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NAR Top Articles - Gene Regulation, Chromatin and Epigenetics

Gene Regulation, Chromatin and Epigenetics

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April 2015

The transcript elongation factor SPT4/SPT5 is involved in auxin-related gene expression in Arabidopsis
Durr, J; Lolas, IB; Sorensen, BB; Schubert, V; Houben, A; Melzer, M; Deutzmann, R; Grasser, M; Grasser, KD
Nucleic Acids Res. 2014, 42, 4332-4347
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The heterodimeric complex SPT4/SPT5 is a transcript elongation factor (TEF) that directly interacts with RNA polymerase II (RNAPII) to regulate messenger RNA synthesis in the chromatin context. We provide biochemical evidence that in Arabidopsis, SPT4 occurs in a complex with SPT5, demonstrating that the SPT4/SPT5 complex is conserved in plants. Each subunit is encoded by two genes SPT4-1/2 and SPT5-1/2. A mutant affected in the tissue-specifically expressed SPT5-1 is viable, whereas inactivation of the generally expressed SPT5-2 is homozygous lethal. RNAi-mediated downregulation of SPT4 decreases cell proliferation and causes growth reduction and developmental defects. These plants display especially auxin signalling phenotypes. Consistently, auxin-related genes, most strikingly AUX/IAA genes, are downregulated in SPT4-RNAi plants that exhibit an enhanced auxin response. In Arabidopsis nuclei, SPT5 clearly localizes to the transcriptionally active euchromatin, and essentially co-localizes with transcribing RNAPII. Typical for TEFs, SPT5 is found over the entire transcription unit of RNAPII-transcribed genes...

The eIF2{alpha}/ATF4 pathway is essential for stress-induced autophagy gene expression
B'chir, W; Maurin, AC; Carraro, V; Averous, J; Jousse, C; Muranishi, Y; Parry, L; Stepien, G; Fafournoux, P; Bruhat, A
Nucleic Acids Res. 2013, 41, 7683-7699
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In response to different environmental stresses, eIF2 alpha 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 eIF2 alpha/ATF4 pathway directs an autophagy gene transcriptional program in response to amino acid starvation or endoplasmic reticulum stress. The eIF2 alpha-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 eIF2 alpha-ATF4 pathway in the fine-tuning of the autophagy gene transcription program...

Evidence of efficient stop codon readthrough in four mammalian genes
Loughran, G; Chou, MY; Ivanov, IP; Jungreis, I; Kellis, M; Kiran, AM; Baranov, PV; Atkins, JF
Nucleic Acids Res. 2014, 42, 8928-8938
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Stop codon readthrough is used extensively by viruses to expand their gene expression. Until recent discoveries in Drosophila, only a very limited number of readthrough cases in chromosomal genes had been reported. Analysis of conserved protein coding signatures that extend beyond annotated stop codons identified potential stop codon readthrough of four mammalian genes. Here we use a modified targeted bioinformatic approach to identify a further three mammalian readthrough candidates. All seven genes were tested experimentally using reporter constructs transfected into HEK-293T cells. Four displayed efficient stop codon readthrough, and these have UGA immediately followed by CUAG. Comparative genomic analysis revealed that in the four readthrough candidates containing UGA-CUAG, this motif is conserved not only in mammals but throughout vertebrates with the first six of the seven nucleotides being universally conserved. The importance of the CUAG motif was confirmed using a systematic mutagenesis approach. One gene, OPRL1, encoding an opiate receptor, displayed extremely efficient levels of readthrough (similar to 31%) in HEK-293T cells...

TET1 is a maintenance DNA demethylase that prevents methylation spreading in differentiated cells
Jin, CL; Lu, Y; Jelinek, J; Liang, SD; Estecio, MRH; Barton, MC; Issa, JPJ
Nucleic Acids Res. 2014, 42, 6956-6971
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TET1 is a 5-methylcytosine dioxygenase and its DNA demethylating activity has been implicated in pluripotency and reprogramming. However, the precise role of TET1 in DNA methylation regulation outside of developmental reprogramming is still unclear. Here, we show that overexpression of the TET1 catalytic domain but not full length TET1 (TET1-FL) induces massive global DNA demethylation in differentiated cells. Genome-wide mapping reveals that 5-hydroxymethylcytosine production by TET1-FL is inhibited as DNA methylation increases, which can be explained by the preferential binding of TET1-FL to unmethylated CpG islands (CGIs) through its CXXC domain. TET1-FL specifically accumulates 5-hydroxymethylcytosine at the edges of hypomethylated CGIs, while knockdown of endogenous TET1 induces methylation spreading from methylated edges into hypomethylated CGIs. We also found that gene expression changes after TET1-FL overexpression are relatively small and independent of its dioxygenase function. Thus, our results identify TET1 as a maintenance DNA demethylase that does not purposely decrease methylation levels, but specifically prevents aberrant methylation spreading...

Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors
Hu, JB; Lei, Y; Wong, WK; Liu, SQ; Lee, KC; He, XJ; You, WX; Zhou, R; Guo, JT; Chen, XF; Peng, XL; Sun, H; Huang, H; Zhao, H; Feng, B
Nucleic Acids Res. 2014, 42, 4375-4390
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The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around -120 to -80 bp, while highly effective sgRNAs targeted from -147 to -89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells...

Integrative annotation of chromatin elements from ENCODE data
Hoffman, MM; Ernst, J; Wilder, SP; Kundaje, A; Harris, RS; Libbrecht, M; Giardine, B; Ellenbogen, PM; Bilmes, JA; Birney, E; Hardison, RC; Dunham, I; Kellis, M; Noble, WS
Nucleic Acids Res. 2013, 41, 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, and provide an unbiased approach for evaluating metrics of evolutionary constraint

Chromosome position effects on gene expression in Escherichia coli K-12
Bryant, JA; Sellars, LE; Busby, SJW; Lee, DJ
Nucleic Acids Res. 2014, 42, 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 similar to 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.

Global MEF2 target gene analysis in cardiac and skeletal muscle reveals novel regulation of DUSP6 by p38MAPK-MEF2 signaling
Wales, S; Hashemi, S; Blais, A; McDermott, JC
Nucleic Acids Res. 2014, 42, 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. Furthermore, MEF2D functions as a p38MAPK-dependent repressor of Dusp6 in myoblasts...

Detection of G-quadruplex DNA in mammalian cells
Henderson, A; Wu, YL; Huang, YC; Chavez, EA; Platt, J; Johnson, FB; Brosh, RM; Sen, D; Lansdorp, PM
Nucleic Acids Res. 2014, 42, 860-869
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It has been proposed that guanine-rich DNA forms four-stranded structures in vivo called G-quadruplexes or G4 DNA. G4 DNA has been implicated in several biological processes, but tools to study G4 DNA structures in cells are limited. Here we report the development of novel murine monoclonal antibodies specific for different G4 DNA structures. We show that one of these antibodies designated 1H6 exhibits strong nuclear staining in most human and murine cells. Staining intensity increased on treatment of cells with agents that stabilize G4 DNA and, strikingly, cells deficient in FANCJ, a G4 DNA-specific helicase, showed stronger nuclear staining than controls. Our data strongly support the existence of G4 DNA structures in mammalian cells and indicate that the abundance of such structures is increased in the absence of FANCJ. We conclude that monoclonal antibody 1H6 is a valuable tool for further studies on the role of G4 DNA in cell and molecular biology.

Critical role for p53-serine 15 phosphorylation in stimulating transactivation at p53-responsive promoters
Loughery, J; Cox, M; Smith, LM; Meek, DW
Nucleic Acids Res. 2014, 42, 7666-7680
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The p53 tumour suppressor is induced by various stress stimuli and coordinates an adaptive gene expression programme leading to growth arrest or cell death. Some stimuli, such as DNA damage, lead to rapid and substantial multisite phosphorylation of p53, nucleated initially through phosphorylation of serine 15. Other stimuli, such as hyperproliferation, do not stimulate p53-phosphorylation, raising questions regarding the physiological role for phosphorylation. Here, we show that a basal level of Ser15 phosphorylation occurs in both unstimulated cells and cells stimulated pharmacologically to induce p53. p53 in which Ser15 is substituted by alanine (S15A) fails to mediate p53-dependent transcription or growth arrest but can be rescued by substitution with aspartate (S15D: a phosphomimic). Chromatin immunoprecipitation (ChIP) analyses show that, while wt- and S15A-p53 are detectable on the CDKN1A (p21) promoter (as a representative p53-responsive promoter), S15A-p53 does not stimulate histone acetylation (a measure of chromatin relaxation), nor is its recruitment stimulated, in response to a DNA damage or pharmacological stimulus...

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