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NAR Top Articles - Gene Regulation, Chromatin and Epigenetics

Gene Regulation, Chromatin and Epigenetics

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January 2015

Sudemycin E influences alternative splicing and changes chromatin modifications
P. Convertini, M. Shen, P. M. Potter, G. Palacios, C. Lagisetti, P. de la Grange, C. Horbinski, Y. N. Fondufe-Mittendorf, T. R. Webb and S. Stamm
Nucleic Acids Res. (2014) 42 (8): 4947-4961
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Sudemycin E is an analog of the pre-messenger RNA splicing modulator FR901464 and its derivative spliceostatin A. Sudemycin E causes the death of cancer cells through an unknown mechanism. We found that similar to spliceostatin A, sudemycin E binds to the U2 small nuclear ribonucleoprotein (snRNP) component SF3B1. Native chromatin immunoprecipitations showed that U2 snRNPs physically interact with nucleosomes. Sudemycin E induces a dissociation of the U2 snRNPs and decreases their interaction with nucleosomes. To determine the effect on gene expression, we performed genome-wide array analysis. Sudemycin E first causes a rapid change in alternative pre-messenger RNA splicing, which is later followed by changes in overall gene expression and arrest in the G2 phase of the cell cycle. The changes in alternative exon usage correlate with a loss of the H3K36me3 modification in chromatin encoding these exons. We propose that sudemycin E interferes with the ability of U2 snRNP to maintain an H3K36me3 modification in actively transcribed genes...

Chromosome position effects on gene expression in Escherichia coli K-12
J. A. Bryant, L. E. Sellars, S. J. Busby and D. J. Lee
Nucleic Acids Res. (2014) 42 (18): 11383-11392
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In eukaryotes, the location of a gene on the chromosome is known to affect its expression, but such position effects are poorly understood in bacteria. Here, using Escherichia coli K-12, we demonstrate that expression of a reporter gene cassette, comprised of the model E. coli lac promoter driving expression of gfp, varies by approximately 300-fold depending on its precise position on the chromosome. At some positions, expression was more than 3-fold higher than at the natural lac promoter locus, whereas at several other locations, the reporter cassette was completely silenced: effectively overriding local lac promoter control. These effects were not due to differences in gene copy number, caused by partially replicated genomes. Rather, the differences in gene expression occur predominantly at the level of transcription and are mediated by several different features that are involved in chromosome organization. Taken together, our findings identify a tier of gene regulation above local promoter control and highlight the importance of chromosome position effects on gene expression profiles in bacteria.

The eIF2{alpha}/ATF4 pathway is essential for stress-induced autophagy gene expression
W. B'Chir, A. C. Maurin, V. Carraro, J. Averous, C. Jousse, Y. Muranishi, L. Parry, G. Stepien, P. Fafournoux and A. Bruhat
Nucleic Acids Res. (2013) 41 (16): 7683-7699
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In response to different environmental stresses, eIF2alpha phosphorylation represses global translation coincident with preferential translation of ATF4, a master regulator controlling the transcription of key genes essential for adaptative functions. Here, we establish that the eIF2alpha/ATF4 pathway directs an autophagy gene transcriptional program in response to amino acid starvation or endoplasmic reticulum stress. The eIF2alpha-kinases GCN2 and PERK and the transcription factors ATF4 and CHOP are also required to increase the transcription of a set of genes implicated in the formation, elongation and function of the autophagosome. We also identify three classes of autophagy genes according to their dependence on ATF4 and CHOP and the binding of these factors to specific promoter cis elements. Furthermore, different combinations of CHOP and ATF4 bindings to target promoters allow the trigger of a differential transcriptional response according to the stress intensity. Overall, this study reveals a novel regulatory role of the eIF2alpha-ATF4 pathway in the fine-tuning of the autophagy gene transcription program...

Aberrant transcriptional regulations in cancers: genome, transcriptome and epigenome analysis of lung adenocarcinoma cell lines
A. Suzuki, H. Makinoshima, H. Wakaguri, H. Esumi, S. Sugano, T. Kohno, K. Tsuchihara and Y. Suzuki
Nucleic Acids Res. (2014) 42 (22): 13557-13572
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Here we conducted an integrative multi-omics analysis to understand how cancers harbor various types of aberrations at the genomic, epigenomic and transcriptional levels. In order to elucidate biological relevance of the aberrations and their mutual relations, we performed whole-genome sequencing, RNA-Seq, bisulfite sequencing and ChIP-Seq of 26 lung adenocarcinoma cell lines. The collected multi-omics data allowed us to associate an average of 536 coding mutations and 13,573 mutations in promoter or enhancer regions with aberrant transcriptional regulations. We detected the 385 splice site mutations and 552 chromosomal rearrangements, representative cases of which were validated to cause aberrant transcripts. Averages of 61, 217, 3687 and 3112 mutations are located in the regulatory regions which showed differential DNA methylation, H3K4me3, H3K4me1 and H3K27ac marks, respectively. We detected distinct patterns of aberrations in transcriptional regulations depending on genes. We found that the irregular histone marks were characteristic to EGFR and CDKN1A...

Tls1 regulates splicing of shelterin components to control telomeric heterochromatin assembly and telomere length
J. Wang, X. Tadeo, H. Hou, S. Andrews, J. J. Moresco, J. R. Yates, 3rd, P. L. Nagy and S. Jia
Nucleic Acids Res. (2014) 42 (18): 11419-11432
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Heterochromatin preferentially forms at repetitive DNA elements through RNAi-mediated targeting of histone-modifying enzymes. It was proposed that splicing factors interact with the RNAi machinery or regulate the splicing of repeat transcripts to directly participate in heterochromatin assembly. Here, by screening the fission yeast deletion library, we comprehensively identified factors required for telomeric heterochromatin assembly, including a novel gene tls1+. Purification of Tls1 and mass spectrometry analysis of its interacting proteins show that Tls1 associates with the spliceosome subunit Brr2. RNA sequencing analysis shows that the splicing of a subset of mRNAs are affected in tls1Delta cells, including mRNAs of shelterin components rap1+ and poz1+. Importantly, replacing rap1+ and poz1+ with their cDNAs significantly alleviated heterochromatin defects of tls1Delta cells, suggesting that the missplicing of shelterin components is the cause of such defects, and that splicing factors regulate telomeric heterochromatin through the proper splicing of heterochromatin factors...

AGO2 and SETDB1 cooperate in promoter-targeted transcriptional silencing of the androgen receptor gene
S. Cho, J. S. Park and Y. K. Kang
Nucleic Acids Res. (2014) 42 (22): 13545-13556
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In mammals, RNA interference is primarily a post-transcriptional mechanism. Evidence has accumulated for additional role in transcriptional gene silencing (TGS) but the question for a good paradigm for small interfering antigene RNA (agRNA)-induced chromatin modification remains unanswered. Here, we show that SETDB1, a histone H3-lysine 9 (H3K9)-specific methyltransferase, cooperates with Argonaute-2 (AGO2) and plays an essential role in agRNA-induced TGS. The androgen receptor (AR) gene was transcriptionally silenced by agRNA targeted to its promoter, and we show that this repression was mitigated by knockdown of SETDB1 or AGO2. Chromatin immunoprecipitation demonstrated that agRNA-driven AGO2 was first targeted to the AR promoter, followed by SETDB1. SIN3A and HDAC1/2, the components of the SIN3-HDAC complex, immunoprecipitated with SETDB1, and localized at the agRNA-targeted promoter. Agreeing with the presence of SETDB1, trimethyl-H3K9 was enriched in the AR promoter. Both EZH2 and trimethyl-H3K27 were also present in the targeted locus; accordingly, EZH2 immunoprecipitated with SETDB1...

Explicit DNase sequence bias modeling enables high-resolution transcription factor footprint detection
G. G. Yardimci, C. L. Frank, G. E. Crawford and U. Ohler
Nucleic Acids Res. (2014) 42 (19): 11865-11878
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DNaseI footprinting is an established assay for identifying transcription factor (TF)-DNA interactions with single base pair resolution. High-throughput DNase-seq assays have recently been used to detect in vivo DNase footprints across the genome. Multiple computational approaches have been developed to identify DNase-seq footprints as predictors of TF binding. However, recent studies have pointed to a substantial cleavage bias of DNase and its negative impact on predictive performance of footprinting. To assess the potential for using DNase-seq to identify individual binding sites, we performed DNase-seq on deproteinized genomic DNA and determined sequence cleavage bias. This allowed us to build bias corrected and TF-specific footprint models. The predictive performance of these models demonstrated that predicted footprints corresponded to high-confidence TF-DNA interactions. DNase-seq footprints were absent under a fraction of ChIP-seq peaks, which we show to be indicative of weaker binding, indirect TF-DNA interactions or possible ChIP artifacts. The modeling approach was also able to detect variation in the consensus motifs that TFs bind to...

Integrative annotation of chromatin elements from ENCODE data
M. M. Hoffman, J. Ernst, S. P. Wilder, A. Kundaje, R. S. Harris, M. Libbrecht, B. Giardine, P. M. Ellenbogen, J. A. Bilmes, E. Birney, R. C. Hardison, I. Dunham, M. Kellis and W. S. Noble
Nucleic Acids Res. (2013) 41 (2): 827-841
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The ENCODE Project has generated a wealth of experimental information mapping diverse chromatin properties in several human cell lines. Although each such data track is independently informative toward the annotation of regulatory elements, their interrelations contain much richer information for the systematic annotation of regulatory elements. To uncover these interrelations and to generate an interpretable summary of the massive datasets of the ENCODE Project, we apply unsupervised learning methodologies, converting dozens of chromatin datasets into discrete annotation maps of regulatory regions and other chromatin elements across the human genome. These methods rediscover and summarize diverse aspects of chromatin architecture, elucidate the interplay between chromatin activity and RNA transcription, and reveal that a large proportion of the genome lies in a quiescent state, even across multiple cell types. The resulting annotation of non-coding regulatory elements correlate strongly with mammalian evolutionary constraint...

Alternative Lengthening of Telomeres is characterized by reduced compaction of telomeric chromatin
H. Episkopou, I. Draskovic, A. Van Beneden, G. Tilman, M. Mattiussi, M. Gobin, N. Arnoult, A. Londono-Vallejo and A. Decottignies
Nucleic Acids Res. (2014) 42 (7): 4391-4405
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Proper telomeric chromatin configuration is thought to be essential for telomere homeostasis and stability. Previous studies in mouse suggested that loss of heterochromatin marks at telomeres might favor onset of Alternative Lengthening of Telomeres (ALT) pathway, by promoting homologous recombination. However, analysis of chromatin status at human ALT telomeres has never been reported. Here, using isogenic human cell lines and cellular hybrids, which rely either on telomerase or ALT to maintain telomeres, we show that chromatin compaction is reduced at ALT telomeres and this is associated with a global decrease in telomeric H3K9me3. This, subsequently, leads to upregulation of telomere transcription. Accordingly, restoration of a more condensed telomeric chromatin through telomerase-dependent elongation of short ALT telomeres reduces telomere transcription. We further show that loss of ATRX chromatin remodeler function, a frequent characteristic of ALT cells, is not sufficient to decrease chromatin condensation at telomeres nor to increase the expression of telomeric RNA species...

Global MEF2 target gene analysis in cardiac and skeletal muscle reveals novel regulation of DUSP6 by p38MAPK-MEF2 signaling
S. Wales, S. Hashemi, A. Blais and J. C. McDermott
Nucleic Acids Res. (2014) 42 (18): 11349-11362
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MEF2 plays a profound role in the regulation of transcription in cardiac and skeletal muscle lineages. To define the overlapping and unique MEF2A genomic targets, we utilized ChIP-exo analysis of cardiomyocytes and skeletal myoblasts. Of the 2783 and 1648 MEF2A binding peaks in skeletal myoblasts and cardiomyocytes, respectively, 294 common binding sites were identified. Genomic targets were compared to differentially expressed genes in RNA-seq analysis of MEF2A depleted myogenic cells, revealing two prominent genetic networks. Genes largely associated with muscle development were down-regulated by loss of MEF2A while up-regulated genes reveal a previously unrecognized function of MEF2A in suppressing growth/proliferative genes. Several up-regulated (Tprg, Mctp2, Kitl, Prrx1, Dusp6) and down-regulated (Atp1a2, Hspb7, Tmem182, Sorbs2, Lmod3) MEF2A target genes were chosen for further investigation. Interestingly, siRNA targeting of the MEF2A/D heterodimer revealed a somewhat divergent role in the regulation of Dusp6, a MAPK phosphatase, in cardiac and skeletal myogenic lineages...

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