NAR Top Articles - Gene Regulation, Chromatin and Epigenetics
The eIF2α/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...
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...
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...
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.
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.
ETS1 is a genome-wide effector of RAS/ERK signaling in epithelial cells
Plotnik, JP; Budka, JA; Ferris, MW; Hollenhorst, PC
Nucleic Acids Res. 2014, 42, 11928-11940
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The RAS/ERK pathway is commonly activated in carcinomas and promotes oncogenesis by altering transcriptional programs. However, the array of cis-regulatory elements and trans-acting factors that mediate these transcriptional changes is still unclear. Our genome-wide analysis determined that a sequence consisting of neighboring ETS and AP-1 transcription factor binding sites is enriched near cell migration genes activated by RAS/ERK signaling in epithelial cells. In vivo screening of candidate ETS proteins revealed that ETS1 is specifically required for migration of RAS/ERK activated cells. Furthermore, both migration and transcriptional activation through ETS/AP-1 required ERK phosphorylation of ETS1. Genome-wide mapping of multiple ETS proteins demonstrated that ETS1 binds specifically to enhancer ETS/AP-1 sequences. ETS1 occupancy, and its role in cell migration, was conserved in epithelial cells derived from multiple tissues, consistent with a chromatin organization common to epithelial cell lines. Genome-wide expression analysis showed that ETS1 was required for activation of RAS-regulated cell migration genes, but also identified a surprising role for ETS1 in the repression of genes such as DUSP4...
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...
Small molecule induced reactivation of mutant p53 in cancer cells
Liu, XR; Wilcken, R; Joerger, AC; Chuckowree, IS; Amin, J; Spencer, J; Fersht, AR
Nucleic Acids Res. 2013, 41, 6034-6044
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The p53 cancer mutant Y220C is an excellent paradigm for rescuing the function of conformationally unstable p53 mutants because it has a unique surface crevice that can be targeted by small-molecule stabilizers. Here, we have identified a compound, PK7088, which is active in vitro: PK7088 bound to the mutant with a dissociation constant of 140 mu M and raised its melting temperature, and we have determined the binding mode of a close structural analogue by X-ray crystallography. We showed that PK7088 is biologically active in cancer cells carrying the Y220C mutant by a battery of tests. PK7088 increased the amount of folded mutant protein with wild-type conformation, as monitored by immunofluorescence, and restored its transcriptional functions. It induced p53-Y220C-dependent growth inhibition, cell-cycle arrest and apoptosis. Most notably, PK7088 increased the expression levels of p21 and the proapoptotic NOXA protein. PK7088 worked synergistically with Nutlin-3 on up-regulating p21 expression, whereas Nutlin-3 on its own had no effect, consistent with its mechanism of action. PK7088 also restored non-transcriptional apoptotic functions of p53 by triggering nuclear export of BAX...
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...
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