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Epigenetics News

September 2015

Active promoters give rise to false positive 'Phantom Peaks' in ChIP-seq experiments
In this issue of Nucleic Acid Research, researchers in the Becker Lab identify a series of “phantom peaks” that commonly appear in ChIP-Seq experiments including modENCODE ChIP-Seq profiles. According to the authors, “phantom peaks” are false positive signals, most likely caused by non-specific immunoprecipitation. The authors also suggest that these profiles and other ChIP-Seq data featuring prominent "phantom peaks", must be validated with chromatin from cells in which the protein of interest has been depleted.
Becker et al. (2015) Nucleic Acids Res. doi:10.1093/nar/gkv637
 
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MicroRNA-224 promotes tumor progression in nonsmall cell lung cancer
In this study, Ri Cui and his collaborators explore the functional link between miR-224 and tumor progression in nonsmall cell lung cancer (NSCLC). Despite recent advancements, the survival rate of lung cancer patients remains extremely poor and the molecular mechanism of NSCLC invasion remains poorly understood. The results of this study indicate that miR-224 promotes cellular migratory, invasive, and proliferative capacity and tumor growth both in vitro and in vivo. Additionally, the authors identify TNFα-induced protein 1 and SMAD4 as targets of miR-224 and suggest that targeting miR-224 might be a promising therapeutic strategy in the treatment of NSCLC.
Croce et al. (2015) PNAS. doi:10.1073/pnas.1502068112

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A CTCF Code for 3D Genome Architecture
The architectural protein CTCF, as part of the cohesion complex, plays an important role in higher order chromosomal organization by contributing to the formation of loops, especially between distal enhancers and target promoters. In this current paper, the authors emphasize the importance of the orientation of CTCF binding sites (CBS) in 3D genome architecture and propose a model for orientation-specific loop formation.
Corces, V. G., & M.H. Nichols (2015) Cell. doi:10.1016/j.cell.2015.07.053

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August 2015

Single-cell chromatin accessibility mapping by scATAC-seq
In this recent Nature paper, Will Greenleaf and his colleagues at Stanford detail the single-cell ATAC-seq (scATAC-seq) method, which they‘ve recently developed for mapping the accessible genome of individual cells. By integrating an assay for transposase-accessible chromatin using sequencing (ATAC-seq) with a programmable microfluidics platform, Greenleaf and his colleagues have been able to gather single-cell ATAC-seq (scATAC-seq) maps from hundreds of single cells and assemble an aggregate of these accessibility profiles. The results of which provide new insight into the cellular variation of the ‘regulome’.
Greenleaf et al. (2015) Nature. 10.1038/nature14590.
Abstract.
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Putting Non-coding RNA on Display with CRISPR
In this recent issue of Nature Methods, Shechner et al. reported the development of CRISPR Display (CRISP-Disp), which is a sophisticated, flexible, modular, and multiplexable platform for targeting different types of non-coding RNAs (ncRNAs) to genomic loci. CRISP-Disp will facilitate synthetic-biology applications and enable the elucidation of ncRNA functions.
Rinn et al. (2015) Nature Methods. 10.1038/nmeth.3433.
Abstract.
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5hmdC and 5fdC recycling as a new therapeutic target in cancer
In this recent paper, Kriaucionis et al. set out to understand what happens to 5-hydroxymethyldeoxycytidine (5hmdC) and 5-formyldeoxycytidine (5fdC) during nucleotide recycling. The results indicate that although such nucleotides are normally discarded, their recycling and incorporation into DNA in certain cancers constitutes a new therapeutically-exploitable avenue for cancer research.
Kriaucionis et al. (2015) Nature. doi: 10.1038/nature14948
Abstract.
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July 2015

Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells
Forward genetic screens in Drosophila melanogaster for modifiers of position-effect variegation have revealed the basis of much of our understanding of heterochromatin. We took an analogous approach to identify genes required for epigenetic repression in human cells. A nonlethal forward genetic screen in near-haploid KBM7 cells identified the HUSH (human silencing hub) complex, comprising three poorly characterized proteins, TASOR, MPP8, and periphilin; this complex is absent from Drosophila but is conserved from fish to humans. Loss of HUSH components resulted in decreased H3K9me3 both at endogenous genomic loci and at retroviruses integrated into heterochromatin. Our results suggest that the HUSH complex is recruited to genomic loci rich in H3K9me3, where subsequent recruitment of the methyltransferase SETDB1 is required for further H3K9me3 deposition to maintain transcriptional silencing.
Tchasovnikarova et al. (2015) Science. doi: 10.1126/science.aaa7227
Abstract.
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Multiplex single-cell profiling of chromatin accessibility by combinatorial cellular indexing
Technical advances have enabled the collection of genome and transcriptome data sets with single-cell resolution. However, single-cell characterization of the epigenome has remained challenging. Furthermore, because cells must be physically separated before biochemical processing, conventional single-cell preparatory methods scale linearly. We applied combinatorial cellular indexing to measure chromatin accessibility in thousands of single cells per assay, circumventing the need for compartmentalization of individual cells. We report chromatin accessibility profiles from more than 15,000 single cells and use these data to cluster cells on the basis of chromatin accessibility landscapes. We identify modules of coordinately regulated chromatin accessibility at the level of single cells both between and within cell types, with a scalable method that may accelerate progress toward a human cell atlas.
Cusanovich et al. (2015) Science. 10.1126/science.aab1601
Abstract.
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Mass Spectrometric Quantification of Histone Post-translational Modifications by a Hybrid Chemical Labeling Method
Mass spectrometry is a powerful alternative to antibody-based methods for the analysis of histone post-translational modifications (marks). A key development in this approach was the deliberate propionylation of histones to improve sequence coverage across the lysine-rich and hydrophilic tails that bear most modifications. Several marks continue to be problematic however, particularly di- and tri-methylated lysine 4 of histone H3 which we found to be subject to substantial and selective losses during sample preparation and liquid chromatography-mass spectrometry. We developed a new method employing a “one-pot” hybrid chemical derivatization of histones, whereby an initial conversion of free lysines to their propionylated forms under mild aqueous conditions is followed by trypsin digestion and labeling of new peptide N termini with phenyl isocyanate. High resolution mass spectrometry was used to collect qualitative and quantitative data, and a novel web-based software application (Fishtones) was developed for viewing and quantifying histone marks in the resulting data sets. Recoveries of 53 methyl, acetyl, and phosphoryl marks on histone H3.1 were improved by an average of threefold overall, and over 50-fold for H3K4 di- and tri-methyl marks. The power of this workflow for epigenetic research and drug discovery was demonstrated by measuring quantitative changes in H3K4 trimethylation induced by small molecule inhibitors of lysine demethylases and siRNA knockdown of epigenetic modifiers ASH2L and WDR5.
Maile et al. (2015) Molecular and Cellular Proteomics. 10.1074/mcp.O114.046573
Abstract.
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June 2015

DNA Methylation on N6-Adenine in C. elegans
In mammalian cells, DNA methylation on the fifth position of cytosine (5mC) plays an important role as an epigenetic mark. However, DNA methylation was considered to be absent in C. elegans because of the lack of detectable 5mC, as well as homologs of the cytosine DNA methyltransferases. Here, using multiple approaches, we demonstrate the presence of adenine N6-methylation (6mA) in C. elegans DNA. We further demonstrate that this modification increases trans-generationally in a paradigm of epigenetic inheritance. Importantly, we identify a DNA demethylase, NMAD-1, and a potential DNA methyltransferase, DAMT-1, which regulate 6mA levels and crosstalk between methylations of histone H3K4 and adenines and control the epigenetic inheritance of phenotypes associated with the loss of the H3K4me2 demethylase spr-5. Together, these data identify a DNA modification in C. elegans and raise the exciting possibility that 6mA may be a carrier of heritable epigenetic information in eukaryotes.
Greer et al. (2015) Cell. doi:10.1016/j.cell.2015.04.005
Abstract.
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Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells
DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Here we inactivated all three catalytically active DNA methyltransferases (DNMTs) in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing to further investigate the roles and genomic targets of these enzymes. Disruption of DNMT3A or DNMT3B individually as well as of both enzymes in tandem results in viable, pluripotent cell lines with distinct effects on the DNA methylation landscape, as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to findings in mouse, deletion of DNMT1 resulted in rapid cell death in human ESCs. To overcome this immediate lethality, we generated a doxycycline-responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1-mutant lines. However, doxycycline-mediated repression of exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive cell death. Our data provide a comprehensive characterization of DNMT-mutant ESCs, including single-base genome-wide maps of the targets of these enzymes.
Liao et al. (2015) Nature Genetics. doi:10.1038/ng.3258
Abstract.
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Experience-dependent DNA methylation regulates plasticity in the developing visual cortex
DNA methylation is an epigenetic repressor mark for transcription dynamically regulated in neurons. We analyzed visual experience regulation of DNA methylation in mice and its involvement in ocular dominance plasticity of the developing visual cortex. Monocular deprivation modulated the expression of factors controlling DNA methylation and exerted opposite effects on DNA methylation and hydroxymethylation in specific plasticity genes. Inhibition of DNA methyltrasferase (DNMT) blocked molecular and functional effects of monocular deprivation, partially reversing the monocular deprivation transcriptional program.
Tognini et al. (2015) Nature. doi:10.1038/nn.4026
Abstract.
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May 2015

Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation.
DNA methylation is an epigenetic modification associated with transcriptional repression of promoters and is essential for mammalian development. Establishment of DNA methylation is mediated by the de novo DNA methyltransferases DNMT3A and DNMT3B, whereas DNMT1 ensures maintenance of methylation through replication. Absence of these enzymes is lethal, and somatic mutations in these genes have been associated with several human diseases. How genomic DNA methylation patterns are regulated remains poorly understood, as the mechanisms that guide recruitment and activity of DNMTs in vivo are largely unknown. To gain insights into this matter we determined genomic binding and site-specific activity of the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B. We show that both enzymes localize to methylated, CpG-dense regions in mouse stem cells, yet are excluded from active promoters and enhancers. By specifically measuring sites of de novo methylation, we observe that enzymatic activity reflects binding. De novo methylation increases with CpG density, yet is excluded from nucleosomes. Notably, we observed selective binding of DNMT3B to the bodies of transcribed genes, which leads to their preferential methylation. This targeting to transcribed sequences requires SETD2-mediated methylation of lysine 36 on histone H3 and a functional PWWP domain of DNMT3B. Together these findings reveal how sequence and chromatin cues guide de novo methyltransferase activity to ensure methylome integrity.
Baubec et al. (2015) Nature. doi:10.1038/nature14176
Abstract.
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Single-Cell DNA Methylome Sequencing and Bioinformatic Inference of Epigenomic Cell-State Dynamics
Methods for single-cell genome and transcriptome sequencing have contributed to our understanding of cellular heterogeneity, whereas methods for single-cell epigenomics are much less established. Here, we describe a whole-genome bisulfite sequencing (WGBS) assay that enables DNA methylation mapping in very small cell populations (μWGBS) and single cells (scWGBS). Our assay is optimized for profiling many samples at low coverage, and we describe a bioinformatic method that analyzes collections of single-cell methylomes to infer cell-state dynamics. Using these technological advances, we studied epigenomic cell-state dynamics in three in vitro models of cellular differentiation and pluripotency, where we observed characteristic patterns of epigenome remodeling and cell-to-cell heterogeneity. The described method enables single-cell analysis of DNA methylation in a broad range of biological systems, including embryonic development, stem cell differentiation, and cancer. It can also be used to establish composite methylomes that account for cell-to-cell heterogeneity in complex tissue samples.
Farlik et al. (2015) Cell Reports. doi:10.1016/j.celrep.2015.02.001
Abstract.
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A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depends on histone recycling in transcribed chromatin
Nucleosome composition actively contributes to chromatin structure and accessibility. Cells have developed mechanisms to remove or recycle histones, generating a landscape of differentially aged nucleosomes. This study aimed to create a high-resolution genome-wide map of nucleosome turnover in Schizosaccharomyces pombe. The recombination-induced tag exchange (RITE) method was used to study replication-independent nucleosome turnover through the appearance of new histone H3 and the disappearance or preservation of old histone H3. The genome-wide location of histones was determined by chromatin immunoprecipitation-exonuclease methodology (ChIP-exo). The findings were compared with diverse chromatin marks, including histone variant H2A.Z, post-translational histone modifications, and Pol II binding. Finally genome-wide mapping of the methylation states of H4K20 was performed to determine the relationship between methylation (mono, di and tri) of this residue and nucleosome turnover. Our analysis showed that histone recycling resulted in low nucleosome turnover in the coding regions of active genes, stably expressed at intermediate levels. High levels of transcription resulted in the incorporation of new histones primarily at the end of transcribed units. H4K20 was methylated in low-turnover nucleosomes in euchromatic regions, notably in the coding regions of long genes that were expressed at low levels. This transcription-dependent accumulation of histone methylation was dependent on the histone chaperone complex FACT. Our data showed that nucleosome turnover is highly dynamic in the genome and several mechanisms are at play to either maintain or suppress stability. In particular, we found that FACT-associated transcription conserves histones by recycling them and is required for progressive H4K20 methylation.
Svensson et al. (2015) Genome Research. doi:10.1101/gr.188870.114
Abstract.
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April 2015

Disruption of DNA-methylation-dependent long gene repression in Rett syndrome
Disruption of the MECP2 gene leads to Rett syndrome (RTT), a severe neurological disorder with features of autism. MECP2 encodes a methyl-DNA-binding protein that has been proposed to function as a transcriptional repressor, but despite numerous mouse studies examining neuronal gene expression in Mecp2 mutants, no clear model has emerged for how MeCP2 protein regulates transcription. Here we identify a genome-wide length-dependent increase in gene expression in MeCP2 mutant mouse models and human RTT brains. We present evidence that MeCP2 represses gene expression by binding to methylated CA sites within long genes, and that in neurons lacking MeCP2, decreasing the expression of long genes attenuates RTT-associated cellular deficits. In addition, we find that long genes as a population are enriched for neuronal functions and selectively expressed in the brain. These findings suggest that mutations in MeCP2 may cause neurological dysfunction by specifically disrupting long gene expression in the brain.
Gabel et al. (2015) Nature. doi:10.1038/nature14319
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m6A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation
Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N6-methyladenosine (m6A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout preimplantation epiblasts and naïve embryonic stem cells are depleted for m6A in mRNAs, yet are viable. However, they fail to adequately terminate their naïve state and, subsequently, undergo aberrant and restricted lineage priming at the postimplantation stage, which leads to early embryonic lethality. m6A predominantly and directly reduces mRNA stability, including that of key naïve pluripotency-promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo and identifies regulatory modules that functionally influence naïve and primed pluripotency in an opposing manner.
Geula et al. (2015) Science. doi:10.1126/science.1261417
Abstract.
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Jarid2 Methylation via the PRC2 Complex Regulates H3K27me3 Deposition during Cell Differentiation
Polycomb Group (PcG) proteins maintain transcriptional repression throughout development, mostly by regulating chromatin structure. Polycomb Repressive Complex 2 (PRC2), a component of the Polycomb machinery, is responsible for the methylation of histone H3 lysine 27 (H3K27me2/3). Jarid2 was previously identified as a cofactor of PRC2, regulating PRC2 targeting to chromatin and its enzymatic activity. Deletion of Jarid2 leads to impaired orchestration of gene expression during cell lineage commitment. Here, we reveal an unexpected crosstalk between Jarid2 and PRC2, with Jarid2 being methylated by PRC2. This modification is recognized by the Eed core component of PRC2 and triggers an allosteric activation of PRC2’s enzymatic activity. We show that Jarid2 methylation is important to promote PRC2 activity at a locus devoid of H3K27me3 and for the correct deposition of this mark during cell differentiation. Our results uncover a regulation loop where Jarid2 methylation fine-tunes PRC2 activity depending on the chromatin context.
Sanulli et al. (2015) Molecular Cell. doi:10.1016/j.molcel.2014.12.020
Abstract.
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March 2015

Integrative analysis of 111 reference human epigenomes
The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.
Kundaje et al. (2015) Nature. doi:10.1038/nature14248
Abstract.
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Chromatin architecture reorganization during stem cell differentiation
Higher-order chromatin structure is emerging as an important regulator of gene expression. Although dynamic chromatin structures have been identified in the genome, the full scope of chromatin dynamics during mammalian development and lineage specification remains to be determined. By mapping genome-wide chromatin interactions in human embryonic stem (ES) cells and four human ES-cell-derived lineages, we uncover extensive chromatin reorganization during lineage specification. We observe that although self-associating chromatin domains are stable during differentiation, chromatin interactions both within and between domains change in a striking manner, altering 36% of active and inactive chromosomal compartments throughout the genome. By integrating chromatin interaction maps with haplotype-resolved epigenome and transcriptome data sets, we find widespread allelic bias in gene expression correlated with allele-biased chromatin states of linked promoters and distal enhancers. Our results therefore provide a global view of chromatin dynamics and a resource for studying long-range control of gene expression in distinct human cell lineages.
Dixon et al. (2015) Nature. doi:10.1038/nature14222
Abstract.
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Cell-of-origin chromatin organization shapes the mutational landscape of cancer
Cancer is a disease potentiated by mutations in somatic cells. Cancer mutations are not distributed uniformly along the human genome. Instead, different human genomic regions vary by up to fivefold in the local density of cancer somatic mutations, posing a fundamental problem for statistical methods used in cancer genomics. Epigenomic organization has been proposed as a major determinant of the cancer mutational landscape. However, both somatic mutagenesis and epigenomic features are highly cell-type-specific. We investigated the distribution of mutations in multiple independent samples of diverse cancer types and compared them to cell-type-specific epigenomic features. Here we show that chromatin accessibility and modification, together with replication timing, explain up to 86% of the variance in mutation rates along cancer genomes. The best predictors of local somatic mutation density are epigenomic features derived from the most likely cell type of origin of the corresponding malignancy. Moreover, we find that cell-of-origin chromatin features are much stronger determinants of cancer mutation profiles than chromatin features of matched cancer cell lines. Furthermore, we show that the cell type of origin of a cancer can be accurately determined based on the distribution of mutations along its genome. Thus, the DNA sequence of a cancer genome encompasses a wealth of information about the identity and epigenomic features of its cell of origin.
Polak et al. (2015) Nature. doi:10.1038/nature14221
Abstract.
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