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NAR Top Articles - Gene Regulation, Chromatin and Epigenetics

Gene Regulation, Chromatin and Epigenetics

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December 2013


NF-kappaB P50/P65 hetero-dimer mediates differential regulation of CD166/ALCAM expression via interaction with micoRNA-9 after serum deprivation, providing evidence for a novel negative auto-regulatory loop
Wang, JY; Gu, ZD; Ni, PH; Qiao, YX; Chen, CQ; Liu, XF; Lin, JF; Chen, N; Fan, QS
Nucleic Acids Res. (2011) 39 (15): 6440-6455
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CD166/ALCAM plays an important role in tumor aggression and progression as well as protecting cancer cells against apoptosis and autophagy. However, the mechanism by which pro-cell death signals control CD166 expression remains unclear. Here we show that following serum deprivation (SD), upregulation of CD166 protein is shorter than that of CD166 mRNA. Molecular analysis revealed both CD166 and miR-9-1 as two novel NF-kappa B target genes in hepatoma cells. In vivo activation and translocation of the NF-kappa B P50/P65 heterodimer into the nucleus following the phosphorylation and accompanied degradation of its inhibitor, I kappa B alpha, contributes to efficient transcription of both genes following SD. We show that following serum starvation, delayed up-regulation of miR-9 represses translation of CD166 protein through its target sites in the 3'-UTR of CD166 mRNA. We also propose that miR-9 promotes cell migration largely due to inhibition of CD166...

Histone H2A variants in nucleosomes and chromatin: more or less stable?
Bonisch, C; Hake, SB
Nucleic Acids Res. (2012) 40 (21): 10719-10741
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In eukaryotes, DNA is organized together with histones and non-histone proteins into a highly complex nucleoprotein structure called chromatin, with the nucleosome as its monomeric subunit. Various interconnected mechanisms regulate DNA accessibility, including replacement of canonical histones with specialized histone variants. Histone variant incorporation can lead to profound chromatin structure alterations thereby influencing a multitude of biological processes ranging from transcriptional regulation to genome stability. Among core histones, the H2A family exhibits highest sequence divergence, resulting in the largest number of variants known. Strikingly, H2A variants differ mostly in their C-terminus, including the docking domain, strategically placed at the DNA entry/exit site and implicated in interactions with the (H3-H4)(2)-tetramer within the nucleosome and in the L1 loop, the interaction interface of H2A-H2B dimers. Moreover, the acidic patch, important for internucleosomal contacts and higher-order chromatin structure, is altered between different H2A variants. Consequently, H2A variant incorporation has the potential to strongly regulate DNA organization on several levels resulting in meaningful biological output.

ChIP-seq analysis reveals distinct H3K27me3 profiles that correlate with transcriptional activity
Young, MD; Willson, TA; Wakefield, MJ; Trounson, E; Hilton, DJ; Blewitt, ME; Oshlack, A; Majewski, IJ
Nucleic Acids Res. (2011) 39 (17): 7415-7427
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Transcriptional control is dependent on a vast network of epigenetic modifications. One epigenetic mark of particular interest is tri-methylation of lysine 27 on histone H3 (H3K27me3), which is catalysed and maintained by Polycomb Repressive Complex 2 (PRC2). Although this histone mark is studied widely, the precise relationship between its local pattern of enrichment and regulation of gene expression is currently unclear. We have used ChIP-seq to generate genome-wide maps of H3K27me3 enrichment, and have identified three enrichment profiles with distinct regulatory consequences. First, a broad domain of H3K27me3 enrichment across the body of genes corresponds to the canonical view of H3K27me3 as inhibitory to transcription. Second, a peak of enrichment around the transcription start site (TSS) is commonly associated with 'bivalent' genes, where H3K4me3 also marks the TSS. Finally and most surprisingly, we identified an enrichment profile with a peak in the promoter of genes that is associated with active transcription...

Next-generation insights into regulatory T cells: expression profiling and FoxP3 occupancy in Human
Birzele, F; Fauti, T; Stahl, H; Lenter, MC; Simon, E; Knebel, D; Weith, A; Hildebrandt, T; Mennerich, D
Nucleic Acids Res. (2011) 39 (18): 7946-7960
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Regulatory T-cells (Treg) play an essential role in the negative regulation of immune answers by developing an attenuated cytokine response that allows suppressing proliferation and effector function of T-cells (CD4(+) Th). The transcription factor FoxP3 is responsible for the regulation of many genes involved in the Treg gene signature. Its ablation leads to severe immune deficiencies in human and mice. Recent developments in sequencing technologies have revolutionized the possibilities to gain insights into transcription factor binding by ChiP-seq and into transcriptome analysis by mRNA-seq. We combine FoxP3 ChiP-seq and mRNA-seq in order to understand the transcriptional differences between primary human CD4(+) T helper and regulatory T-cells, as well as to study the role of FoxP3 in generating those differences. We show, that mRNA-seq allows analyzing the transcriptomal landscape of T-cells including the expression of specific splice variants at much greater depth than previous approaches, whereas 50% of transcriptional regulation events have not been described before by using diverse array technologies...

Integration of Hi-C and ChIP-seq data reveals distinct types of chromatin linkages
Lan, X; Witt, H; Katsumura, K; Ye, ZQ; Wang, QB; Bresnick, EH; Farnham, PJ; Jin, VX
Nucleic Acids Res. (2012) 40 (16): 7690-7704
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We have analyzed publicly available K562 Hi-C data, which enable genome-wide unbiased capturing of chromatin interactions, using a Mixture Poisson Regression Model and a power-law decay background to define a highly specific set of interacting genomic regions. We integrated multiple ENCODE Consortium resources with the Hi-C data, using DNase-seq data and ChIP-seq data for 45 transcription factors and 9 histone modifications. We classified 12 different sets (clusters) of interacting loci that can be distinguished by their chromatin modifications and which can be categorized into two types of chromatin linkages. The different clusters of loci display very different relationships with transcription factor-binding sites. As expected, many of the transcription factors show binding patterns specific to clusters composed of interacting loci that encompass promoters or enhancers. However, cluster 9, which is distinguished by marks of open chromatin but not by active enhancer or promoter marks, was not bound by most transcription factors but was highly enriched for three transcription factors (GATA1, GATA2 and c-Jun) and three chromatin modifiers...

Dynamic hydroxymethylation of deoxyribonucleic acid marks differentiation-associated enhancers
Serandour, AA; Avner, S; Oger, F; Bizot, M; Percevault, F; Lucchetti-Miganeh, C; Palierne, G; Gheeraert, C; Barloy-Hubler, F; Le Peron, C; Madigou, T; Durand, E; Froguel, P; Staels, B; Lefebvre, P; Metivier, R; Eeckhoute, J; Salbert, G
Nucleic Acids Res. (2012) 40 (17): 8255-8265
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Enhancers are developmentally controlled transcriptional regulatory regions whose activities are modulated through histone modifications or histone variant deposition. In this study, we show by genome-wide mapping that the newly discovered deoxyribonucleic acid (DNA) modification 5-hydroxymethylcytosine (5hmC) is dynamically associated with transcription factor binding to distal regulatory sites during neural differentiation of mouse P19 cells and during adipocyte differentiation of mouse 3T3-L1 cells. Functional annotation reveals that regions gaining 5hmC are associated with genes expressed either in neural tissues when P19 cells undergo neural differentiation or in adipose tissue when 3T3-L1 cells undergo adipocyte differentiation. Furthermore, distal regions gaining 5hmC together with H3K4me2 and H3K27ac in P19 cells behave as differentiation-dependent transcriptional enhancers. Identified regions are enriched in motifs for transcription factors regulating specific cell fates...

Mitochondrial DNA copy number is regulated in a tissue specific manner by DNA methylation of the nuclear-encoded DNA polymerase gamma A
Kelly, RDW; Mahmud, A; McKenzie, M; Trounce, IA; St John, JC
Nucleic Acids Res. (2012) 40 (20): 10124-10138
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DNA methylation is an essential mechanism controlling gene expression during differentiation and development. We investigated the epigenetic regulation of the nuclear-encoded, mitochondrial DNA (mtDNA) polymerase gamma catalytic subunit (PolgA) by examining the methylation status of a CpG island within exon 2 of PolgA. Bisulphite sequencing identified low methylation levels (< 10%) within exon 2 of mouse oocytes, blastocysts and embryonic stem cells (ESCs), while somatic tissues contained significantly higher levels (> 40%). In contrast, induced pluripotent stem (iPS) cells and somatic nuclear transfer ESCs were hypermethylated (> 20%), indicating abnormal epigenetic reprogramming. Real time PCR analysis of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) immunoprecipitated DNA suggests active DNA methylation and demethylation within exon 2 of PolgA. Moreover, neural differentiation of ESCs promoted de novo methylation and demethylation at the exon 2 locus. Regression analysis demonstrates that cell-specific PolgA expression levels were negatively correlated with DNA methylation within exon 2 and mtDNA copy number...

Neural stem cells exposed to BrdU lose their global DNA methylation and undergo astrocytic differentiation
Schneider, L; di Fagagna, FD
Nucleic Acids Res. (2012) 40 (12): 5332-5342
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Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU) is a halogenated nucleotide of low toxicity commonly used to monitor DNA replication. It is considered a valuable tool for in vitro and in vivo studies, including the detection of the small population of neural stem cells (NSC) in the mammalian brain. Here, we show that NSC grown in self-renewing conditions in vitro, when exposed to BrdU, lose the expression of stem cell markers like Nestin, Sox2 and Pax6 and undergo glial differentiation, strongly up-regulating the astrocytic marker GFAP. The onset of GFAP expression in BrdU exposed NSC was paralleled by a reduced expression of key DNA methyltransferases (DNMT) and a rapid loss of global DNA CpG methylation, as we determined by our specially developed analytic assay. Remarkably, a known DNA demethylating compound, 5-aza-2'-deoxycytidine (Decitabine), had similar effect on demethylation and differentiation of NSC. Since our key findings apply also to NSC derived from murine forebrain, our observations strongly suggest more caution in BrdU uses in stem cells research. We also propose that BrdU and its related substances may also open new opportunities for differentiation therapy in oncology.

Genomic mapping of 5-hydroxymethylcytosine in the human brain
Jin, SG; Wu, XW; Li, AX; Pfeifer, GP
Nucleic Acids Res. (2011) 39 (12): 5015-5024
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Methylation at the 5-position of cytosine is a well-studied epigenetic pathway. In addition to 5-methylcytosine (5mC), substantial amounts of 5-hydroxymethylcytosine (5hmC) also referred to as the sixth DNA base have been detected in certain tissues, most notably the brain. However, the genomic distribution of this cytosine modification is unknown. Here, we have used an immunoprecipitation technique (5hmC-IP) to examine the occurrence of 5hmC in DNA from human brain frontal lobe tissue. The distribution of 5hmC was compared to that of 5mC. We show that 5hmC is more selectively targeted to genes than is 5mC. 5hmC is particularly enriched at promoters and in intragenic regions (gene bodies) but is largely absent from non-gene regions. 5hmC peaks at transcription start sites did not correlate with gene expression levels for promoters with intermediate or high CpG content. However, the presence of 5hmC in gene bodies was more positively correlated with gene expression levels than was the presence of 5mC. Promoters of testis-specific genes showed strong 5mC peaks in brain DNA but were almost completely devoid of 5hmC. Our data provide an overview of the genomic distribution of 5hmC in human brain...

Fine tuning of RFX/DAF-19-regulated target gene expression through binding to multiple sites in Caenorhabditis elegans
Chu, JSC; Tarailo-Graovac, M; Zhang, D; Wang, J; Uyar, B; Tu, D; Trinh, J; Baillie, DL; Chen, NS
Nucleic Acids Res. (2012) 40 (1): 53-64
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In humans, mutations of a growing list of regulatory factor X (RFX) target genes have been associated with devastating genetics disease conditions including ciliopathies. However, mechanisms underlying RFX transcription factors (TFs)-mediated gene expression regulation, especially differential gene expression regulation, are largely unknown. In this study, we explore the functional significance of the co-existence of multiple X-box motifs in regulating differential gene expression in Caenorhabditis elegans. We hypothesize that the effect of multiple X-box motifs is not a simple summation of binding effect to individual X-box motifs located within a same gene. To test this hypothesis, we identified eight C. elegans genes that contain two or more X-box motifs using comparative genomics. We examined one of these genes, F25B4.2, which contains two 15-bp X-box motifs. F25B4.2 expression in ciliated neurons is driven by the proximal motif and its expression is repressed by the distal motif. Our data suggest that two X-box motifs cooperate together to regulate the expression of F25B4.2 in location and intensity...

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