvejg K.; Kristensen, Michael; Kudsk, Per

    2016-01-01

    Herbicide resistance is a ubiquitous challenge to herbicide sustainability and a looming threat to control weeds in crops. Recently four genes were found constituently over-expressed in herbicide resistant individuals of Lolium rigidum, a close relative of Lolium multiflorum. These include two cytochrome P450s, one nitronate monooxygenase and one glycosyl-transferase. Higher expressions of these four herbicide metabolism related (HMR) genes were also observed after herbicides exposure in the gene expression databases, indicating them as reliable markers. In order to get an overview of herbicidal resistance status of L. multiflorum L, 19 field populations were collected. Among these populations, four populations were found to be resistant to acetolactate synthase (ALS) inhibitors while three exhibited resistance to acetyl-CoA carboxylase (ACCase) inhibitors in our initial screening and dose response study. The genotyping showed the presence of mutations Trp-574-Leu and Ile-2041-Asn in ALS and ACCase, respectively, and qPCR experiments revealed the enhanced expression of HMR genes in individuals of certain resistant populations. Moreover, co-expression networks and promoter analyses of HMR genes in O. sativa and A. thaliana resulted in the identification of a cis-regulatory motif and zinc finger transcription factors. The identified transcription factors were highly expressed similar to HMR genes in response to xenobiotics whereas the identified motif is known to play a vital role in coping with environmental stresses and maintaining genome stability. Overall, our findings provide an important step forward toward a better understanding of metabolism-based herbicide resistance that can be utilized to devise novel strategies of weed management. PMID:27547209

  16. GAGA factor binding to DNA via a single trinucleotide sequence element.

    PubMed Central

    Wilkins, R C; Lis, J T

    1998-01-01

    GAGA transcription factor (GAF) is an essential protein in Drosophila , important for the transcriptional regulation of numerous genes. GAF binds to GA repeats in the promoters of these genes via a DNA-binding domain containing a single zinc finger. While GAF binding sites are typically composed of 3.5 GA repeats, the Drosophila hsp70 gene contains much smaller elements, some of which are as little as three bases (GAG) in length. Interestingly, the binding of GAF to more distant trinucleotide elements is relatively strong and not appreciably affected by the removal of larger GA arrays in the promoter. Moreover, a simple synthetic GAG sequence is sufficient to bind GAF in vitro . Here we directly compare the affinity of GAF for different sequence elements by immunoprecipitation and gel mobility shift analysis. Furthermore, our measures of the concentration of GAF in vivo indicate that it is a highly abundant nuclear protein, prevalent enough to occupy a sizable fraction of correspondingly abundant trinucleotide sites. PMID:9592153

  17. Integrative Modeling of eQTLs and Cis-Regulatory Elements Suggests Mechanisms Underlying Cell Type Specificity of eQTLs

    PubMed Central

    Brown, Christopher D.; Mangravite, Lara M.; Engelhardt, Barbara E.

    2013-01-01

    Genetic variants in cis-regulatory elements or trans-acting regulators frequently influence the quantity and spatiotemporal distribution of gene transcription. Recent interest in expression quantitative trait locus (eQTL) mapping has paralleled the adoption of genome-wide association studies (GWAS) for the analysis of complex traits and disease in humans. Under the hypothesis that many GWAS associations tag non-coding SNPs with small effects, and that these SNPs exert phenotypic control by modifying gene expression, it has become common to interpret GWAS associations using eQTL data. To fully exploit the mechanistic interpretability of eQTL-GWAS comparisons, an improved understanding of the genetic architecture and causal mechanisms of cell type specificity of eQTLs is required. We address this need by performing an eQTL analysis in three parts: first we identified eQTLs from eleven studies on seven cell types; then we integrated eQTL data with cis-regulatory element (CRE) data from the ENCODE project; finally we built a set of classifiers to predict the cell type specificity of eQTLs. The cell type specificity of eQTLs is associated with eQTL SNP overlap with hundreds of cell type specific CRE classes, including enhancer, promoter, and repressive chromatin marks, regions of open chromatin, and many classes of DNA binding proteins. These associations provide insight into the molecular mechanisms generating the cell type specificity of eQTLs and the mode of regulation of corresponding eQTLs. Using a random forest classifier with cell specific CRE-SNP overlap as features, we demonstrate the feasibility of predicting the cell type specificity of eQTLs. We then demonstrate that CREs from a trait-associated cell type can be used to annotate GWAS associations in the absence of eQTL data for that cell type. We anticipate that such integrative, predictive modeling of cell specificity will improve our ability to understand the mechanistic basis of human complex phenotypic

  18. Structure of an RNA dimer of a regulatory element from human thymidylate synthase mRNA

    SciTech Connect

    Dibrov, Sergey; McLean, Jaime; Hermann, Thomas

    2011-09-27

    A sequence around the start codon of the mRNA of human thymidylate synthase (TS) folds into a secondary-structure motif in which the initiation site is sequestered in a metastable hairpin. Binding of the protein to its own mRNA at the hairpin prevents the production of TS through a translation-repression feedback mechanism. Stabilization of the mRNA hairpin by other ligands has been proposed as a strategy to reduce TS levels in anticancer therapy. Rapidly proliferating cells require high TS activity to maintain the production of thymidine as a building block for DNA synthesis. The crystal structure of a model oligonucleotide (TS1) that represents the TS-binding site of the mRNA has been determined. While fluorescence studies showed that the TS1 RNA preferentially adopts a hairpin structure in solution, even at high RNA concentrations, an asymmetric dimer of two hybridized TS1 strands was obtained in the crystal. The TS1 dimer contains an unusual S-turn motif that also occurs in the 'off' state of the human ribosomal decoding site RNA.

  19. Identification and characterization of a cis-regulatory element for zygotic gene expression in Chlamydomonas reinhardtii

    DOE PAGESBeta

    Hamaji, Takashi; Lopez, David; Pellegrini, Matteo; Umen, James

    2016-03-26

    Upon fertilization Chlamydomonas reinhardtii zygotes undergo a program of differentiation into a diploid zygospore that is accompanied by transcription of hundreds of zygote-specific genes. We identified a distinct sequence motif we term a zygotic response element (ZYRE) that is highly enriched in promoter regions of C. reinhardtii early zygotic genes. A luciferase reporter assay was used to show that native ZYRE motifs within the promoter of zygotic gene ZYS3 or intron of zygotic gene DMT4 are necessary for zygotic induction. A synthetic luciferase reporter with a minimal promoter was used to show that ZYRE motifs introduced upstream are sufficient tomore » confer zygotic upregulation, and that ZYRE-controlled zygotic transcription is dependent on the homeodomain transcription factor GSP1. Furthermore, we predict that ZYRE motifs will correspond to binding sites for the homeodomain proteins GSP1-GSM1 that heterodimerize and activate zygotic gene expression in early zygotes.« less

  20. Identification and Characterization of a cis-Regulatory Element for Zygotic Gene Expression in Chlamydomonas reinhardtii.

    PubMed

    Hamaji, Takashi; Lopez, David; Pellegrini, Matteo; Umen, James

    2016-01-01

    Upon fertilization Chlamydomonas reinhardtii zygotes undergo a program of differentiation into a diploid zygospore that is accompanied by transcription of hundreds of zygote-specific genes. We identified a distinct sequence motif we term a zygotic response element (ZYRE) that is highly enriched in promoter regions of C reinhardtii early zygotic genes. A luciferase reporter assay was used to show that native ZYRE motifs within the promoter of zygotic gene ZYS3 or intron of zygotic gene DMT4 are necessary for zygotic induction. A synthetic luciferase reporter with a minimal promoter was used to show that ZYRE motifs introduced upstream are sufficient to confer zygotic upregulation, and that ZYRE-controlled zygotic transcription is dependent on the homeodomain transcription factor GSP1. We predict that ZYRE motifs will correspond to binding sites for the homeodomain proteins GSP1-GSM1 that heterodimerize and activate zygotic gene expression in early zygotes. PMID:27172209

  1. The ubiquitous yybP-ykoY riboswitch is a manganese-responsive regulatory element

    PubMed Central

    Updegrove, Taylor B.; Anantharaman, Vivek; Aravind, L.; Waters, Lauren S.; Storz, Gisela

    2015-01-01

    SUMMARY The highly-structured, cis-encoded RNA elements known as riboswitches modify gene expression upon binding a wide range of molecules. The yybP-ykoY motif was one of the most broadly distributed and numerous bacterial riboswitch whose cognate ligand was unknown. Using a combination of in vivo reporter and in vitro expression assays, equilibrium dialysis and northern analysis, we show that the yybP-ykoY motif responds directly to manganese ions in both Escherichia coli and Bacillus subtilis. The identification of the yybP-ykoY motif as a manganese ion sensor suggests the genes that are preceded by this motif, and encode a diverse set of poorly characterized membrane proteins, have roles in metal homeostasis. PMID:25794618

  2. Identification and Characterization of a cis-Regulatory Element for Zygotic Gene Expression in Chlamydomonas reinhardtii

    PubMed Central

    Hamaji, Takashi; Lopez, David; Pellegrini, Matteo; Umen, James

    2016-01-01

    Upon fertilization Chlamydomonas reinhardtii zygotes undergo a program of differentiation into a diploid zygospore that is accompanied by transcription of hundreds of zygote-specific genes. We identified a distinct sequence motif we term a zygotic response element (ZYRE) that is highly enriched in promoter regions of C. reinhardtii early zygotic genes. A luciferase reporter assay was used to show that native ZYRE motifs within the promoter of zygotic gene ZYS3 or intron of zygotic gene DMT4 are necessary for zygotic induction. A synthetic luciferase reporter with a minimal promoter was used to show that ZYRE motifs introduced upstream are sufficient to confer zygotic upregulation, and that ZYRE-controlled zygotic transcription is dependent on the homeodomain transcription factor GSP1. We predict that ZYRE motifs will correspond to binding sites for the homeodomain proteins GSP1-GSM1 that heterodimerize and activate zygotic gene expression in early zygotes. PMID:27172209

  3. Residue Substitutions Near the Redox Center of Bacillus subtilis Spx Affect RNA Polymerase Interaction, Redox Control, and Spx-DNA Contact at a Conserved cis-Acting Element

    PubMed Central

    Lin, Ann A.; Walthers, Don

    2013-01-01

    Spx, a member of the ArsC protein family, is a regulatory factor that interacts with RNA polymerase (RNAP). It is highly conserved in Gram-positive bacteria and controls transcription on a genome-wide scale in response to oxidative stress. The structural requirements for RNAP interaction and promoter DNA recognition by Spx were examined through mutational analysis. Residues near the CxxC redox disulfide center of Spx functioned in RNAP α subunit interaction and in promoter DNA binding. R60E and C10A mutants were shown previously to confer defects in transcriptional activation, but both were able to interact with RNAP. R92, which is conserved in ArsC-family proteins, is likely involved in redox control of Spx, as the C10A mutation, which blocks disulfide formation, was epistatic to the R92A mutation. The R91A mutation reduced transcriptional activation and repression, suggesting a defect in RNAP interaction, which was confirmed by interaction assays using an epitope-tagged mutant protein. Protein-DNA cross-linking detected contact between RNAP-bound Spx and the AGCA element at −44 that is conserved in Spx-controlled genes. This interaction caused repositioning of the RNAP σA subunit from a −35-like element upstream of the trxB (thioredoxin reductase) promoter to positions −36 and −11 of the core promoter. The study shows that RNAP-bound Spx contacts a conserved upstream promoter sequence element when bound to RNAP. PMID:23813734

  4. The extended regulatory networks of SXT/R391 integrative and conjugative elements and IncA/C conjugative plasmids

    PubMed Central

    Poulin-Laprade, Dominic; Carraro, Nicolas; Burrus, Vincent

    2015-01-01

    Nowadays, healthcare systems are challenged by a major worldwide drug resistance crisis caused by the massive and rapid dissemination of antibiotic resistance genes and associated emergence of multidrug resistant pathogenic bacteria, in both clinical and environmental settings. Conjugation is the main driving force of gene transfer among microorganisms. This mechanism of horizontal gene transfer mediates the translocation of large DNA fragments between two bacterial cells in direct contact. Integrative and conjugative elements (ICEs) of the SXT/R391 family (SRIs) and IncA/C conjugative plasmids (ACPs) are responsible for the dissemination of a broad spectrum of antibiotic resistance genes among diverse species of Enterobacteriaceae and Vibrionaceae. The biology, diversity, prevalence and distribution of these two families of conjugative elements have been the subject of extensive studies for the past 15 years. Recently, the transcriptional regulators that govern their dissemination through the expression of ICE- or plasmid-encoded transfer genes have been described. Unrelated repressors control the activation of conjugation by preventing the expression of two related master activator complexes in both types of elements, i.e., SetCD in SXT/R391 ICEs and AcaCD in IncA/C plasmids. Finally, in addition to activating ICE- or plasmid-borne genes, these master activators have been shown to specifically activate phylogenetically unrelated mobilizable genomic islands (MGIs) that also disseminate antibiotic resistance genes and other adaptive traits among a plethora of pathogens such as Vibrio cholerae and Salmonella enterica. PMID:26347724

  5. Epstein-Barr virus transcription factor Zta acts through distal regulatory elements to directly control cellular gene expression.

    PubMed

    Ramasubramanyan, Sharada; Osborn, Kay; Al-Mohammad, Rajaei; Naranjo Perez-Fernandez, Ijiel B; Zuo, Jianmin; Balan, Nicolae; Godfrey, Anja; Patel, Harshil; Peters, Gordon; Rowe, Martin; Jenner, Richard G; Sinclair, Alison J

    2015-04-20

    Lytic replication of the human gamma herpes virus Epstein-Barr virus (EBV) is an essential prerequisite for the spread of the virus. Differential regulation of a limited number of cellular genes has been reported in B-cells during the viral lytic replication cycle. We asked whether a viral bZIP transcription factor, Zta (BZLF1, ZEBRA, EB1), drives some of these changes. Using genome-wide chromatin immunoprecipitation coupled to next-generation DNA sequencing (ChIP-seq) we established a map of Zta interactions across the human genome. Using sensitive transcriptome analyses we identified 2263 cellular genes whose expression is significantly changed during the EBV lytic replication cycle. Zta binds 278 of the regulated genes and the distribution of binding sites shows that Zta binds mostly to sites that are distal to transcription start sites. This differs from the prevailing view that Zta activates viral genes by binding exclusively at promoter elements. We show that a synthetic Zta binding element confers Zta regulation at a distance and that distal Zta binding sites from cellular genes can confer Zta-mediated regulation on a heterologous promoter. This leads us to propose that Zta directly reprograms the expression of cellular genes through distal elements. PMID:25779048

  6. Multiple mechanisms contribute to osmotic inducibility of proU operon expression in Escherichia coli: demonstration of two osmoresponsive promoters and of a negative regulatory element within the first structural gene.

    PubMed Central

    Dattananda, C S; Rajkumari, K; Gowrishankar, J

    1991-01-01

    Transcription of the proU operon in Escherichia coli is induced several hundredfold upon growth of cells in media of elevated osmolarity. A low-copy-number promoter-cloning plasmid vector, with lacZ as the reporter gene, was used for assaying the osmoresponsive promoter activity of each of various lengths of proU DNA, generated by cloning of discrete restriction fragments and by an exonuclease III-mediated deletion approach. The results indicate that expression of proU in E. coli is directed from two promoters, one (P2) characterized earlier by other workers with the start site of transcription 60 nucleotides upstream of the initiation codon of the first structural gene (proV), and the other (P1) situated 250 nucleotides upstream of proV. Furthermore, a region of DNA within proV was shown to be involved in negative regulation of proU transcription; phage Mu dII1681-generated lac fusions in the early region of proV also exhibited partial derepression of proU regulation, in comparison with fusions further downstream in the operon. Sequences around promoter P1, sequences around P2, and the promoter-downstream negative regulatory element, respectively, conferred approximately 5-, 8-, and 25-fold osmoresponsivity on proU expression. Within the region genetically defined to encode the negative regulatory element, there is a 116-nucleotide stretch that is absolutely conserved between the proU operons of E. coli and Salmonella typhimurium and has the capability of exhibiting alternative secondary structure. Insertion of this region of DNA into each of two different plasmid vectors was associated with a marked reduction in the mean topological linking number in plasmid molecules isolated from cultures grown in high-osmolarity medium. We propose that this region of DNA undergoes reversible transition to an underwound DNA conformation under high-osmolarity growth conditions and that this transition mediates its regulatory effect on proU expression. Images FIG. 2 FIG. 3 PMID

  7. Regulatory elements in the first intron contribute to transcriptional control of the human. cap alpha. 1(I) collagen gene

    SciTech Connect

    Bornstein, P.; McKay, J.; Morishima, J.K.; Devarayalu, S.; Gelinas, R.E.

    1987-12-01

    Several lines of evidence have suggested that the regulation of type I collagen gene transcription is complex and that important regulatory elements reside 5' to, and within, the first intron of the ..cap alpha..1(I) gene. The authors therefore sequenced a 2.3-kilobase HindIII fragment that encompasses 804 base pairs of 5' flanking sequence, the first exon, and most of the first intron of the ..cap alpha..1(I) human collagen gene. A 274-base-pair intronic sequence, flanked by Ava I sites (A274), contained a sequence identical to a high-affinity decanucleotide binding site for transcription factor Sp1 and a viral core enhancer sequence. DNase I protection experiments indicated zones of protection that corresponded to these motifs. When A274 was cloned 5' to the chloramphenicol acetyltransferase (CAT) gene, driven by an ..cap alpha..1(I) collagen promoter sequence, and expression was assessed by transfection, significant orientation-specific inhibition of CAT activity was observed. This effect was most apparent in chicken tendon fibroblasts, which modulate their level of collagen synthesis in culture. They propose that normal regulation of ..cap alpha..1(I) collagen gene transcription results from an interplay of positive and negative elements present in the promoter region and within the first intron.

  8. Piperine Induces Hepatic Low-Density Lipoprotein Receptor Expression through Proteolytic Activation of Sterol Regulatory Element-Binding Proteins

    PubMed Central

    Ochiai, Ayasa; Miyata, Shingo; Shimizu, Makoto; Inoue, Jun; Sato, Ryuichiro

    2015-01-01

    Elevated plasma low-density lipoprotein (LDL) cholesterol is considered as a risk factor for atherosclerosis. Because the hepatic LDL receptor (LDLR) uptakes plasma lipoproteins and lowers plasma LDL cholesterol, the activation of LDLR is a promising drug target for atherosclerosis. In the present study, we identified the naturally occurring alkaloid piperine, as an inducer of LDLR gene expression by screening the effectors of human LDLR promoter. The treatment of HepG2 cells with piperine increased LDLR expression at mRNA and protein levels and stimulated LDL uptake. Subsequent luciferase reporter gene assays revealed that the mutation of sterol regulatory element-binding protein (SREBP)-binding element abolished the piperine-mediated induction of LDLR promoter activity. Further, piperine treatments increased mRNA levels of several SREBP targets and mature forms of SREBPs. However, the piperine-mediated induction of the mature forms of SREBPs was not observed in SRD–15 cells, which lack insulin-induced gene–1 (Insig–1) and Insig–2. Finally, the knockdown of SREBPs completely abolished the piperine-meditated induction of LDLR gene expression in HepG2 cells, indicating that piperine stimulates the proteolytic activation of SREBP and subsequent induction of LDLR expression and activity. PMID:26431033

  9. Detecting the limits of regulatory element conservation anddivergence estimation using pairwise and multiple alignments

    SciTech Connect

    Pollard, Daniel A.; Moses, Alan M.; Iyer, Venky N.; Eisen,Michael B.

    2006-08-14

    Background: Molecular evolutionary studies of noncodingsequences rely on multiple alignments. Yet how multiple alignmentaccuracy varies across sequence types, tree topologies, divergences andtools, and further how this variation impacts specific inferences,remains unclear. Results: Here we develop a molecular evolutionsimulation platform, CisEvolver, with models of background noncoding andtranscription factor binding site evolution, and use simulated alignmentsto systematically examine multiple alignment accuracy and its impact ontwo key molecular evolutionary inferences: transcription factor bindingsite conservation and divergence estimation. We find that the accuracy ofmultiple alignments is determined almost exclusively by the pairwisedivergence distance of the two most diverged species and that additionalspecies have a negligible influence on alignment accuracy. Conservedtranscription factor binding sites align better than surroundingnoncoding DNA yet are often found to be misaligned at relatively shortdivergence distances, such that studies of binding site gain and losscould easily be confounded by alignment error. Divergence estimates frommultiple alignments tend to be overestimated at short divergencedistances but reach a tool specific divergence at which they cease toincrease, leading to underestimation at long divergences. Our moststriking finding was that overall alignment accuracy, binding sitealignment accuracy and divergence estimation accuracy vary greatly acrossbranches in a tree and are most accurate for terminal branches connectingsister taxa and least accurate for internal branches connectingsub-alignments. Conclusions: Our results suggest that variation inalignment accuracy can lead to errors in molecular evolutionaryinferences that could be construed as biological variation. Thesefindings have implications for which species to choose for analyses, whatkind of errors would be expected for a given set of species and howmultiple alignment tools and

  10. Transcriptional induction of IFN-gamma-responsive genes is modulated by DNA surrounding the interferon stimulation response element.

    PubMed Central

    Strehlow, I; Decker, T

    1992-01-01

    The 9/27 and GBP mRNAs are both inducible by Interferon-gamma (IFN-gamma). The promoters of both genes contain an Interferon Stimulation Response Element (ISRE), but while the GBP gene is strongly induced transcriptionally by IFN-gamma the response of the 9/27 promoter is very weak. We investigated the molecular basis for this difference. The different IFN-gamma-responsiveness was found to have more than one reason. First, 9/27 promoter DNA was unable to bind the Gamma Interferon Activation Factor (GAF) with a single high affinity site. It efficiently competed for the association of the GAF with the GBP promoter but this competition was due to the presence of two low affinity sites, the ISRE and an ISRE-like sequence, suggesting that the GAS and ISRE, though both having clear preferences for specific proteins, may nevertheless share a certain degree of structural homology. Second, the 9/27 and GBP ISREs differed markedly in their affinities for regulatory proteins (ISGFs 1,2,3) and the GBP ISRE was more potent in mediating IFN-gamma-induced promoter activity in transient transfection. Third and most importantly, however, the strong difference between the IFN-gamma response of the two promoters was mainly due to the sequences surrounding the ISRE: the positive-acting GAS on one side and sequences with silencing properties 5' and 3' of the 9/27 ISRE on the other side. The data thus show mechanisms to both up- and down-regulate the activity of the ISRE. Images PMID:1508672

  11. A Repetitive DNA Element Regulates Expression of the Helicobacter pylori Sialic Acid Binding Adhesin by a Rheostat-like Mechanism

    PubMed Central

    Vallström, Anna; Olofsson, Annelie; Öhman, Carina; Rakhimova, Lena; Borén, Thomas; Engstrand, Lars; Brännström, Kristoffer; Arnqvist, Anna

    2014-01-01

    During persistent infection, optimal expression of bacterial factors is required to match the ever-changing host environment. The gastric pathogen Helicobacter pylori has a large set of simple sequence repeats (SSR), which constitute contingency loci. Through a slipped strand mispairing mechanism, the SSRs generate heterogeneous populations that facilitate adaptation. Here, we present a model that explains, in molecular terms, how an intergenically located T-tract, via slipped strand mispairing, operates with a rheostat-like function, to fine-tune activity of the promoter that drives expression of the sialic acid binding adhesin, SabA. Using T-tract variants, in an isogenic strain background, we show that the length of the T-tract generates multiphasic output from the sabA promoter. Consequently, this alters the H. pylori binding to sialyl-Lewis x receptors on gastric mucosa. Fragment length analysis of post-infection isolated clones shows that the T-tract length is a highly variable feature in H. pylori. This mirrors the host-pathogen interplay, where the bacterium generates a set of clones from which the best-fit phenotypes are selected in the host. In silico and functional in vitro analyzes revealed that the length of the T-tract affects the local DNA structure and thereby binding of the RNA polymerase, through shifting of the axial alignment between the core promoter and UP-like elements. We identified additional genes in H. pylori, with T- or A-tracts positioned similar to that of sabA, and show that variations in the tract length likewise acted as rheostats to modulate cognate promoter output. Thus, we propose that this generally applicable mechanism, mediated by promoter-proximal SSRs, provides an alternative mechanism for transcriptional regulation in bacteria, such as H. pylori, which possesses a limited repertoire of classical trans-acting regulatory factors. PMID:24991812

  12. DNA damage-induced regulatory interplay between DAXX, p53, ATM kinase and Wip1 phosphatase

    PubMed Central

    Brazina, Jan; Svadlenka, Jan; Macurek, Libor; Andera, Ladislav; Hodny, Zdenek; Bartek, Jiri; Hanzlikova, Hana

    2015-01-01

    Death domain-associated protein 6 (DAXX) is a histone chaperone, putative regulator of apoptosis and transcription, and candidate modulator of p53-mediated gene expression following DNA damage. DAXX becomes phosphorylated upon DNA damage, however regulation of this modification, and its relationship to p53 remain unclear. Here we show that in human cells exposed to ionizing radiation or genotoxic drugs etoposide and neocarzinostatin, DAXX became rapidly phosphorylated in an ATM kinase-dependent manner. Our deletion and site-directed mutagenesis experiments identified Serine 564 (S564) as the dominant ATM-targeted site of DAXX, and immunofluorescence experiments revealed localization of S564-phosphorylated DAXX to PML nuclear bodies. Furthermore, using a panel of human cell types, we identified the p53-regulated Wip1 protein phosphatase as a key negative regulator of DAXX phosphorylation at S564, both in vitro and in cells. Consistent with the emerging oncogenic role of Wip1, its DAXX-dephosphorylating impact was most apparent in cancer cell lines harboring gain-of-function mutant and/or overexpressed Wip1. Unexpectedly, while Wip1 depletion increased DAXX phosphorylation both before and after DNA damage and increased p53 stability and transcriptional activity, knock-down of DAXX impacted neither p53 stabilization nor p53-mediated expression of Gadd45a, Noxa, Mdm2, p21, Puma, Sesn2, Tigar or Wip1. Consistently, analyses of cells with genetic, TALEN-mediated DAXX deletion corroborated the notion that neither phosphorylated nor non-phosphorylated DAXX is required for p53-mediated gene expression upon DNA damage. Overall, we identify ATM kinase and Wip1 phosphatase as opposing regulators of DAXX-S564 phosphorylation, and propose that the role of DAXX phosphorylation and DAXX itself are independent of p53-mediated gene expression. PMID:25659035

  13. Origin of the human L1 elements: proposed progenitor genes deduced from a consensus DNA sequence.

    PubMed

    Scott, A F; Schmeckpeper, B J; Abdelrazik, M; Comey, C T; O'Hara, B; Rossiter, J P; Cooley, T; Heath, P; Smith, K D; Margolet, L

    1987-10-01

    A consensus sequence for the human long interspersed repeated DNA element, L1Hs (LINE or KpnI sequence), is presented. The sequence contains two open reading frames (ORFs) which are homologous to ORFs in corresponding regions of L1 elements in other species. The L1Hs ORFs are separated by a small evolutionarily nonconserved region. The 5' end of the consensus contains frequent terminators in all three reading frames and has a relatively high GC content with numerous stretches of weak homology with AluI repeats. The 5' ORF extends for a minimum of 723 bp (241 codons). The 3' ORF is 3843 bp (1281 codons) and predicts a protein of 149 kD which has regions of weak homology to the polymerase domain of various reverse transcriptases. The 3' end of the consensus has a 208-bp nonconserved region followed by an adenine-rich end. The organization of the L1Hs consensus sequence resembles the structure of eukaryotic mRNAs except for the noncoding region between ORFs. However, due to base substitutions or truncation most elements appear incapable of producing mRNA that can be translated. Our observation that individual elements cluster into subfamilies on the basis of the presence or absence of blocks of sequence, or by the linkage of alternative bases at multiple positions, suggests that most L1 sequences were derived from a small number of structural genes. An estimate of the mammalian L1 substitution rate was derived and used to predict the age of individual human elements. From this it follows that the majority of human L1 sequences have been generated within the last 30 million years. The human elements studied here differ from each other, yet overall the L1Hs sequences demonstrate a pattern of species-specificity when compared to the L1 families of other mammals. Possible mechanisms that may account for the origin and evolution of the L1 family are discussed. These include pseudogene formation (retroposition), transposition, gene conversion, and RNA recombination. PMID

  14. A novel transcriptional element in circular DNA monomers of the duck hepatitis B virus.

    PubMed

    Beckel-Mitchener, A; Summers, J

    1997-10-01

    We report the presence of two elements, pet and net, that are required for proper transcription of the duck hepatitis B virus (DHBV). These regions were previously identified by using plasmid clones of the virus in transient expression assays (M. Huang and J. Summers, J. Virol. 68:1564-1572, 1994). In this study, we further analyzed these regions by using in vitro-synthesized circular DHBV DNA monomers to mimic the authentic transcriptional template. We observed that pet was required for pregenome transcription from circular viral monomers, and in the absence of pet-dependent transcription, expression of the viral envelope genes was increased. We found that deletion of net in circularized DNA monomers led to the production of abnormally long transcripts due to a failure to form 3' ends during transcription. In addition, we report the presence of a net-like region in the mammalian hepadnavirus woodchuck hepatitis virus. These results are consistent with a model that net is a region involved in transcription termination and that in DHBV, pet is required for transcription complexes to read through this region during the first pass through net. PMID:9311882

  15. Chromatin immunoprecipitation reveals that the 180-bp satellite repeat is the key functional DNA element of Arabidopsis thaliana centromeres.

    PubMed Central

    Nagaki, Kiyotaka; Talbert, Paul B; Zhong, Cathy Xiaoyan; Dawe, R Kelly; Henikoff, Steven; Jiang, Jiming

    2003-01-01

    The centromeres of Arabidopsis thaliana chromosomes contain megabases of complex DNA consisting of numerous types of repetitive DNA elements. We developed a chromatin immunoprecipitation (ChIP) technique using an antibody against the centromeric H3 histone, HTR12, in Arabidopsis. ChIP assays showed that the 180-bp centromeric satellite repeat was precipitated with the antibody, suggesting that this repeat is the key component of the centromere/kinetochore complex in Arabidopsis. PMID:12663558

  16. Regulatory elements necessary for termination of transcription within the immunoglobulin heavy chain gene locus

    SciTech Connect

    Moore, B.B.

    1992-01-01

    Previous experimentation demonstrated that regulation of the IgM only phenotype in both pre-B and immature B cells was primarily at the transcriptional level. Expression of IgD mRNA involves transcription of the entire 29 kilobase rearranged [mu]-[delta] locus. Mature B cells transcribe the [beta] exons at approximately half the level that they transcribe the [delta] gene. Early B cells however, transcribe the [mu] gene with approximately 90% more efficiency than they do the [delta] gene. Specifically, early B cells show a transcription termination event occurring within a 1 kilobase region of the [mu]-[delta] intron. This dissertation analyzes the sequence elements necessary to encode the transcription termination event within the [mu]-[delta] intron. This work shows that the termination motif consists of specific sequences within the [mu]m poly(A) site as well as a region of the [mu]-[delta] intron contained within a 1200 base pair fragment. The 1200 base pair fragment extends from the Pst I site within the intron and ends just prior to the C[delta]1 exon. This fragment contains a 162 base pair unique sequence inverted repeat (USIR). Furthermore, the [mu]m site is specifically required because the [mu]s site was unable to substitute, despite extensive usage. In addition, the USIR-containing intron functions in an orientation-dependent manner. Analysis of this termination motif in a variety of lymphoid and non-lymphoid cells suggests that this motif is an intrinsic polymerase II termination motif. This implies that transcription termination in early B cells is by a default model and that active regulation of this motif involves an anti-termination event in mature B cells.

  17. Sequence, Genomic Distribution and DNA Modification of a Mu1 Element from Non-Mutator Maize Stocks

    PubMed Central

    Chandler, V. L.; Talbert, L. E.; Raymond, F.

    1988-01-01

    The increased mutation rate of Mutator stocks of maize has been shown to be the result of transposition of Mu elements. One element, Mu1, is present in 10-60 copies in Mutator stocks and approximately 0-3 copies in non-Mutator stocks. The sequence, structure and genomic distribution of an intact Mu1 element cloned from the non-Mutator inbred line B37 has been determined. The sequence of this element, termed Mu1.4-B37, is identical to Mu1 and it is flanked by 9-bp direct repeats indicative of a target site duplication. Mu1.4-B37 is not in the same genomic location in all stocks, which further suggests that it transposed into its genomic location in B37. We previously reported that in genomic DNA this element is modified such that certain methylation-sensitive restriction enzymes will not cut sites within the element. This is similar to that observed for Mu elements in Mutator stocks that have lost activity. We report herein that the Mu1.4-B37 element loses its modification and becomes accessible to digestion when placed in an active Mutator stock by genetic crosses. This suggests that factors conditioning unmodified elements are dominant in the initial cross between Mutator and non-Mutator stocks. In F(2) individuals that have subsequently lost Mutator activity the Mu1.4-B37 element again becomes modified as do most of the Mu elements in the stock. Thus, the modification state of the Mu1.4-B37 element and the other Mu1-like elements correlates with Mutator activity. We hypothesize that factor(s) within an active Mutator stock may inhibit the modification of Mu elements, and that this activity is missing in non-Mutator stocks and may become limiting in certain Mutator stocks resulting in DNA modification. PMID:2842229

  18. Turnover of R1 (Type I) and R2 (Type Ii) Retrotransposable Elements in the Ribosomal DNA of Drosophila Melanogaster

    PubMed Central

    Jakubczak, J. L.; Zenni, M. K.; Woodruff, R. C.; Eickbush, T. H.

    1992-01-01

    R1 and R2 are distantly related non-long terminal repeat retrotransposable elements each of which inserts into a specific site in the 28S rRNA genes of most insects. We have analyzed aspects of R1 and R2 abundance and sequence variation in 27 geographical isolates of Drosophila melanogaster. The fraction of 28S rRNA genes containing these elements varied greatly between strains, 17-67% for R1 elements and 2-28% for R2 elements. The total percentage of the rDNA repeats inserted ranged from 32 to 77%. The fraction of the rDNA repeats that contained both of these elements suggested that R1 and R2 exhibit neither an inhibition of nor preference for insertion into a 28S gene already containing the other type of element. Based on the conservation of restriction sites in the elements of all strains, and sequence analysis of individual elements from three strains, nucleotide divergence is very low for R1 and R2 elements within or between strains (<0.6%). This sequence uniformity is the expected result of the forces of concerted evolution (unequal crossovers and gene conversion) which act on the rRNA genes themselves. Evidence for the role of retrotransposition in the turnover of R1 and R2 was obtained by using naturally occurring 5' length polymorphisms of the elements as markers for independent transposition events. The pattern of these different length 5' truncations of R1 and R2 was found to be diverse and unique to most strains analyzed. Because recombination can only, with time, amplify or eliminate those length variants already present, the diversity found in each strain suggests that retrotransposition has played a critical role in maintaining these elements in the rDNA repeats of D. melanogaster. PMID:1317313

  19. Binding of estrogen receptors to switch sites and regulatory elements in the immunoglobulin heavy chain locus of activated B cells suggests a direct influence of estrogen on antibody expression.

    PubMed

    Jones, Bart G; Penkert, Rhiannon R; Xu, Beisi; Fan, Yiping; Neale, Geoff; Gearhart, Patricia J; Hurwitz, Julia L

    2016-09-01

    Females and males differ in antibody isotype expression patterns and in immune responses to foreign- and self-antigens. For example, systemic lupus erythematosus is a condition that associates with the production of isotype-skewed anti-self antibodies, and exhibits a 9:1 female:male disease ratio. To explain differences between B cell responses in males and females, we sought to identify direct interactions of the estrogen receptor (ER) with the immunoglobulin heavy chain locus. This effort was encouraged by our previous identification of estrogen response elements (ERE) in heavy chain switch (S) regions. We conducted a full-genome chromatin immunoprecipitation analysis (ChIP-seq) using DNA from LPS-activated B cells and an ERα-specific antibody. Results revealed ER binding to a wide region of DNA, spanning sequences from the JH cluster to Cδ, with peaks in Eμ and Sμ sites. Additional peaks of ERα binding were coincident with hs1,2 and hs4 sites in the 3' regulatory region (3'RR) of the heavy chain locus. This first demonstration of direct binding of ER to key regulatory elements in the immunoglobulin locus supports our hypothesis that estrogen and other nuclear hormone receptors and ligands may directly influence antibody expression and class switch recombination (CSR). Our hypothesis encourages the conduct of new experiments to evaluate the consequences of ER binding. A better understanding of ER:DNA interactions in the immunoglobulin heavy chain locus, and respective mechanisms, may ultimately translate to better control of antibody expression, better protection against pathogens, and prevention of pathologies caused by auto-immune disease. PMID:27494228

  20. Stress induced gene expression drives transient DNA methylation changes at adjacent repetitive elements

    PubMed Central

    Secco, David; Wang, Chuang; Shou, Huixia; Schultz, Matthew D; Chiarenza, Serge; Nussaume, Laurent; Ecker, Joseph R; Whelan, James; Lister, Ryan

    2015-01-01

    Cytosine DNA methylation (mC) is a genome modification that can regulate the expression of coding and non-coding genetic elements. However, little is known about the involvement of mC in response to environmental cues. Using whole genome bisulfite sequencing to assess the spatio-temporal dynamics of mC in rice grown under phosphate starvation and recovery conditions, we identified widespread phosphate starvation-induced changes in mC, preferentially localized in transposable elements (TEs) close to highly induced genes. These changes in mC occurred after changes in nearby gene transcription, were mostly DCL3a-independent, and could partially be propagated through mitosis, however no evidence of meiotic transmission was observed. Similar analyses performed in Arabidopsis revealed a very limited effect of phosphate starvation on mC, suggesting a species-specific mechanism. Overall, this suggests that TEs in proximity to environmentally induced genes are silenced via hypermethylation, and establishes the temporal hierarchy of transcriptional and epigenomic changes in response to stress. DOI: http://dx.doi.org/10.7554/eLife.09343.001 PMID:26196146

  1. sup 1 H NMR studies of DNA recognition by the glucocorticoid receptor: Complex of the DNA binding domain with a half-site response element

    SciTech Connect

    Remerowski, M.L.; Kellenbach, E.; Boelens, R.; Kaptein, R. ); van der Marel, G.A.; van Boom, J.H. ); Maler, B.A.; Yamamoto, K.R. )

    1991-12-17

    The complex of the rat glucocorticoid receptor (GR) DNA binding domain (DBD) and half-site sequence of the consensus glucocorticoid response element (GRE) has been studied by two-dimensional {sup 1}H NMR spectroscopy. The DNA fragment is a 10 base-pair oligonucleotide, 5{prime}d(GCTGTTCTGC)3{prime}{center dot}5{prime}d-(GCAGAACAGC)3{prime}, containing the stronger binding GRE half-site hexamer, with GC base pairs at each end. The 93-residue GR-DBD contains an 86-residue segment corresponding to residues 440-525 of the rat GR. Eleven NOE cross peaks between the protein and DNA have been identified, and changes in the chemical shift of the DNA protons upon complex formation have been analyzed. Using these protein-DNA contact points, it can be concluded that (1) the 'recognition helix' formed by residues C460-E469 lies in the major groove of the DNA; (2) the GR-DBD is oriented on the GRE half-site such that residues A477-D481, forming the so-called D-loop, are available for protein-protein interaction in the GR-DBD dimer on the intact consensus GRE; and (3) the 5-methyl of the second thymine in the half-site and valine 462 interact, confirming indirect evidence that both play an important role in GR-DBD DNA binding.

  2. Regulatory elements in the introns of the human HPRT gene are necessary for its expression in embryonic stem cells.

    PubMed Central

    Reid, L H; Gregg, R G; Smithies, O; Koller, B H

    1990-01-01

    We have examined the expression of transfected human hypoxanthine phosphoribosyltransferase minigenes (HPRT) in mouse embryonic stem (ES) cells. cDNA constructs of this gene that have been successfully used in somatic cell lines failed to confer hypoxanthine/aminopterin/thymidine (HAT) resistance in ES cells. In contrast, constructs containing introns 1 and 2 from the HPRT gene produced a high frequency of HAT-resistant colonies. This observation allowed us to identify two sequences in these introns that influence expression of the HPRT gene in ES cells. One element, located in intron 2, is required for effective HPRT expression in these cells; the other element, located in intron 1, acts as an enhancer of HPRT expression. Using this information, we have constructed an HPRT minigene that can be used for either positive or negative selection in ES cell experiments. This dual capability allows the design of "in-out" procedures to create subtle changes in target genes by homologous recombination with the aid of this selectable minigene. PMID:2349238

  3. Identification of conserved regulatory elements in upstream promoter regions of mammals at relaxed thresholds by comparative genomics - a case study using PEPCK

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background Comparative genomics is the primary method to discover regulatory elements by identifying conserved sequences due to evolutionary constraints by cross-species genome comparison. Except for the most conserved and prominent transcription factor binding sites (TFBS), there is a general lack ...

  4. DNA methylation as a regulatory mechanism in rat gamma-crystallin gene expression.

    PubMed Central

    Peek, R; Niessen, R W; Schoenmakers, J G; Lubsen, N H

    1991-01-01

    We have investigated the methylation state of the rat gamma-crystallin genes in DNA from lens cells at different developmental stages as well as from kidney and heart cells. A clear correlation between the extent of demethylation of the promoter and 5' gene regions and the expression of these genes was observed. No change in the methylation state of the far upstream or 3' regions of the genes was seen. The demethylation of the promoter region was shown to occur during the differentiation from the lens epithelial to the lens fiber cell. The effect of cytosine methylation on gamma-crystallin promoter activity was tested by measuring gamma-crystallin promoter/chloramphenicol acetyltransferase fusion gene expression after in vitro primed repair synthesis of the promoter region in the presence of either dCTP or 5mdCTP. The hemimethylated promoter was no longer capable of promoting high CAT activity after introduction into lens-like cells. Taken together, our data suggest that DNA demethylation may be the determining step in the developmental stage-specific expression of the rat gamma-crystallin genes. Images PMID:2011513

  5. Identification of regulatory elements in the AGT1 promoter of ale and lager strains of brewer's yeast.

    PubMed

    Vidgren, Virve; Kankainen, Matti; Londesborough, John; Ruohonen, Laura

    2011-08-01

    Agt1 is an interesting α-glucoside transporter for the brewing industry, as it efficiently transports maltotriose, a sugar often remaining partly unused during beer fermentation. It has been shown that on maltose the expression level of AGT1 is much higher in ale strains than in lager strains, and that glucose represses the expression, particularly in the ale strains. In the present study the regulatory elements of the AGT1 promoter of one ale and two lager strains were identified by computational methods. Promoter regions up to 1.9 kbp upstream of the AGT1 gene were sequenced from the three brewer's yeast strains and the laboratory yeast strain CEN.PK-1D. The promoter sequence of the laboratory strain was identical to the AGT1 promoter of strain S288c of the Saccharomyces Genome Database, whereas the promoter sequences of the industrial strains diverged markedly from the S288c strain. The AGT1 promoter regions of the ale and lager strains were for the most part identical to each other, except for one 22 bp deletion and two 94 and 95 bp insertions in the ale strain. Computational analyses of promoter elements revealed that the promoter sequences contained several Mig1- and MAL-activator binding sites, as was expected. However, some of the Mig1 and MAL-activator binding sites were located on the two insertions of the ale strain, and thus offered a plausible explanation for the different expression pattern of the AGT1 gene in the ale strains. Accordingly, functional analysis of A60 ale and A15 lager strain AGT1 promoters fused to GFP (encoding the green fluorescent protein) showed a significant difference in the ability of these two promoters to drive GFP expression. Under the control of the AGT1 promoter of the ale strain the emergence of GFP was strongly induced by maltose, whereas only a low level of GFP was detected with the construct carrying the AGT1 promoter of the lager strain. Thus, the extra MAL-activator binding element, present in the AGT1 promoter of

  6. Germ line and embryonic expression of Fex, a member of the Drosophila F-element retrotransposon family, is mediated by an internal cis-regulatory control region.

    PubMed Central

    Kerber, B; Fellert, S; Taubert, H; Hoch, M

    1996-01-01

    The F elements of Drosophila melanogaster belong to the superfamily of long interspersed nucleotide element retrotransposons. To date, F-element transcription has not been detected in flies. Here we describe the isolation of a member of the F-element family, termed Fex, which is transcribed in specific cells of the female and male germ lines and in various tissues during embryogenesis of D. melanogaster. Sequence analysis revealed that this element contains two complete open reading frames coding for a putative nucleic acid-binding protein and a putative reverse transcriptase. Functional analysis of the 5' region, using germ line transformation of Fex-lacZ reporter gene constructs, demonstrates that major aspects of tissue-specific Fex expression are controlled by internal cis-acting elements that lie in the putative coding region of open reading frame 1. These sequences mediate dynamic gene expression in eight expression domains during embryonic and germ line development. The capacity of the cis-regulatory region of the Fex element to mediate such complex expression patterns is unique among members of the long interspersed nucleotide element superfamily of retrotransposons and is reminiscent of regulatory regions of developmental control genes. PMID:8649411

  7. The Zinc Finger Protein ZNF658 Regulates the Transcription of Genes Involved in Zinc Homeostasis and Affects Ribosome Biogenesis through the Zinc Transcriptional Regulatory Element

    PubMed Central

    Ogo, Ogo A.; Tyson, John; Cockell, Simon J.; Howard, Alison; Valentine, Ruth A.

    2015-01-01

    We previously identified the ZTRE (zinc transcriptional regulatory element) in genes involved in zinc homeostasis and showed that it mediates transcriptional repression in response to zinc. We now report that ZNF658 acts at the ZTRE. ZNF658 was identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry of a band excised after electrophoretic mobility shift assay using a ZTRE probe. The protein contains a KRAB domain and 21 zinc fingers. It has similarity with ZAP1 from Saccharomyces cerevisiae, which regulates the response to zinc restriction, including a conserved DNA binding region we show to be functional also in ZNF658. Small interfering RNA (siRNA) targeted to ZNF658 abrogated the zinc-induced, ZTRE-dependent reduction in SLC30A5 (ZnT5 gene), SLC30A10 (ZnT10 gene), and CBWD transcripts in human Caco-2 cells and the ability of zinc to repress reporter gene expression from corresponding promoter-reporter constructs. Microarray analysis of the effect of reducing ZNF658 expression by siRNA uncovered a large decrease in rRNA. We find that ZTREs are clustered within the 45S rRNA precursor. We also saw effects on expression of multiple ribosomal proteins. ZNF658 thus links zinc homeostasis with ribosome biogenesis, the most active transcriptional, and hence zinc-demanding, process in the cell. ZNF658 is thus a novel transcriptional regulator that plays a fundamental role in the orchestrated cellular response to zinc availability. PMID:25582195

  8. Control of human PLP1 expression through transcriptional regulatory elements and alternatively spliced exons in intron 1.

    PubMed

    Hamdan, Hamdan; Kockara, Neriman T; Jolly, Lee Ann; Haun, Shirley; Wight, Patricia A

    2015-01-01

    Although the myelin proteolipid protein gene (PLP1) encodes the most abundant protein in central nervous system (CNS) myelin, not much is known about the mechanisms that govern expression of the human gene (hPLP1). Much more is known about the processes that regulate Plp1 gene expression in rodents. From studies with Plp1-lacZ transgenic mice, it was determined that the first intron of mouse Plp1 (mPlp1) is required to attain high levels of expression in brain, concurrent with the active myelination period. Other studies have suggested that within mPlp1 intron 1 (>8 kb) lie several regions with enhancer-like activity. To test whether these sequences (and possibly others) in hPLP1 intron 1 are functional, deletion-transfection analysis was performed with hPLP1-lacZ constructs that contain various portions of the intron, or lack it altogether. Results presented here demonstrate the importance of hPLP1 intron 1 in achieving maximal levels of expression in the immortalized oligodendroglial cell line, Oli-neu. Deletion analysis indicates that the intron contains multiple positive regulatory elements which are active in Oli-neu cells. Some of these elements appear to be functionally conserved between human and mouse, while others are not. Furthermore, our studies demonstrate that multiple splice variants can be formed due to inclusion of extra (supplementary) exons from what is classically thought of as hPLP1 intron 1. Thus, splicing of these novel exons (which are not recognized as such in mPlp1 due to lack of conserved splice sites) must utilize factors common to both human and mouse since Oli-neu cells are of mouse origin. PMID:25694552

  9. Xanthohumol Improves Diet-induced Obesity and Fatty Liver by Suppressing Sterol Regulatory Element-binding Protein (SREBP) Activation*

    PubMed Central

    Miyata, Shingo; Inoue, Jun; Shimizu, Makoto; Sato, Ryuichiro

    2015-01-01

    Sterol regulatory element-binding proteins (SREBPs) are key transcription factors that stimulate the expression of genes involved in fatty acid and cholesterol biosynthesis. Here, we demonstrate that a prenylated flavonoid in hops, xanthohumol (XN), is a novel SREBP inactivator that reduces the de novo synthesis of fatty acid and cholesterol. XN independently suppressed the maturation of SREBPs of insulin-induced genes in a manner different from sterols. Our results suggest that XN impairs the endoplasmic reticulum-to-Golgi translocation of the SREBP cleavage-activating protein (SCAP)-SREBP complex by binding to Sec23/24 and blocking SCAP/SREBP incorporation into common coated protein II vesicles. Furthermore, in diet-induced obese mice, dietary XN suppressed SREBP-1 target gene expression in the liver accompanied by a reduction of the mature form of hepatic SREBP-1, and it inhibited the development of obesity and hepatic steatosis. Altogether, our data suggest that XN attenuates the function of SREBP-1 by repressing its maturation and that it has the potential of becoming a nutraceutical food or pharmacological agent for improving metabolic syndrome. PMID:26140926

  10. Pi class glutathione S-transferase genes are regulated by Nrf 2 through an evolutionarily conserved regulatory element in zebrafish

    PubMed Central

    Suzuki, Takafumi; Takagi, Yaeko; Osanai, Hitoshi; Li, Li; Takeuchi, Miki; Katoh, Yasutake; Kobayashi, Makoto; Yamamoto, Masayuki

    2005-01-01

    Pi class GSTs (glutathione S-transferases) are a member of the vertebrate GST family of proteins that catalyse the conjugation of GSH to electrophilic compounds. The expression of Pi class GST genes can be induced by exposure to electrophiles. We demonstrated previously that the transcription factor Nrf 2 (NF-E2 p45-related factor 2) mediates this induction, not only in mammals, but also in fish. In the present study, we have isolated the genomic region of zebrafish containing the genes gstp1 and gstp2. The regulatory regions of zebrafish gstp1 and gstp2 have been examined by GFP (green fluorescent protein)-reporter gene analyses using microinjection into zebrafish embryos. Deletion and point-mutation analyses of the gstp1 promoter showed that an ARE (antioxidant-responsive element)-like sequence is located 50 bp upstream of the transcription initiation site which is essential for Nrf 2 transactivation. Using EMSA (electrophoretic mobility-shift assay) analysis we showed that zebrafish Nrf 2–MafK heterodimer specifically bound to this sequence. All the vertebrate Pi class GST genes harbour a similar ARE-like sequence in their promoter regions. We propose that this sequence is a conserved target site for Nrf 2 in the Pi class GST genes. PMID:15654768

  11. Regulation of steroid 5-{alpha} reductase type 2 (Srd5a2) by sterol regulatory element binding proteins and statin

    SciTech Connect

    Seo, Young-Kyo; Zhu, Bing; Jeon, Tae-Il; Osborne, Timothy F.

    2009-11-01

    In this study, we show that sterol regulatory element binding proteins (SREBPs) regulate expression of Srd5a2, an enzyme that catalyzes the irreversible conversion of testosterone to dihydroxytestosterone in the male reproductive tract and is highly expressed in androgen-sensitive tissues such as the prostate and skin. We show that Srd5a2 is induced in livers and prostate from mice fed a chow diet supplemented with lovastatin plus ezitimibe (L/E), which increases the activity of nuclear SREBP-2. The three fold increase in Srd5a2 mRNA mediated by L/E treatment was accompanied by the induction of SREBP-2 binding to the Srd5a2 promoter detected by a ChIP-chip assay in liver. We identified a SREBP-2 responsive region within the first 300 upstream bases of the mouse Srd5a2 promoter by co-transfection assays which contain a site that bound SREBP-2 in vitro by an EMSA. Srd5a2 protein was also induced in cells over-expressing SREBP-2 in culture. The induction of Srd5a2 through SREBP-2 provides a mechanistic explanation for why even though statin therapy is effective in reducing cholesterol levels in treating hypercholesterolemia it does not compromise androgen production in clinical studies.

  12. Cis-Regulatory Elements Determine Germline Specificity and Expression Level of an Isopentenyltransferase Gene in Sperm Cells of Arabidopsis.

    PubMed

    Zhang, Jinghua; Yuan, Tong; Duan, Xiaomeng; Wei, Xiaoping; Shi, Tao; Li, Jia; Russell, Scott D; Gou, Xiaoping

    2016-03-01

    Flowering plant sperm cells transcribe a divergent and complex complement of genes. To examine promoter function, we chose an isopentenyltransferase gene known as PzIPT1. This gene is highly selectively transcribed in one sperm cell morphotype of Plumbago zeylanica, which preferentially fuses with the central cell during fertilization and is thus a founding cell of the primary endosperm. In transgenic Arabidopsis (Arabidopsis thaliana), PzIPT1 promoter displays activity in both sperm cells and upon progressive promoter truncation from the 5'-end results in a progressive decrease in reporter production, consistent with occurrence of multiple enhancer sites. Cytokinin-dependent protein binding motifs are identified in the promoter sequence, which respond with stimulation by cytokinin. Expression of PzIPT1 promoter in sperm cells confers specificity independently of previously reported Germline Restrictive Silencer Factor binding sequence. Instead, a cis-acting regulatory region consisting of two duplicated 6-bp Male Gamete Selective Activation (MGSA) motifs occurs near the site of transcription initiation. Disruption of this sequence-specific site inactivates expression of a GFP reporter gene in sperm cells. Multiple copies of the MGSA motif fused with the minimal CaMV35S promoter elements confer reporter gene expression in sperm cells. Similar duplicated MGSA motifs are also identified from promoter sequences of sperm cell-expressed genes in Arabidopsis, suggesting selective activation is possibly a common mechanism for regulation of gene expression in sperm cells of flowering plants. PMID:26739233

  13. Combining Hi-C data with phylogenetic correlation to predict the target genes of distal regulatory elements in human genome

    PubMed Central

    Lu, Yulan; Zhou, Yuanpeng; Tian, Weidong

    2013-01-01

    Defining the target genes of distal regulatory elements (DREs), such as enhancer, repressors and insulators, is a challenging task. The recently developed Hi-C technology is designed to capture chromosome conformation structure by high-throughput sequencing, and can be potentially used to determine the target genes of DREs. However, Hi-C data are noisy, making it difficult to directly use Hi-C data to identify DRE–target gene relationships. In this study, we show that DREs–gene pairs that are confirmed by Hi-C data are strongly phylogenetic correlated, and have thus developed a method that combines Hi-C read counts with phylogenetic correlation to predict long-range DRE–target gene relationships. Analysis of predicted DRE–target gene pairs shows that genes regulated by large number of DREs tend to have essential functions, and genes regulated by the same DREs tend to be functionally related and co-expressed. In addition, we show with a couple of examples that the predicted target genes of DREs can help explain the causal roles of disease-associated single-nucleotide polymorphisms located in the DREs. As such, these predictions will be of importance not only for our understanding of the function of DREs but also for elucidating the causal roles of disease-associated noncoding single-nucleotide polymorphisms. PMID:24003029

  14. Gentiana manshurica Kitagawa reverses acute alcohol-induced liver steatosis through blocking sterol regulatory element-binding protein-1 maturation.

    PubMed

    Lian, Li-Hua; Wu, Yan-Ling; Song, Shun-Zong; Wan, Ying; Xie, Wen-Xue; Li, Xin; Bai, Ting; Ouyang, Bing-Qing; Nan, Ji-Xing

    2010-12-22

    This study was undertaken to investigate the protective effects of Gentiana manshurica Kitagawa (GM) on acute alcohol-induced fatty liver. Mice were treated with ethanol (5 g/kg of body weight) by gavage every 12 h for a total of three doses to induce acute fatty liver. Methanol extract of GM (50, 100, or 200 mg/kg) or silymarin (100 mg/kg) was gavaged simultaneously with ethanol for three doses. GM administration significantly reduced the increases in serum ALT and AST levels, the serum and hepatic triglyceride levels, at 4 h after the last ethanol administration. GM was also found to prevent ethanol-induced hepatic steatosis and necrosis, as indicated by liver histopathological studies. Additionally, GM suppressed the elevation of malondialdehyde (MDA) levels, restored the glutathione (GSH) levels, and enhanced the superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities. The concurrent administration of GM efficaciously abrogated cytochrome P450 2E1 (CYP2E1) induction. Moreover, GM significantly reduced the nuclear translocation of sterol regulatory element-binding protein-1 (nSREBP-1) in ethanol-treated mice. These data indicated that GM possessed the ability to prevent ethanol-induced acute liver steatosis, possibly through blocking CYP2E1-mediated free radical scavenging effects and SREBP-1-regulated fatty acid synthesis. Especially, GM may be developed as a potential therapeutic candidate for ethanol-induced oxidative damage in liver. PMID:21105651

  15. Sterol Regulatory Element Binding Protein Regulates the Expression and Metabolic Functions of Wild-Type and Oncogenic IDH1.

    PubMed

    Ricoult, Stéphane J H; Dibble, Christian C; Asara, John M; Manning, Brendan D

    2016-09-15

    Sterol regulatory element binding protein (SREBP) is a major transcriptional regulator of the enzymes underlying de novo lipid synthesis. However, little is known about the SREBP-mediated control of processes that indirectly support lipogenesis, for instance, by supplying reducing power in the form of NAPDH or directing carbon flux into lipid precursors. Here, we characterize isocitrate dehydrogenase 1 (IDH1) as a transcriptional target of SREBP across a spectrum of cancer cell lines and human cancers. IDH1 promotes the synthesis of lipids specifically from glutamine-derived carbons. Neomorphic mutations in IDH1 occur frequently in certain cancers, leading to the production of the oncometabolite 2-hydroxyglutarate (2-HG). We found that SREBP induces the expression of oncogenic IDH1 and influences 2-HG production from glucose. Treatment of cells with 25-hydroxycholesterol or statins, which respectively inhibit or activate SREBP, further supports SREBP-mediated regulation of IDH1 and, in cells with oncogenic IDH1, carbon flux into 2-HG. PMID:27354064

  16. Computational discovery of soybean promoter cis-regulatory elements for the construction of soybean cyst nematode-inducible synthetic promoters.

    PubMed

    Liu, Wusheng; Mazarei, Mitra; Peng, Yanhui; Fethe, Michael H; Rudis, Mary R; Lin, Jingyu; Millwood, Reginald J; Arelli, Prakash R; Stewart, Charles Neal

    2014-10-01

    Computational methods offer great hope but limited accuracy in the prediction of functional cis-regulatory elements; improvements are needed to enable synthetic promoter design. We applied an ensemble strategy for de novo soybean cyst nematode (SCN)-inducible motif discovery among promoters of 18 co-expressed soybean genes that were selected from six reported microarray studies involving a compatible soybean-SCN interaction. A total of 116 overlapping motif regions (OMRs) were discovered bioinformatically that were identified by at least four out of seven bioinformatic tools. Using synthetic promoters, the inducibility of each OMR or motif itself was evaluated by co-localization of gain of function of an orange fluorescent protein reporter and the presence of SCN in transgenic soybean hairy roots. Among 16 OMRs detected from two experimentally confirmed SCN-inducible promoters, 11 OMRs (i.e. 68.75%) were experimentally confirmed to be SCN-inducible, leading to the discovery of 23 core motifs of 5- to 7-bp length, of which 14 are novel in plants. We found that a combination of the three best tools (i.e. SCOPE, W-AlignACE and Weeder) could detect all 23 core motifs. Thus, this strategy is a high-throughput approach for de novo motif discovery in soybean and offers great potential for novel motif discovery and synthetic promoter engineering for any plant and trait in crop biotechnology. PMID:24893752

  17. Enhancing Transgene Expression from Recombinant AAV8 Vectors in Different Tissues Using Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element

    PubMed Central

    Wang, Lizheng; Wang, Zixuan; Zhang, Fangfang; Zhu, Rui; Bi, Jinpeng; Wu, Jiaxin; Zhang, Haihong; Wu, Hui; Kong, Wei; Yu, Bin; Yu, Xianghui

    2016-01-01

    Adeno-associated virus (AAV) vectors have been utilized extensively in gene therapy and gene function studies, as strong transgene expression is a prerequisite for positive outcomes. AAV8 was reported as the most efficient AAV serotype for transduction of the liver, brain and muscle compared with other serotypes. However, AAV8-mediated transduction of human hepatocytes is rather poor with approximately 20-fold lower efficiency compared with that of mouse hepatocytes. Therefore, we applied the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to enhance AAV8-mediated transgene expression driven by a combination promoter (CAG promoter) with a CMV-IE enhancer and chicken beta-actin promoter for a more efficient viral vector. Transgene expression from recombinant AAV8 (rAAV8) vectors harboring a red fluorescent protein (RFP) reporter gene with or without WPRE were evaluated in vitro and in vivo. The results demonstrated that WPRE improved AAV8-mediated RFP expression in different cell lines with clear increases of transgene expression in the liver, brain or muscle of animals. The findings of this study will help to substantially reduce the quantity of viral particles that must be injected in order to reach a therapeutic level of transgene expression in gene therapy. Consequently, such dose reductions may lessen the potential risks associated with high doses of viral vectors. PMID:27076785

  18. Expression of the CDR1 efflux pump in clinical Candida albicans isolates is controlled by a negative regulatory element

    SciTech Connect

    Gaur, Naseem Akhtar; Manoharlal, Raman; Saini, Preeti; Prasad, Tulika; Mukhopadhyay, Gauranga; Hoefer, Milan; Morschhaeuser, Joachim; Prasad, Rajendra . E-mail: rp47@hotmail.com

    2005-06-24

    Resistance to azole antifungal drugs in clinical isolates of the human fungal pathogen Candida albicans is often caused by constitutive overexpression of the CDR1 gene, which encodes a multidrug efflux pump of the ABC transporter superfamily. To understand the relevance of a recently identified negative regulatory element (NRE) in the CDR1 promoter for the control of CDR1 expression in the clinical scenario, we investigated the effect of mutation or deletion of the NRE on CDR1 expression in two matched pairs of azole-sensitive and resistant clinical isolates of C. albicans. Expression of GFP or lacZ reporter genes from the wild type CDR1 promoter was much higher in the azole-resistant C. albicans isolates than in the azole-susceptible isolates, reflecting the known differences in CDR1 expression in these strains. Deletion or mutation of the NRE resulted in enhanced reporter gene expression in azole-sensitive strains, but did not further increase the already high CDR1 promoter activity in the azole-resistant strains. In agreement with these findings, electrophoretic mobility shift assays showed a reduced binding to the NRE of nuclear extracts from the resistant C. albicans isolates as compared with extracts from the sensitive isolates. These results demonstrate that the NRE is involved in maintaining CDR1 expression at basal levels and that this repression is overcome in azole-resistant clinical C. albicans isolates, resulting in constitutive CDR1 overexpression and concomitant drug resistance.

  19. Retrotransposable elements R1 and R2 in the rDNA units of Drosophila mercatorum: abnormal abdomen revisited.

    PubMed Central

    Malik, H S; Eickbush, T H

    1999-01-01

    R1 and R2 retrotransposable elements are stable components of the 28S rRNA genes of arthropods. While each retrotransposition event leads to incremental losses of rDNA unit expression, little is known about the selective consequences of these elements on the host genome. Previous reports suggested that in the abnormal abdomen (aa) phenotype of Drosophila mercatorum, high levels of rDNA insertions (R1) in conjunction with the under-replication locus (ur), enable the utilization of different ecological conditions via a population level shift to younger age. We have sequenced the R1 and R2 elements of D. mercatorum and show that the levels of R1- and R2-inserted rDNA units were inaccurately scored in the original studies of aa, leading to several misinterpretations. In particular, contrary to earlier reports, aa flies differentially underreplicate R1- and R2-inserted rDNA units, like other species of Drosophila. However, aa flies do not undergo the lower level of underreplication of their functional rDNA units (general underreplication) that is seen in wild-type strains. The lack of general underreplication is expected to confer a selective advantage and, thus, can be interpreted as an adaptation to overcome high levels of R1 and R2 insertions. These results allow us to reconcile some of the apparently contradictory effects of aa and the bobbed phenotype found in other species of Drosophila. PMID:9927458

  20. Dynamics of R1 and R2 elements in the rDNA locus of Drosophila simulans.

    PubMed Central

    Pérez-González, C E; Eickbush, T H

    2001-01-01

    The mobile elements R1 and R2 insert specifically into the rRNA gene locus (rDNA locus) of arthropods, a locus known to undergo concerted evolution, the recombinational processes that preserve the sequence homogeneity of all repeats. To monitor how rapidly individual R1 and R2 insertions are turned over in the rDNA locus by these processes, we have taken advantage of the many 5' truncation variants that are generated during the target-primed reverse transcription mechanism used by these non-LTR retrotransposons for their integration. A simple PCR assay was designed to reveal the pattern of the 5' variants present in the rDNA loci of individual X chromosomes in a population of Drosophila simulans. Each rDNA locus in this population was found to have a large, unique collection of 5' variants. Each variant was present at low copy number, usually one copy per chromosome, and was seldom distributed to other chromosomes in the population. The failure of these variants to spread to other units in the same rDNA locus suggests a strong recombinational bias against R1 and R2 that results in the individual copies of these elements being rapidly lost from the rDNA locus. This bias suggests a significantly higher frequency of R1 and R2 retrotransposition than we have previously suggested. PMID:11514447

  1. HIV-1 p24(gag) derived conserved element DNA vaccine increases the breadth of immune response in mice.

    PubMed

    Kulkarni, Viraj; Rosati, Margherita; Valentin, Antonio; Ganneru, Brunda; Singh, Ashish K; Yan, Jian; Rolland, Morgane; Alicea, Candido; Beach, Rachel Kelly; Zhang, Gen-Mu; Le Gall, Sylvie; Broderick, Kate E; Sardesai, Niranjan Y; Heckerman, David; Mothe, Beatriz; Brander, Christian; Weiner, David B; Mullins, James I; Pavlakis, George N; Felber, Barbara K

    2013-01-01

    Viral diversity is considered a major impediment to the development of an effective HIV-1 vaccine. Despite this diversity, certain protein segments are nearly invariant across the known HIV-1 Group M sequences. We developed immunogens based on the highly conserved elements from the p24(gag) region according to two principles: the immunogen must (i) include strictly conserved elements of the virus that cannot mutate readily, and (ii) exclude both HIV regions capable of mutating without limiting virus viability, and also immunodominant epitopes located in variable regions. We engineered two HIV-1 p24(gag) DNA immunogens that express 7 highly Conserved Elements (CE) of 12-24 amino acids in length and differ by only 1 amino acid in each CE ('toggle site'), together covering >99% of the HIV-1 Group M sequences. Altering intracellular trafficking of the immunogens changed protein localization, stability, and also the nature of elicited immune responses. Immunization of C57BL/6 mice with p55(gag) DNA induced poor, CD4(+) mediated cellular responses, to only 2 of the 7 CE; in contrast, vaccination with p24CE DNA induced cross-clade reactive, robust T cell responses to 4 of the 7 CE. The responses were multifunctional and composed of both CD4(+) and CD8(+) T cells with mature cytotoxic phenotype. These findings provide a method to increase immune response to universally conserved Gag epitopes, using the p24CE immunogen. p24CE DNA vaccination induced humoral immune responses similar in magnitude to those induced by p55(gag), which recognize the virus encoded p24(gag) protein. The inclusion of DNA immunogens composed of conserved elements is a promising vaccine strategy to induce broader immunity by CD4(+) and CD8(+) T cells to additional regions of Gag compared to vaccination with p55(gag) DNA, achieving maximal cross-clade reactive cellular and humoral responses. PMID:23555935

  2. Alteration of C-MYB DNA binding to cognate responsive elements in HL-60 variant cells

    PubMed Central

    Gaillard, C; Le Rouzic, E; Créminon, C; Perbal, B

    2002-01-01

    Aims: To establish whether the MYB protein expressed in HL-60 variant cells, which are cells resistant to 12-O-tetradecanoylphorbol-13-acetate (TPA) induced differentiation, is able to bind MYB recognition elements (MREs) involved in the transcriptional regulation of myb target genes. In addition, to determine whether alterations in the binding of the MYB protein to MREs affects HL-60 cell proliferation and differentiation. Methods: Nuclear extracts of HL-60 variant cells exhibiting different degrees of resistance to TPA induced monocytic differentiation were used in electrophoretic mobility shift experiments (EMSAs), bandshift experiments performed with labelled oliogonucleotides containing the MYB consensus binding sequences. Results: The MYB protein contained in nuclear extracts from HL-60 variant cells did not bind efficiently to the MYB recognition elements identified in the mim-1 and PR264 promoters. Molecular cloning of the myb gene and analysis of the MYB protein expressed in the HL-60 variant cells established that the lack of binding did not result from a structural alteration of MYB in these cells. The lack of MRE binding did not abrogate the ability of variant HL-60s to proliferate and to undergo differentiation. Furthermore, the expression of the PR264/SC35 splicing factor was not affected as a result of the altered MYB DNA binding activity. Conclusions: Because the MYB protein expressed in HL-60 variant cells did not appear to be structurally different from the MYB protein expressed in parental HL-60 cells, it is possible that the HL-60 variant cells contain a MYB binding inhibitory factor (MBIF) that interferes with MYB binding on MREs. The increased proliferation rate of HL-60 variant cells and their reduced serum requirement argues against the need for direct MYB binding in the regulation of cell growth. PMID:12354938

  3. A baculovirus photolyase with DNA repair activity and circadian clock regulatory function.

    PubMed

    Biernat, Magdalena A; Eker, André P M; van Oers, Monique M; Vlak, Just M; van der Horst, Gijsbertus T J; Chaves, Inês

    2012-02-01

    Cryptochromes and photolyases belong to the same family of flavoproteins but, despite being structurally conserved, display distinct functions. Photolyases use visible light to repair ultraviolet-induced DNA damage. Cryptochromes, however, function as blue-light receptors, circadian photoreceptors, or repressors of the CLOCK/BMAL1 heterodimer, the transcription activator controlling the molecular circadian clock. Here, we present evidence that the functional divergence between cryptochromes and photolyases is not so univocal. Chrysodeixis chalcites nucleopolyhedrovirus possesses 2 photolyase-like genes: phr1 and phr2. We show that PHR1 and PHR2 are able to bind the CLOCK protein. Only for PHR2, however, the physical interaction with CLOCK represses CLOCK/BMAL1-driven transcription. This result shows that binding of photolyase per se is not sufficient to inhibit the CLOCK/BMAL1 heterodimer. PHR2, furthermore, affects the oscillation of immortalized mouse embryonic fibroblasts, suggesting that PHR2 can regulate the molecular circadian clock. These findings are relevant for further understanding the evolution of cryptochromes and photolyases as well as behavioral changes induced in insects by baculoviruses. PMID:22306969

  4. Retroposons do jump: a B2 element recently integrated in an 18S rDNA gene.

    PubMed Central

    Oberbäumer, I

    1992-01-01

    Several cDNA clones were isolated from cDNA libraries constructed with mRNA longer than 28S RNA from the murine cell line PYS-2/12. The plasmids have inserts containing 1-1.2 kb of the ribosomal 5' external transcribed spacer followed by nearly 700 nt of sequence for 18S rRNA and ending with a B2 element (retroposon). The cloned sequence differed in a few positions from published ribosomal sequences. The 3' adjacent genomic sequence was obtained by polymerase chain reaction (PCR) and showed that the B2 element has a poly(A) tail of about 50 nt and is surrounded by perfect direct repeats of 15 nt. Analysis of genomic DNA from several murine cell lines revealed that PYS cells contain at least one copy of 18S RNA with the B2 element which is not present in the genome of other murine cell lines derived from the same teratocarcinoma. Similarly, rRNA transcripts containing the B2 element were only detected in PYS cells. According to the publication dates of the different cell lines, the B2 element must have been integrated into an rRNA transcription unit during the years 1970 through 1974 thus proving that retroposons (SINEs) can still be inserted into the genome in our times. Images PMID:1311830

  5. A novel cis-acting element required for DNA damage-inducible expression of yeast DIN7

    SciTech Connect

    Yoshitani, Ayako; Yoshida, Minoru; Ling Feng

    2008-01-04

    Din7 is a DNA damage-inducible mitochondrial nuclease that modulates the stability of mitochondrial DNA (mtDNA) in Saccharomyces cerevisiae. How DIN7 gene expression is regulated, however, has remained largely unclear. Using promoter sequence alignment, we found a highly conserved 19-bp sequence in the promoter regions of DIN7 and NTG1, which encodes an oxidative stress-inducible base-excision-repair enzyme. Deletion of the 19-bp sequence markedly reduced the hydroxyurea (HU)-enhanced DIN7 promoter activity. In addition, nuclear fractions prepared from HU-treated cells were used in in vitro band shift assays to reveal the presence of currently unidentified trans-acting factor(s) that preferentially bound to the 19-bp region. These results suggest that the 19-bp sequence is a novel cis-acting element that is required for the regulation of DIN7 expression in response to HU-induced DNA damage.

  6. An active DNA transposon nDart causing leaf variegation and mutable dwarfism and its related elements in rice.

    PubMed

    Tsugane, Kazuo; Maekawa, Masahiko; Takagi, Kyoko; Takahara, Hiroyuki; Qian, Qian; Eun, Chang-Ho; Iida, Shigeru

    2006-01-01

    While characterized mutable alleles caused by DNA transposons have been abundant in maize since the discovery of Dissociation conferring variegation by Barbara McClintock, only a few mutable alleles have been described in rice even though the rice genome contains various transposons. Here, we show that a spontaneous mutable virescent allele, pyl-v, is caused by the disruption of the nuclear-coded essential chloroplast protease gene, OsClpP5, due to insertion of a 607-bp non-autonomous DNA transposon, non-autonomous DNA-based active rice transposon one (nDart1), belonging to the hAT superfamily. The transposition of nDart1 can be induced by crossing with a line containing an autonomous element, aDart, and stabilized by segregating out of aDart. We also identified a novel mutable dwarf allele thl-m caused by an insertion of nDart1. The japonica cultivar Nipponbare carries no aDart, although it contains epigenetically silenced Dart element(s), which can be activated by 5-azacytidine. Nipponbare bears four subgroups of about 3.6-kb Dart-like sequences, three of which contain potential transposase genes, and around 3.6-kb elements without an apparent transposase gene, as well as three subgroups of about 0.6-kb nDart1-related elements that are all internal deletions of the Dart-like sequences. Both nDart1 and 3.6-kb Dart-like elements were also present in indica varieties 93-11 and Kasalath. nDart1 appears to be the most active mutagen among nDart1-related elements contributing to generating natural variations. A candidate for an autonomous element, aDart, and a possible application of nDart1 for transposon tagging are discussed. PMID:16367953

  7. Regulated tissue-specific alternative splicing of enhanced green fluorescent protein transgenes conferred by alpha-tropomyosin regulatory elements in transgenic mice.

    PubMed

    Ellis, Peter D; Smith, Christopher W J; Kemp, Paul

    2004-08-27

    The mutually exclusive exons 2 and 3 of alpha-tropomyosin (alphaTM) have been used as a model system for strictly regulated alternative splicing. Exon 2 inclusion is only observed at high levels in smooth muscle (SM) tissues, whereas striated muscle and non-muscle cells use predominantly exon 3. Experiments in cell culture have shown that exon 2 selection results from repression of exon 3 and that this repression is mediated by regulatory elements flanking exon 3. We have now tested the cell culture-derived model in transgenic mice. We show that by harnessing the intronic splicing regulatory elements, expression of an enhanced green fluorescent protein transgene with a constitutively active promoter can be restricted to SM cells. Splicing of both endogenous alphaTM and a series of transgenes carrying regulatory element mutations was analyzed by reverse transcriptasePCR. These studies indicated that although SM-rich tissues are equipped to regulate splicing of high levels of endogenous or transgene alphaTM RNA, other non-SM tissues such as spleen, which express lower amounts of alphaTM, also splice significant proportions of exon 2, and this splicing pattern can be recapitulated by transgenes expressed at low levels. We confirm the importance in vivo of the negatively acting regulatory elements for regulated skipping of exon 3. Moreover, we provide evidence that some of the regulatory factors responsible for exon 3 skipping appear to be titratable, with loss of regulated splicing sometimes being associated with high transgene expression levels. PMID:15194683

  8. Tumorigenesis by Meis1 overexpression is accompanied by a change of DNA target-sequence specificity which allows binding to the AP-1 element.

    PubMed

    Dardaei, Leila; Penkov, Dmitry; Mathiasen, Lisa; Bora, Pranami; Morelli, Marco J; Blasi, Francesco

    2015-09-22

    Meis1 overexpression induces tumorigenicity but its activity is inhibited by Prep1 tumor suppressor. Why does overexpression of Meis1 cause cancer and how does Prep1 inhibit? Tumor profiling and ChIP-sequencing data in a genetically-defined set of cell lines show that: 1) The number of Meis1 and Prep1 DNA binding sites increases linearly with their concentration resulting in a strong increase of "extra" target genes. 2) At high concentration, Meis1 DNA target specificity changes such that the most enriched consensus becomes that of the AP-1 regulatory element, whereas the specific OCTA consensus is not enriched because diluted within the many extra binding sites. 3) Prep1 inhibits Meis1 tumorigenesis preventing the binding to many of the "extra" genes containing AP-1 sites. 4) The overexpression of Prep1, but not of Meis1, changes the functional genomic distribution of the binding sites, increasing seven fold the number of its "enhancer" and decreasing its "promoter" targets. 5) A specific Meis1 "oncogenic" and Prep1 "tumor suppressing" signature has been identified selecting from the pool of genes bound by each protein those whose expression was modified uniquely by the "tumor-inducing" Meis1 or tumor-inhibiting Prep1 overexpression. In both signatures, the enriched gene categories are the same and are involved in signal transduction. However, Meis1 targets stimulatory genes while Prep1 targets genes that inhibit the tumorigenic signaling pathways. PMID:26259236

  9. Development of two highly sensitive forensic sex determination assays based on human DYZ1 and Alu repetitive DNA elements.

    PubMed

    Fazi, Amanda; Gobeski, Brianne; Foran, David

    2014-11-01

    Sex determination is a critical component of forensic identification, the standard genetic method for which is detection of the single copy amelogenin gene that has differing homologues on the X and Y chromosomes. However, this assay may not be sensitive enough when DNA samples are minute or highly compromised, thus other strategies for sex determination are needed. In the current research, two ultrasensitive sexing assays, based on real-time PCR and pyrosequencing, were developed targeting the highly repetitive elements DYZ1 on the Y chromosome and Alu on the autosomes. The DYZ1/Alu strategy was compared to amelogenin for overall sensitivity based on high molecular weight and degraded DNA, followed by assaying the sex of 34 touch DNA samples and DNA from 30 hair shafts. The real-time DYZ1/Alu assay proved to be approximately 1500 times more sensitive than its amelogenin counterpart based on high molecular weight DNA, and even more sensitive when sexing degraded DNA. The pyrosequencing DYZ1/Alu assay correctly sexed 26 of the touch DNAs, compared to six using amelogenin. Hair shaft DNAs showed equally improved sexing results using the DYZ1/Alu assays. Overall, both DYZ1/Alu assays were far more sensitive and accurate than was the amelogenin assay, and thus show great utility for sexing poor quality and low quantity DNA evidence. PMID:25168471

  10. Identification of Predictive Cis-Regulatory Elements Using a Discriminative Objective Function and a Dynamic Search Space

    PubMed Central

    Karnik, Rahul; Beer, Michael A.

    2015-01-01

    The generation of genomic binding or accessibility data from massively parallel sequencing technologies such as ChIP-seq and DNase-seq continues to accelerate. Yet state-of-the-art computational approaches for the identification of DNA binding motifs often yield motifs of weak predictive power. Here we present a novel computational algorithm called MotifSpec, designed to find predictive motifs, in contrast to over-represented sequence elements. The key distinguishing feature of this algorithm is that it uses a dynamic search space and a learned threshold to find discriminative motifs in combination with the modeling of motifs using a full PWM (position weight matrix) rather than k-mer words or regular expressions. We demonstrate that our approach finds motifs corresponding to known binding specificities in several mammalian ChIP-seq datasets, and that our PWMs classify the ChIP-seq signals with accuracy comparable to, or marginally better than motifs from the best existing algorithms. In other datasets, our algorithm identifies novel motifs where other methods fail. Finally, we apply this algorithm to detect motifs from expression datasets in C. elegans using a dynamic expression similarity metric rather than fixed expression clusters, and find novel predictive motifs. PMID:26465884

  11. Identification of Predictive Cis-Regulatory Elements Using a Discriminative Objective Function and a Dynamic Search Space.

    PubMed

    Karnik, Rahul; Beer, Michael A

    2015-01-01

    The generation of genomic binding or accessibility data from massively parallel sequencing technologies such as ChIP-seq and DNase-seq continues to accelerate. Yet state-of-the-art computational approaches for the identification of DNA binding motifs often yield motifs of weak predictive power. Here we present a novel computational algorithm called MotifSpec, designed to find predictive motifs, in contrast to over-represented sequence elements. The key distinguishing feature of this algorithm is that it uses a dynamic search space and a learned threshold to find discriminative motifs in combination with the modeling of motifs using a full PWM (position weight matrix) rather than k-mer words or regular expressions. We demonstrate that our approach finds motifs corresponding to known binding specificities in several mammalian ChIP-seq datasets, and that our PWMs classify the ChIP-seq signals with accuracy comparable to, or marginally better than motifs from the best existing algorithms. In other datasets, our algorithm identifies novel motifs where other methods fail. Finally, we apply this algorithm to detect motifs from expression datasets in C. elegans using a dynamic expression similarity metric rather than fixed expression clusters, and find novel predictive motifs. PMID:26465884

  12. Two regulatory elements of similar structure and placed in tandem account for the repressive activity of the first intron of the human apolipoprotein A-II gene.

    PubMed Central

    Bossu, J P; Chartier, F L; Fruchart, J C; Auwerx, J; Staels, B; Laine, B

    1996-01-01

    Recent reports indicate that apolipoprotein (apo) A-II, the second most abundant protein of high-density lipoproteins, plays a crucial role in counteracting the beneficial effect of apo A-I against atherogenesis. Transcription of the human apo A-II gene is controlled by an enhancer comprising 14 regulatory elements located upstream of its promoter whereas the first intron of this gene behaves as a silencer. Here we show that two sequence elements account for the repressive activity of this intron and correspond to negative regulatory elements termed NRE I and NRE II. The activity of intron I and the nuclear proteins binding to NRE I and II are encountered in hepatic cells but not in non-hepatic cells studied here. Both NREs form nucleoprotein complexes of very similar physicochemical characteristics and bind the same or closely related proteins. Site-directed mutagenesis, transient transfection and gel-shift analysis experiments indicate that both NREs exhibit similar structures, being composed of two sites required for maximal activity and optimal binding of transcription factors. Therefore two negative regulatory elements of similar structure and function, placed in tandem, account for the repressive activity of the first intron of the human apo A-II gene. These NREs do not exhibit structural similarity with known NREs of other genes. PMID:8809045

  13. Cellular localization of the embryo-specific hybrid PRP from Zea mays, and characterization of promoter regulatory elements of its gene.

    PubMed

    Jose-Estanyol, M; Puigdomènech, P

    2012-10-01

    The expression, regulation and cellular localization of ZmHyPRP, a gene marker of embryo differentiation whose expression declines after ABA induction, was studied. ZmHyPRP is a proline-rich protein with a C-terminal domain having eight cysteines in a CM8 pattern. Transient expression in onion epidermal cells, transformed with a 2x35S::ZmHyPRP-GFP construction, indicated the protein is present in vesicles lining the membrane of the cell. The ZmHyPRP gene expression is under the control of classic promoter seed-specific regulatory elements such as Sph/RY and G-boxes, suggesting regulation by B3 and b-ZIP transcription factors. Promoter deletion analysis, by particle-bombardment transient transformation of maize immature embryos with serial deletions of the promoter fused to GUS, showed the presence of two negative regulatory elements, NE1 (-2070 to -1280) and NE2 (-232 to -178), in the ZmHyPRP promoter. By selective deletion or mutation of ZmHyPRP regulatory promoter elements we conclude that the promoter expression is attenuated by the NE2 element as well as by the G-box2 and the Sph1-2 box together with the G-box2. PMID:22915319

  14. The organization structure and regulatory elements of Chlamydomonas histone genes reveal features linking plant and animal genes.

    PubMed

    Fabry, S; Müller, K; Lindauer, A; Park, P B; Cornelius, T; Schmitt, R

    1995-09-01

    The genome of the green alga Chlamydomonas reinhardtii contains approximately 15 gene clusters of the nucleosomal (or core) histone H2A, H2B, H3 and H4 genes and at least one histone H1 gene. Seven non-allelic histone gene loci were isolated from a genomic library, physically mapped, and the nucleotide sequences of three isotypes of each core histone gene species and one linked H1 gene determined. The core histone genes are organized in clusters of H2A-H2B and H3-H4 pairs, in which each gene pair shows outwardly divergent transcription from a short (< 300 bp) intercistronic region. These intercistronic regions contain typically conserved promoter elements, namely a TATA-box and the three motifs TGGCCAG-G(G/C)-CGAG, CGTTGACC and CGGTTG. Different from the genes of higher plants, but like those of animals and the related alga Volvox, the 3' untranslated regions contain no poly A signal, but a palindromic sequence (3' palindrome) essential for mRNA processing is present. One single H1 gene was found in close linkage to a H2A-H2B pair. The H1 upstream region contains the octameric promoter element GGTTGACC (also found upstream of the core histone genes) and two specific sequence motifs that are shared only with the Volvox H1 promoters. This suggests differential transcription of the H1 and the core histone genes. The H1 gene is interrupted by two introns. Unlike Volvox H3 genes, the three sequenced H3 isoforms are intron-free. Primer-directed PCR of genomic DNA demonstrated, however, that at least 8 of the about 15 H3 genes do contain one intron at a conserved position. In synchronized C. reinhardtii cells, H4 mRNA levels (representative of all core histone mRNAs) peak during cell division, suggesting strict replication-dependent gene control. The derived peptide sequences place C. reinhardtii core histones closer to plants than to animals, except that the H2A histones are more animal-like. The peptide sequence of histone H1 is closely related to the V. carteri VH1-II

  15. Regulatory T Cells Suppress Natural Killer Cells during Plasmid DNA Vaccination in Mice, Blunting the CD8+ T Cell Immune Response by the Cytokine TGFβ

    PubMed Central

    Frimpong-Boateng, Kwesi; van Rooijen, Nico; Geiben-Lynn, Ralf

    2010-01-01

    Background CD4+CD25+ regulatory T cells (Tregs) suppress adaptive T cell-mediated immune responses to self- and foreign-antigens. Tregs may also suppress early innate immune responses to vaccine antigens and might decrease vaccine efficacy. NK and NKT cells are the first responders after plasmid DNA vaccination and are found at the site of inoculation. Earlier reports demonstrated that NKT cells could improve plasmid DNA efficacy, a phenomenon not found for NK cells. In fact, it has been shown that under certain disease conditions, NK cells are suppressed by Tregs via their release of IL-10 and/or TGFβ. Therefore, we tested the hypothesis that NK cell function is suppressed by Tregs in the setting of plasmid DNA vaccination. Methodology/Principal Findings In this study we show that Tregs directly inhibit NK cell function during plasmid DNA vaccination by suppressing the potentially 10-fold, NK cell-mediated, augmentation of plasmid DNA antigen-specific CD8+ T cells. We found that this phenomenon is dependent on the secretion of cytokine TGFβ by Tregs, and independent of IL-10. Conclusions Our data indicate a crucial function for Tregs in blocking plasmid DNA vaccine-elicited immune responses, revealing potentially novel strategies for improving the efficiency of plasmid DNA vaccines including chemical- or antibody-induced localized blockage of Treg-mediated suppression of NK cells at the site of plasmid DNA vaccine inoculation. PMID:20808850

  16. Chromatin Heterogeneity and Distribution of Regulatory Elements in the Late-Replicating Intercalary Heterochromatin Domains of Drosophila melanogaster Chromosomes

    PubMed Central

    Khoroshko, Varvara A.; Levitsky, Viktor G.; Zykova, Tatyana Yu.; Antonenko, Oksana V.; Belyaeva, Elena S.; Zhimulev, Igor F.

    2016-01-01

    Late-replicating domains (intercalary heterochromatin) in the Drosophila genome display a number of features suggesting their organization is quite unique. Typically, they are quite large and encompass clusters of functionally unrelated tissue-specific genes. They correspond to the topologically associating domains and conserved microsynteny blocks. Our study aims at exploring further details of molecular organization of intercalary heterochromatin and has uncovered surprising heterogeneity of chromatin composition in these regions. Using the 4HMM model developed in our group earlier, intercalary heterochromatin regions were found to host chromatin fragments with a particular epigenetic profile. Aquamarine chromatin fragments (spanning 0.67% of late-replicating regions) are characterized as a class of sequences that appear heterogeneous in terms of their decompactization. These fragments are enriched with enhancer sequences and binding sites for insulator proteins. They likely mark the chromatin state that is related to the binding of cis-regulatory proteins. Malachite chromatin fragments (11% of late-replicating regions) appear to function as universal transitional regions between two contrasting chromatin states. Namely, they invariably delimit intercalary heterochromatin regions from the adjacent active chromatin of interbands. Malachite fragments also flank aquamarine fragments embedded in the repressed chromatin of late-replicating regions. Significant enrichment of insulator proteins CP190, SU(HW), and MOD2.2 was observed in malachite chromatin. Neither aquamarine nor malachite chromatin types appear to correlate with the positions of highly conserved non-coding elements (HCNE) that are typically replete in intercalary heterochromatin. Malachite chromatin found on the flanks of intercalary heterochromatin regions tends to replicate earlier than the malachite chromatin embedded in intercalary heterochromatin. In other words, there exists a gradient of

  17. Chromatin Heterogeneity and Distribution of Regulatory Elements in the Late-Replicating Intercalary Heterochromatin Domains of Drosophila melanogaster Chromosomes.

    PubMed

    Khoroshko, Varvara A; Levitsky, Viktor G; Zykova, Tatyana Yu; Antonenko, Oksana V; Belyaeva, Elena S; Zhimulev, Igor F

    2016-01-01

    Late-replicating domains (intercalary heterochromatin) in the Drosophila genome display a number of features suggesting their organization is quite unique. Typically, they are quite large and encompass clusters of functionally unrelated tissue-specific genes. They correspond to the topologically associating domains and conserved microsynteny blocks. Our study aims at exploring further details of molecular organization of intercalary heterochromatin and has uncovered surprising heterogeneity of chromatin composition in these regions. Using the 4HMM model developed in our group earlier, intercalary heterochromatin regions were found to host chromatin fragments with a particular epigenetic profile. Aquamarine chromatin fragments (spanning 0.67% of late-replicating regions) are characterized as a class of sequences that appear heterogeneous in terms of their decompactization. These fragments are enriched with enhancer sequences and binding sites for insulator proteins. They likely mark the chromatin state that is related to the binding of cis-regulatory proteins. Malachite chromatin fragments (11% of late-replicating regions) appear to function as universal transitional regions between two contrasting chromatin states. Namely, they invariably delimit intercalary heterochromatin regions from the adjacent active chromatin of interbands. Malachite fragments also flank aquamarine fragments embedded in the repressed chromatin of late-replicating regions. Significant enrichment of insulator proteins CP190, SU(HW), and MOD2.2 was observed in malachite chromatin. Neither aquamarine nor malachite chromatin types appear to correlate with the positions of highly conserved non-coding elements (HCNE) that are typically replete in intercalary heterochromatin. Malachite chromatin found on the flanks of intercalary heterochromatin regions tends to replicate earlier than the malachite chromatin embedded in intercalary heterochromatin. In other words, there exists a gradient of

  18. Solution structure of the Zβ domain of human DNA-dependent activator of IFN-regulatory factors and its binding modes to B- and Z-DNAs

    PubMed Central

    Kim, Kyungmin; Khayrutdinov, Bulat I.; Lee, Chung-Kyung; Cheong, Hae-Kap; Kang, Sung Wook; Park, Hyejin; Lee, Sangho; Kim, Yang-Gyun; Jee, JunGoo; Rich, Alexander; Kim, Kyeong Kyu; Jeon, Young Ho

    2011-01-01

    The DNA-dependent activator of IFN-regulatory factors (DAI), also known as DLM-1/ZBP1, initiates an innate immune response by binding to foreign DNAs in the cytosol. For full activation of the immune response, three DNA binding domains at the N terminus are required: two Z-DNA binding domains (ZBDs), Zα and Zβ, and an adjacent putative B-DNA binding domain. The crystal structure of the Zβ domain of human DAI (hZβDAI) in complex with Z-DNA revealed structural features distinct from other known Z-DNA binding proteins, and it was classified as a group II ZBD. To gain structural insights into the DNA binding mechanism of hZβDAI, the solution structure of the free hZβDAI was solved, and its bindings to B- and Z-DNAs were analyzed by NMR spectroscopy. Compared to the Z-DNA–bound structure, the conformation of free hZβDAI has notable alterations in the α3 recognition helix, the “wing,” and Y145, which are critical in Z-DNA recognition. Unlike some other Zα domains, hZβDAI appears to have conformational flexibility, and structural adaptation is required for Z-DNA binding. Chemical-shift perturbation experiments revealed that hZβDAI also binds weakly to B-DNA via a different binding mode. The C-terminal domain of DAI is reported to undergo a conformational change on B-DNA binding; thus, it is possible that these changes are correlated. During the innate immune response, hZβDAI is likely to play an active role in binding to DNAs in both B and Z conformations in the recognition of foreign DNAs. PMID:21471454

  19. Distal apolipoprotein C-III regulatory elements F to J act as a general modular enhancer for proximal promoters that contain hormone response elements. Synergism between hepatic nuclear factor-4 molecules bound to the proximal promoter and distal enhancer sites.

    PubMed

    Kardassis, D; Tzameli, I; Hadzopoulou-Cladaras, M; Talianidis, I; Zannis, V

    1997-01-01

    Transient transfection assays have shown that the distal apoC-III promoter segments that contain the regulatory elements F to J enhance the strength of the tandemly linked proximal apoA-I promoter 5- to 13-fold in hepatic (HepG2) cells. Activation in intestinal (CaCo-2) cells to levels comparable to those obtained in HepG2 cells requires a larger apoA-I promoter sequence that extends to nucleotide -1500 as well as the presence of hepatic nuclear factor-4 (HNF-4). The distal apoC-III regulatory elements can also enhance 4- to 8-fold the strength of the heterologous apoB promoter in HepG2 and CaCo-2 cells. Finally, these elements in the presence of HNF-4 enhance 14.5- to 18.5-fold the strength of the minimal adenovirus major late promoter linked to two copies of the hormone response element (HRE) AID of apoA-I in both HepG2 and CaCo-2 cells. In vitro mutagenesis of the promoter/enhancer cluster established that the enhancer activity is lost by a mutation in the HRE present in the 3' end of the regulatory element I (-736 to -714) and is reduced significantly by point mutations or deletions in one or more of the regulatory elements F to J of the apoC-III enhancer. The enhancer activity also requires the HREs of the proximal apoA-I promoter. The apoC-III enhancer can also restore the activity of the proximal apoA-I and apoB promoters that have been inactivated by mutations in CCAAT/enhancers binding protein binding sites, indicating that C/EBP may not participate in the synergistic activation of the promoter/enhancer cluster. The findings suggest that the regulatory elements F to J of the apoC-III promoter act as a general modular enhancer that can potentiate the strength of proximal promoters that contain HREs. Such potentiation in the HepG2 cells can be accounted for by synergistic interactions between HNF-4 or other nuclear hormone receptors bound to the proximal and distal HREs and SP1 or other factors bound to the apoC-III enhancer. Additional factors may be

  20. Analysis of Usp DNA binding domain targeting reveals critical determinants of the ecdysone receptor complex interaction with the response element.

    PubMed

    Grad, I; Niedziela-Majka, A; Kochman, M; Ozyhar, A

    2001-07-01

    The steroid hormone, 20-hydroxyecdysone (20E), directs Drosophila metamorphosis via a heterodimeric receptor formed by two members of the nuclear hormone receptors superfamily, the product of the EcR (EcR) and of the ultraspiracle (Usp) genes. Our previous study [Niedziela-Majka, A., Kochman, M., Ozyhar, A. (2000) Eur. J. Biochem. 267, 507-519] on EcR and Usp DNA-binding domains (EcRDBD and UspDBD, respectively) suggested that UspDBD may act as a specific anchor that preferentially binds the 5' half-site of the pseudo-palindromic response element from the hsp27 gene promoter and thus locates the heterocomplex in the defined orientation. Here, we analyzed in detail the determinants of the UspDBD interaction with the hsp27 element. The roles of individual amino acids in the putative DNA recognition alpha helix and the roles of the base pairs of the UspDBD target sequence have been probed by site-directed mutagenesis. The results show how the hsp27 element specifies UspDBD binding and thus the polar assembly of the UspDBD/EcRDBD heterocomplex. It is suggested how possible nucleotide deviations within the 5' half-site of the element may be used for the fine-tuning of the 20E-response element specificity and consequently the physiological response. PMID:11432742

  1. A conserved MCM single-stranded DNA binding element is essential for replication initiation

    PubMed Central

    Froelich, Clifford A; Kang, Sukhyun; Epling, Leslie B; Bell, Stephen P; Enemark, Eric J

    2014-01-01

    The ring-shaped MCM helicase is essential to all phases of DNA replication. The complex loads at replication origins as an inactive double-hexamer encircling duplex DNA. Helicase activation converts this species to two active single hexamers that encircle single-stranded DNA (ssDNA). The molecular details of MCM DNA interactions during these events are unknown. We determined the crystal structure of the Pyrococcus furiosus MCM N-terminal domain hexamer bound to ssDNA and define a conserved MCM-ssDNA binding motif (MSSB). Intriguingly, ssDNA binds the MCM ring interior perpendicular to the central channel with defined polarity. In eukaryotes, the MSSB is conserved in several Mcm2-7 subunits, and MSSB mutant combinations in S. cerevisiae Mcm2-7 are not viable. Mutant Mcm2-7 complexes assemble and are recruited to replication origins, but are defective in helicase loading and activation. Our findings identify an important MCM-ssDNA interaction and suggest it functions during helicase activation to select the strand for translocation. DOI: http://dx.doi.org/10.7554/eLife.01993.001 PMID:24692448

  2. Sterol regulatory element binding protein-dependent regulation of lipid synthesis supports cell survival and tumor growth

    PubMed Central

    2013-01-01

    Background Regulation of lipid metabolism via activation of sterol regulatory element binding proteins (SREBPs) has emerged as an important function of the Akt/mTORC1 signaling axis. Although the contribution of dysregulated Akt/mTORC1 signaling to cancer has been investigated extensively and altered lipid metabolism is observed in many tumors, the exact role of SREBPs in the control of biosynthetic processes required for Akt-dependent cell growth and their contribution to tumorigenesis remains unclear. Results We first investigated the effects of loss of SREBP function in non-transformed cells. Combined ablation of SREBP1 and SREBP2 by siRNA-mediated gene silencing or chemical inhibition of SREBP activation induced endoplasmic reticulum (ER)-stress and engaged the unfolded protein response (UPR) pathway, specifically under lipoprotein-deplete conditions in human retinal pigment epithelial cells. Induction of ER-stress led to inhibition of protein synthesis through increased phosphorylation of eIF2α. This demonstrates for the first time the importance of SREBP in the coordination of lipid and protein biosynthesis, two processes that are essential for cell growth and proliferation. SREBP ablation caused major changes in lipid composition characterized by a loss of mono- and poly-unsaturated lipids and induced accumulation of reactive oxygen species (ROS) and apoptosis. Alterations in lipid composition and increased ROS levels, rather than overall changes to lipid synthesis rate, were required for ER-stress induction. Next, we analyzed the effect of SREBP ablation in a panel of cancer cell lines. Importantly, induction of apoptosis following SREBP depletion was restricted to lipoprotein-deplete conditions. U87 glioblastoma cells were highly susceptible to silencing of either SREBP isoform, and apoptosis induced by SREBP1 depletion in these cells was rescued by antioxidants or by restoring the levels of mono-unsaturated fatty acids. Moreover, silencing of SREBP1

  3. Structured RNA upstream of insect cap distal iron responsive elements enhances iron regulatory protein-mediated control of translation.

    PubMed

    Nichol, Helen; Winzerling, Joy

    2002-12-01

    Iron regulatory protein (IRP) blocks ribosomal assembly by binding to an iron responsive element (IRE) located proximal (<60 nts) to the mRNA cap, thereby repressing translation. Constructs with IREs located 60-100 nts from the cap permit ribosomal assembly but the ribosomes pause at IRE/IRP complexes resulting in partial repression of translation. However, insect ferritin mRNAs have cap-distal IREs located 90-156 nts from the cap. Because iron can be toxic, it seems unlikely that insects would be unable to fully regulate ferritin synthesis at the level of translation. Calpodes ferritin consists of two subunits, S and G. In vitro translation of Calpodes ferritin and IRP1 from fat body mRNA yields only G subunits suggesting that IRP1 more efficiently represses translation of the S subunit than the G. When repression is removed by the addition of IRE competitor RNA, the synthesis of both subunits is greatly increased. S and G ferritin mRNAs have identical IREs in similar far cap-distal positions. While both ferritin mRNAs are predicted to have stem-loops between the IRE and the RNA cap, in general insect S mRNAs have more cap-proximal RNA structure than G mRNAs. Therefore, we examined the effect of upstream secondary structure on ribosomal assembly onto S ferritin mRNA constructs using sucrose gradient analysis of translation initiation complexes. We found no evidence for ribosomal assembly on wild type Calpodes S ferritin mRNA in the presence of IRP1 while constructs lacking the wild type secondary structure showed ribosomal pausing. Constructs with wild type secondary structure preceded by an unstructured upstream leader assemble ribosomes in the presence or absence of IRP1. Sequence and RNA folding analyses of other insect ferritins with cap-distal IREs failed to identify any common sequences or IRE-like structures that might bind to IRP1 with lower affinity or to another RNA binding protein. We propose that stem-loops upstream from the IRE act like pleats that

  4. A genome-wide cis-regulatory element discovery method based on promoter sequences and gene co-expression networks

    PubMed Central

    2013-01-01

    Background Deciphering cis-regulatory networks has become an attractive yet challenging task. This paper presents a simple method for cis-regulatory network discovery which aims to avoid some of the common problems of previous approaches. Results Using promoter sequences and gene expression profiles as input, rather than clustering the genes by the expression data, our method utilizes co-expression neighborhood information for each individual gene, thereby overcoming the disadvantages of current clustering based models which may miss specific information for individual genes. In addition, rather than using a motif database as an input, it implements a simple motif count table for each enumerated k-mer for each gene promoter sequence. Thus, it can be used for species where previous knowledge of cis-regulatory motifs is unknown and has the potential to discover new transcription factor binding sites. Applications on Saccharomyces cerevisiae and Arabidopsis have shown that our method has a good prediction accuracy and outperforms a phylogenetic footprinting approach. Furthermore, the top ranked gene-motif regulatory clusters are evidently functionally co-regulated, and the regulatory relationships between the motifs and the enriched biological functions can often be confirmed by literature. Conclusions Since this method is simple and gene-specific, it can be readily utilized for insufficiently studied species or flexibly used as an additional step or data source for previous transcription regulatory networks discovery models. PMID:23368633

  5. Common and distinct DNA-binding and regulatory activities of the BEN-solo transcription factor family

    PubMed Central

    Dai, Qi; Ren, Aiming; Westholm, Jakub O.; Duan, Hong; Patel, Dinshaw J.

    2015-01-01

    Recently, the BEN (BANP, E5R, and NAC1) domain was recognized as a new class of conserved DNA-binding domain. The fly genome encodes three proteins that bear only a single BEN domain (“BEN-solo” factors); namely, Insensitive (Insv), Bsg25A (Elba1), and CG9883 (Elba2). Insv homodimers preferentially bind CCAATTGG palindromes throughout the genome to mediate transcriptional repression, whereas Bsg25A and Elba2 heterotrimerize with their obligate adaptor, Elba3 (i.e., the ELBA complex), to recognize a CCAATAAG motif in the Fab-7 insulator. While these data suggest distinct DNA-binding properties of BEN-solo proteins, we performed reporter assays that indicate that both Bsg25A and Elba2 can individually recognize Insv consensus sites efficiently. We confirmed this by solving the structure of Bsg25A complexed to the Insv site, which showed that key aspects of the BEN:DNA recognition strategy are similar between these proteins. We next show that both Insv and ELBA proteins are competent to mediate transcriptional repression via Insv consensus sequences but that the ELBA complex appears to be selective for the ELBA site. Reciprocally, genome-wide analysis reveals that Insv exhibits significant cobinding to class I insulator elements, indicating that it may also contribute to insulator function. Indeed, we observed abundant Insv binding within the Hox complexes with substantial overlaps with class I insulators, many of which bear Insv consensus sites. Moreover, Insv coimmunoprecipitates with the class I insulator factor CP190. Finally, we observed that Insv harbors exclusive activity among fly BEN-solo factors with respect to regulation of Notch-mediated cell fate choices in the peripheral nervous system. This in vivo activity is recapitulated by BEND6, a mammalian BEN-solo factor that conserves the Notch corepressor function of Insv but not its capacity to bind Insv consensus sites. Altogether, our data define an array of common and distinct biochemical and functional

  6. Common and distinct DNA-binding and regulatory activities of the BEN-solo transcription factor family.

    PubMed

    Dai, Qi; Ren, Aiming; Westholm, Jakub O; Duan, Hong; Patel, Dinshaw J; Lai, Eric C

    2015-01-01

    Recently, the BEN (BANP, E5R, and NAC1) domain was recognized as a new class of conserved DNA-binding domain. The fly genome encodes three proteins that bear only a single BEN domain ("BEN-solo" factors); namely, Insensitive (Insv), Bsg25A (Elba1), and CG9883 (Elba2). Insv homodimers preferentially bind CCAATTGG palindromes throughout the genome to mediate transcriptional repression, whereas Bsg25A and Elba2 heterotrimerize with their obligate adaptor, Elba3 (i.e., the ELBA complex), to recognize a CCAATAAG motif in the Fab-7 insulator. While these data suggest distinct DNA-binding properties of BEN-solo proteins, we performed reporter assays that indicate that both Bsg25A and Elba2 can individually recognize Insv consensus sites efficiently. We confirmed this by solving the structure of Bsg25A complexed to the Insv site, which showed that key aspects of the BEN:DNA recognition strategy are similar between these proteins. We next show that both Insv and ELBA proteins are competent to mediate transcriptional repression via Insv consensus sequences but that the ELBA complex appears to be selective for the ELBA site. Reciprocally, genome-wide analysis reveals that Insv exhibits significant cobinding to class I insulator elements, indicating that it may also contribute to insulator function. Indeed, we observed abundant Insv binding within the Hox complexes with substantial overlaps with class I insulators, many of which bear Insv consensus sites. Moreover, Insv coimmunoprecipitates with the class I insulator factor CP190. Finally, we observed that Insv harbors exclusive activity among fly BEN-solo factors with respect to regulation of Notch-mediated cell fate choices in the peripheral nervous system. This in vivo activity is recapitulated by BEND6, a mammalian BEN-solo factor that conserves the Notch corepressor function of Insv but not its capacity to bind Insv consensus sites. Altogether, our data define an array of common and distinct biochemical and functional

  7. Distinct cis-Regulatory Elements from the Dlx1/Dlx2 Locus Mark Different Progenitor Cell Populations in the Ganglionic Eminences and Different Subtypes of Adult Cortical Interneurons

    PubMed Central

    Ghanem, Noël; Yu, Man; Long, Jason; Hatch, Gary; Rubenstein, John L. R.; Ekker, Marc

    2016-01-01

    Distinct subtypes of cortical GABAergic interneurons provide inhibitory signals that are indispensable for neural network function. The Dlx homeobox genes have a central role in regulating their development and function. We have characterized the activity of three cis-regulatory sequences involved in forebrain expression of vertebrate Dlx genes: upstream regulatory element 2 (URE2), I12b, and I56i. The three regulatory elements display regional and temporal differences in their activities within the lateral ganglionic eminence (LGE), medial ganglionic eminence (MGE), and caudal ganglionic eminence (CGE) and label distinct populations of tangentially migrating neurons at embryonic day 12.5 (E12.5) and E13.5. We provide evidence that the dorsomedial and ventral MGE are distinct sources of tangentially migrating neurons during midgestation. In the adult cortex, URE2 and I12b/I56i are differentially expressed in parvalbumin-, calretinin-, neuropeptide Y-, and neuronal nitric oxide synthase-positive interneurons; I12b and I56i were specifically active in somatostatin-, vasoactive intestinal peptide-, and calbindin-positive interneurons. These data suggest that interneuron subtypes use distinct combinations of Dlx1/Dlx2 enhancers from the time they are specified through adulthood. PMID:17494687

  8. Separate elements of the TERMINAL FLOWER 1 cis-regulatory region integrate pathways to control flowering time and shoot meristem identity.

    PubMed

    Serrano-Mislata, Antonio; Fernández-Nohales, Pedro; Doménech, María J; Hanzawa, Yoshie; Bradley, Desmond; Madueño, Francisco

    2016-09-15

    TERMINAL FLOWER 1 (TFL1) is a key regulator of Arabidopsis plant architecture that responds to developmental and environmental signals to control flowering time and the fate of shoot meristems. TFL1 expression is dynamic, being found in all shoot meristems, but not in floral meristems, with the level and distribution changing throughout development. Using a variety of experimental approaches we have analysed the TFL1 promoter to elucidate its functional structure. TFL1 expression is based on distinct cis-regulatory regions, the most important being located 3' of the coding sequence. Our results indicate that TFL1 expression in the shoot apical versus lateral inflorescence meristems is controlled through distinct cis-regulatory elements, suggesting that different signals control expression in these meristem types. Moreover, we identified a cis-regulatory region necessary for TFL1 expression in the vegetative shoot and required for a wild-type flowering time, supporting that TFL1 expression in the vegetative meristem controls flowering time. Our study provides a model for the functional organisation of TFL1 cis-regulatory regions, contributing to our understanding of how developmental pathways are integrated at the genomic level of a key regulator to control plant architecture. PMID:27385013

  9. Massively parallel quantification of the regulatory effects of noncoding genetic variation in a human cohort

    PubMed Central

    Vockley, Christopher M.; Guo, Cong; Majoros, William H.; Nodzenski, Michael; Scholtens, Denise M.; Hayes, M. Geoffrey; Lowe, William L.; Reddy, Timothy E.

    2015-01-01

    We report a novel high-throughput method to empirically quantify individual-specific regulatory element activity at the population scale. The approach combines targeted DNA capture with a high-throughput reporter gene expression assay. As demonstration, we measured the activity of more than 100 putative regulatory elements from 95 individuals in a single experiment. In agreement with previous reports, we found that most genetic variants have weak effects on distal regulatory element activity. Because haplotypes are typically maintained within but not between assayed regulatory elements, the approach can be used to identify causal regulatory haplotypes that likely contribute to human phenotypes. Finally, we demonstrate the utility of the method to functionally fine map causal regulatory variants in regions of high linkage disequilibrium identified by expression quantitative trait loci (eQTL) analyses. PMID:26084464

  10. Role of conserved cis-regulatory elements in the post-transcriptional regulation of the human MECP2 gene involved in autism

    PubMed Central

    2013-01-01

    Background The MECP2 gene codes for methyl CpG binding protein 2 which regulates activities of other genes in the early development of the brain. Mutations in this gene have been associated with Rett syndrome, a form of autism. The purpose of this study was to investigate the role of evolutionarily conserved cis-elements in regulating the post-transcriptional expression of the MECP2 gene and to explore their possible correlations with a mutation that is known to cause mental retardation. Results A bioinformatics approach was used to map evolutionarily conserved cis-regulatory elements in the transcribed regions of the human MECP2 gene and its mammalian orthologs. Cis-regulatory motifs including G-quadruplexes, microRNA target sites, and AU-rich elements have gained significant importance because of their role in key biological processes and as therapeutic targets. We discovered in the 5′-UTR (untranslated region) of MECP2 mRNA a highly conserved G-quadruplex which overlapped a known deletion in Rett syndrome patients with decreased levels of MeCP2 protein. We believe that this 5′-UTR G-quadruplex could be involved in regulating MECP2 translation. We mapped additional evolutionarily conserved G-quadruplexes, microRNA target sites, and AU-rich elements in the key sections of both untranslated regions. Our studies suggest the regulation of translation, mRNA turnover, and development-related alternative MECP2 polyadenylation, putatively involving interactions of conserved cis-regulatory elements with their respective trans factors and complex interactions among the trans factors themselves. We discovered highly conserved G-quadruplex motifs that were more prevalent near alternative splice sites as compared to the constitutive sites of the MECP2 gene. We also identified a pair of overlapping G-quadruplexes at an alternative 5′ splice site that could potentially regulate alternative splicing in a negative as well as a positive way in the MECP2 pre

  11. A new large-DNA-fragment delivery system based on integrase activity from an integrative and conjugative element.

    PubMed

    Miyazaki, Ryo; van der Meer, Jan Roelof

    2013-07-01

    During the past few decades, numerous plasmid vectors have been developed for cloning, gene expression analysis, and genetic engineering. Cloning procedures typically rely on PCR amplification, DNA fragment restriction digestion, recovery, and ligation, but increasingly, procedures are being developed to assemble large synthetic DNAs. In this study, we developed a new gene delivery system using the integrase activity of an integrative and conjugative element (ICE). The advantage of the integrase-based delivery is that it can stably introduce a large DNA fragment (at least 75 kb) into one or more specific sites (the gene for glycine-accepting tRNA) on a target chromosome. Integrase recombination activity in Escherichia coli is kept low by using a synthetic hybrid promoter, which, however, is unleashed in the final target host, forcing the integration of the construct. Upon integration, the system is again silenced. Two variants with different genetic features were produced, one in the form of a cloning vector in E. coli and the other as a mini-transposable element by which large DNA constructs assembled in E. coli can be tagged with the integrase gene. We confirmed that the system could successfully introduce cosmid and bacterial artificial chromosome (BAC) DNAs from E. coli into the chromosome of Pseudomonas putida in a site-specific manner. The integrase delivery system works in concert with existing vector systems and could thus be a powerful tool for synthetic constructions of new metabolic pathways in a variety of host bacteria. PMID:23686268

  12. High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation.

    PubMed

    Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra; Meikle, Troy; Jiang, Katherine; Craciun, Gheorghe; Dunham, Maitreya J; Fox, Catherine A

    2016-01-01

    The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid

  13. High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation

    PubMed Central

    Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra; Meikle, Troy; Jiang, Katherine; Craciun, Gheorghe; Dunham, Maitreya J.; Fox, Catherine A.

    2016-01-01

    The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid

  14. Tumorigenesis by Meis1 overexpression is accompanied by a change of DNA target-sequence specificity which allows binding to the AP-1 element

    PubMed Central

    Dardaei, Leila; Penkov, Dmitry; Mathiasen, Lisa; Bora, Pranami; Morelli, Marco J.; Blasi, Francesco

    2015-01-01

    Meis1 overexpression induces tumorigenicity but its activity is inhibited by Prep1 tumor suppressor. Why does overexpression of Meis1 cause cancer and how does Prep1 inhibit? Tumor profiling and ChIP-sequencing data in a genetically-defined set of cell lines show that: 1) The number of Meis1 and Prep1 DNA binding sites increases linearly with their concentration resulting in a strong increase of “extra” target genes. 2) At high concentration, Meis1 DNA target specificity changes such that the most enriched consensus becomes that of the AP-1 regulatory element, whereas the specific OCTA consensus is not enriched because diluted within the many extra binding sites. 3) Prep1 inhibits Meis1 tumorigenesis preventing the binding to many of the “extra” genes containing AP-1 sites. 4) The overexpression of Prep1, but not of Meis1, changes the functional genomic distribution of the binding sites, increasing seven fold the number of its “enhancer” and decreasing its “promoter” targets. 5) A specific Meis1 “oncogenic” and Prep1 “tumor suppressing” signature has been identified selecting from the pool of genes bound by each protein those whose expression was modified uniquely by the “tumor-inducing” Meis1 or tumor-inhibiting Prep1 overexpression. In both signatures, the enriched gene categories are the same and are involved in signal transduction. However, Meis1 targets stimulatory genes while Prep1 targets genes that inhibit the tumorigenic signaling pathways. PMID:26259236

  15. Identification of novel regulatory NFAT and TFII-I binding elements in the calbindin-D28k promoter in response to serum deprivation.

    PubMed

    Hajibeigi, Asghar; Dioum, Elhadji M; Guo, Jianfei; Öz, Orhan K

    2015-09-25

    Calbindin-D28k, a key regulator of calcium homeostasis plays a cytoprotective role in various tissues. We used serum free (SFM) and charcoal stripped serum (csFBS) culture media as models of cellular stress to modulate calbindin D28k expression and identify regulatory cis-elements and trans-acting factors in kidney and beta cells. The murine calbindin-D28k promoter activity was significantly upregulated under SFM or csFBS condition. Promoter analysis revealed evolutionary conserved regulatory cis-elements and deletion of 23 nt from +117/+139 as critical for basal transcription. Bioinformatics analysis of the promoter revealed conserved NFAT and TFII regulators elements. Forced expression of NFAT stimulated promoter activity. Inhibition of NFAT transcriptional activity by FK506 attenuated calbindin-D28k expression. TFII-I was shown to be necessary for basal promoter activity and to act cooperatively with NFAT. Using chromatin immunoprecipitation (ChIP) assays, NFAT was shown to bind to both proximal and distal promoter regions. ChIP assays also revealed recruitment of TFII to the -36/+139 region. Knockdown of TFII-I decreased promoter activity. In summary, calbindin-D28k expression during serum deprivation is partly regulated by NFAT and TF-II. This regulation may be important in vivo during ischemia and growth factor withdrawal to regulate cellular function and maintenance. PMID:26260319

  16. Downstream regulatory elements increase acute and latent herpes simplex virus type 2 latency-associated transcript expression but do not influence recurrence phenotype or establishment of latency.

    PubMed Central

    Yoshikawa, T; Stanberry, L R; Bourne, N; Krause, P R

    1996-01-01

    The role of putative promoter or activator sequences downstream of the herpes simplex virus type 2 latency-associated transcript (LAT) promoter and upstream of the LAT intron was investigated in vivo by constructing and evaluating mutant viruses with deletions in this region. The deletion of LAT promoter sequences upstream of the primary LAT transcript reduced levels of LAT expression during productive infections, compared with the LAT expression level of wild-type virus, and abolished LAT expression during latency. The deletion of the putative downstream regulatory elements reduced but did not eliminate LAT expression during productive and latent infections. The deletion of both regions almost completely eliminated acute LAT transcription, although additional acute LAT-region transcription directed by sequences upstream of either region was detected by reverse transcriptase PCR. The deletion of the downstream elements did not influence the ability of the virus to reactivate from latently infected guinea pigs relative to the ability of the wild-type virus to reactivate; thus, decreased LAT expression did not affect the frequency of recurrence. The deletion of both regions did not affect the ability of the virus to establish latency. We conclude that downstream regulatory elements are necessary for maximal acute LAT expression but do not constitute an independent promoter during latency and do not play an obvious role in the establishment of our reactivation from latency. PMID:8627672

  17. A new glimpse of FadR-DNA crosstalk revealed by deep dissection of the E. coli FadR regulatory protein.

    PubMed

    Zhang, Yongchang; Gao, Rongsui; Ye, Huiyan; Wang, Qingjing; Feng, Youjun

    2014-12-01

    Escherichia coli (E. coli) FadR regulator plays dual roles in fatty acid metabolism, which not only represses the fatty acid degradation (fad) system, but also activates the unsaturated fatty acid synthesis pathway. Earlier structural and biochemical studies of FadR protein have provided insights into interplay between FadR protein with its DNA target and/or ligand, while the missing knowledge gap (esp. residues with indirect roles in DNA binding) remains unclear. Here we report this case through deep mapping of old E. coli fadR mutants accumulated. Molecular dissection of E. coli K113 strain, a fadR mutant that can grow on decanoic acid (C10) as sole carbon sources unexpectedly revealed a single point mutation of T178G in fadR locus (W60G in FadRk113). We also observed that a single genetically-recessive mutation of W60G in FadR regulatory protein can lead to loss of its DNA-binding activity, and thereby impair all the regulatory roles in fatty acid metabolisms. Structural analyses of FadR protein indicated that the hydrophobic interaction amongst the three amino acids (W60, F74 and W75) is critical for its DNA-binding ability by maintaining the configuration of its neighboring two β-sheets. Further site-directed mutagenesis analyses demonstrated that the FadR mutants (F74G and/or W75G) do not exhibit the detected DNA-binding activity, validating above structural reasoning. PMID:25311842

  18. HDA6 Directly Interacts with DNA Methyltransferase MET1 and Maintains Transposable Element Silencing in Arabidopsis1[W][OA

    PubMed Central

    Liu, Xuncheng; Yu, Chun-Wei; Duan, Jun; Luo, Ming; Wang, Koching; Tian, Gang; Cui, Yuhai; Wu, Keqiang

    2012-01-01

    The molecular mechanism of how the histone deacetylase HDA6 participates in maintaining transposable element (TE) silencing in Arabidopsis (Arabidopsis thaliana) is not yet defined. In this study, we show that a subset of TEs was transcriptionally reactivated and that TE reactivation was associated with elevated histone H3 and H4 acetylation as well as increased H3K4Me3 and H3K4Me2 in hda6 mutants. Decreased DNA methylation of the TEs was also detected in hda6 mutants, suggesting that HDA6 silences the TEs by regulating histone acetylation and methylation as well as the DNA methylation status of the TEs. Similarly, transcripts of some of these TEs were also increased in the methyltransferase1 (met1) mutant, with decreased DNA methylation. Furthermore, H4 acetylation, H3K4Me3, H3K4Me2, and H3K36Me2 were enriched at the coregulated TEs in the met1 and hda6 met1 mutants. Protein-protein interaction analysis indicated that HDA6 physically interacts with MET1 in vitro and in vivo, and further deletion analysis demonstrated that the carboxyl-terminal region of HDA6 and the bromo-adjacent homology domain of MET1 were responsible for the interaction. These results suggested that HDA6 and MET1 interact directly and act together to silence TEs by modulating DNA methylation, histone acetylation, and histone methylation status. PMID:21994348

  19. Regulatory roles of tankyrase 1 at telomeres and in DNA repair: suppression of T-SCE and stabilization of DNA-PKcs

    PubMed Central

    Dregalla, Ryan C.; Zhou, Junqing; Idate, Rupa R.; Battaglia, Christine L.R.; Liber, Howard L.; Bailey, Susan M.

    2010-01-01

    Intrigued by the dynamics of the seemingly contradictory yet integrated cellular responses to the requisites of preserving telomere integrity while also efficiently repairing damaged DNA, we investigated roles of the telomere associated poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) tankyrase 1 in both telomere function and the DNA damage response following exposure to ionizing radiation. Tankyrase 1 siRNA knockdown in human cells significantly elevated recombination specifically within telomeres, a phenotype with the potential of accelerating cellular senescence. Additionally, depletion of tankyrase 1 resulted in concomitant and rapid reduction of the nonhomologous end-joining protein DNA-PKcs, while Ku86 and ATM protein levels remained unchanged; DNA-PKcs mRNA levels were also unaffected. We found that the requirement of tankyrase 1 for DNA-PKcs protein stability reflects the necessity of its PARP enzymatic activity. We also demonstrated that depletion of tankyrase 1 resulted in proteasome-mediated DNA-PKcs degradation, explaining the associated defective damage response observed; i.e., increased sensitivity to ionizing radiation-induced cell killing, mutagenesis, chromosome aberration and telomere fusion. We provide the first evidence for regulation of DNA-PKcs by tankyrase 1 PARP activity and taken together, identify roles of tankyrase 1 with implications not only for DNA repair and telomere biology, but also for cancer and aging. PMID:21037379

  20. Terminal deoxynucleotidyl transferase is down-regulated by AP-1-like regulatory elements in human lymphoid cells.

    PubMed

    Peralta-Zaragoza, Oscar; Recillas-Targa, Félix; Madrid-Marina, Vicente

    2004-02-01

    Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that catalyses the incorporation of deoxyribonucleotides into the 3'-hydroxyl end of DNA templates and is thought to increase junctional diversity of antigen receptor genes. TdT is expressed only on immature lymphocytes and acute lymphoblastic leukaemia cells and its transcriptional expression is tightly regulated. We had previously found that protein kinase C (PKC) activation down-regulates TdT expression. PKC-activation induces the synthesis of the Fos and Jun proteins, known as the major components of activation protein 1 (AP-1) transcriptional factor implicated in transcriptional control. Here we report the identification of several DNA-protein interactions within the TdT promoter region in non-TdT expressing human cells. Sequence analysis revealed the presence of a putative AP-1-like DNA-binding site, suggesting that AP-1 may play a relevant role in TdT transcriptional regulation. Using a different source of nuclear extracts and the AP-1-TdT motif as a probe we identified several DNA-protein retarded complexes in electrophoretic mobility shift assays. Super-band shifting analysis using an antibody against c-Jun protein confirmed that the main interaction is produced by a nuclear factor that belongs to the AP-1 family transcription factors. Our findings suggest that the TdT gene expression is down-regulated, at least in part, through AP-1-like transcription factors. PMID:15027905

  1. Synthetic Plant Promoters Containing Defined Regulatory Elements Provide Novel Insights into Pathogen- and Wound-Induced Signaling

    PubMed Central

    Rushton, Paul J.; Reinstädler, Anja; Lipka, Volker; Lippok, Bernadette; Somssich, Imre E.

    2002-01-01

    Pathogen-inducible plant promoters contain multiple cis-acting elements, only some of which may contribute to pathogen inducibility. Therefore, we made defined synthetic promoters containing tetramers of only a single type of element and present evidence that a range of cis-acting elements (boxes W1, W2, GCC, JERE, S, Gst1, and D) can mediate local gene expression in planta after pathogen attack. The expression patterns of the promoters were monitored during interactions with a number of pathogens, including compatible, incompatible, and nonhost interactions. Interestingly, there were major differences in the inducibilities of the various promoters with the pathogens tested as well as differences in the speed of induction and in the basal expression levels. We also show that defense signaling is largely conserved across species boundaries at the cis-acting element level. Many of these promoters also direct local wound-induced expression, and this provides evidence for the convergence of resistance gene, nonhost, and wound responses at the level of the promoter elements. We have used these cis-acting elements to construct improved synthetic promoters and show the effects of varying the number, order, and spacing of such elements. These promoters are valuable additions to the study of signaling and transcriptional activation during plant–pathogen interactions. PMID:11971132

  2. Molecular Cloning and Analysis of a DNA Repetitive Element from the Mouse Genome

    ERIC Educational Resources Information Center

    Geisinger, Adriana; Cossio, Gabriela; Wettstein, Rodolfo

    2006-01-01

    We report the development of a 3-week laboratory activity for an undergraduate molecular biology course. This activity introduces students to the practice of basic molecular techniques such as restriction enzyme digestion, agarose gel electrophoresis, cloning, plasmid DNA purification, Southern blotting, and sequencing. Students learn how to carry…

  3. Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome

    PubMed Central

    DeKelver, Russell C.; Choi, Vivian M.; Moehle, Erica A.; Paschon, David E.; Hockemeyer, Dirk; Meijsing, Sebastiaan H.; Sancak, Yasemin; Cui, Xiaoxia; Steine, Eveline J.; Miller, Jeffrey C.; Tam, Phillip; Bartsevich, Victor V.; Meng, Xiangdong; Rupniewski, Igor; Gopalan, Sunita M.; Sun, Helena C.; Pitz, Kathleen J.; Rock, Jeremy M.; Zhang, Lei; Davis, Gregory D.; Rebar, Edward J.; Cheeseman, Iain M.; Yamamoto, Keith R.; Sabatini, David M.; Jaenisch, Rudolf; Gregory, Philip D.; Urnov, Fyodor D.

    2010-01-01

    Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on human cells. We describe the use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene, a “safe harbor” locus known as AAVS1. ZFNs enable targeted transgenesis at a frequency of up to 15% following transient transfection of both transformed and primary human cells, including fibroblasts and hES cells. When added to this locus, transgenes such as expression cassettes for shRNAs, small-molecule-responsive cDNA expression cassettes, and reporter constructs, exhibit consistent expression and sustained function over 50 cell generations. By avoiding random integration and drug selection, this method allows bona fide isogenic settings for high-throughput functional genomics, proteomics, and regulatory DNA analysis in essentially any transformed human cell type and in primary cells. PMID:20508142

  4. The Regulatory T Cell Lineage Factor Foxp3 Regulates Gene Expression through Several Distinct Mechanisms Mostly Independent of Direct DNA Binding

    PubMed Central

    Andersen, Kristian G.; Hebenstreit, Daniel; Teichmann, Sarah A.; Betz, Alexander G.

    2015-01-01

    The lineage factor Foxp3 is essential for the development and maintenance of regulatory T cells, but little is known about the mechanisms involved. Here, we demonstrate that an N-terminal proline-rich interaction region is crucial for Foxp3’s function. Subdomains within this key region link Foxp3 to several independent mechanisms of transcriptional regulation. Our study suggests that Foxp3, even in the absence of its DNA-binding forkhead domain, acts as a bridge between DNA-binding interaction partners and proteins with effector function permitting it to regulate a large number of genes. We show that, in one such mechanism, Foxp3 recruits class I histone deacetylases to the promoters of target genes, counteracting activation-induced histone acetylation and thereby suppressing their expression. PMID:26107960

  5. Terminal deoxynucleotidyl transferase is down-regulated by AP-1-like regulatory elements in human lymphoid cells

    PubMed Central

    Peralta-Zaragoza, Oscar; Recillas-Targa, Félix; Madrid-Marina, Vicente

    2004-01-01

    Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that catalyses the incorporation of deoxyribonucleotides into the 3′-hydroxyl end of DNA templates and is thought to increase junctional diversity of antigen receptor genes. TdT is expressed only on immature lymphocytes and acute lymphoblastic leukaemia cells and its transcriptional expression is tightly regulated. We had previously found that protein kinase C (PKC) activation down-regulates TdT expression. PKC-activation induces the synthesis of the Fos and Jun proteins, known as the major components of activation protein 1 (AP-1) transcriptional factor implicated in transcriptional control. Here we report the identification of several DNA–protein interactions within the TdT promoter region in non-TdT expressing human cells. Sequence analysis revealed the presence of a putative AP-1-like DNA-binding site, suggesting that AP-1 may play a relevant role in TdT transcriptional regulation. Using a different source of nuclear extracts and the AP-1–TdT motif as a probe we identified several DNA-protein retarded complexes in electrophoretic mobility shift assays. Super-band shifting analysis using an antibody against c-Jun protein confirmed that the main interaction is produced by a nuclear factor that belongs to the AP-1 family transcription factors. Our findings suggest that the TdT gene expression is down-regulated, at least in part, through AP-1-like transcription factors. PMID:15027905

  6. Survey of chimeric IStron elements in bacterial genomes: multiple molecular symbioses between group I intron ribozymes and DNA transposons

    PubMed Central

    Tourasse, Nicolas J.; Stabell, Fredrik B.; Kolstø, Anne-Brit

    2014-01-01

    IStrons are chimeric genetic elements composed of a group I intron associated with an insertion sequence (IS). The group I intron is a catalytic RNA providing the IStron with self-splicing ability, which renders IStron insertions harmless to the host genome. The IS element is a DNA transposon conferring mobility, and thus allowing the IStron to spread in genomes. IStrons are therefore a striking example of a molecular symbiosis between unrelated genetic elements endowed with different functions. In this study, we have conducted the first comprehensive survey of IStrons in sequenced genomes that provides insights into the distribution, diversity, origin and evolution of IStrons. We show that IStrons have a restricted phylogenetic distribution limited to two bacterial phyla, the Firmicutes and the Fusobacteria. Nevertheless, diverse IStrons representing two major groups targeting different insertion site motifs were identified. This taken with the finding that while the intron components of all IStrons belong to the same structural class, they are fused to different IS families, indicates that multiple intron–IS symbioses have occurred during evolution. In addition, introns and IS elements related to those that were at the origin of IStrons were also identified. PMID:25324310

  7. The ribosomal protein L34 gene from the mosquito, Aedes albopictus: exon-intron organization, copy number, and potential regulatory elements.

    PubMed

    Niu, L L; Fallon, A M

    1999-12-01

    We describe the structural analysis of genomic DNA encoding ribosomal protein (rp) L34 from the mosquito, Aedes albopictus. Comparison of genomic DNA sequences encompassing approximately 8 kb with the rpL34 cDNA sequence showed that the gene contains three exons and two introns, encoding a primary transcript with a deduced size of 6196 nucleotides from the transcription start site to the polyadenylation site. Exon 1, which is not translated, measures only 45 bp, and is separated from Exon 2 by a 359 bp intron. Exon 2 measures 78 bp, and contains the AUG translation initiation cod