Epigenetics News

Single-Molecule Analysis of Combinatorial Epigenomic States in Normal and Tumor Cells
Proper placement of epigenetic marks on DNA and histones is fundamental to normal development, and perturbations contribute to a variety of disease states. Combinations of marks act together to control gene expression; therefore, detecting their colocalization is important, but because of technical challenges, such measurements are rarely reported. Instead, measurements of epigenetic marks are typically performed one at a time in a population of cells, and their colocalization is inferred by association. Here, researchers describe a single-molecule analytical approach that can perform direct detection of multiple epigenetic marks simultaneously and use it to identify mechanisms coordinating placement of three gene silencing marks, trimethylated histone H3 lysine 9, lysine 27 (H3K9me3, H3K27me3), and cytosine methylation (mC), in the normal and cancer genome. They show that H3K9me3 and mC are present together on individual chromatin fragments in mouse embryonic stem cells and that half of the H3K9me3 marks require mC for their placement. In contrast, mC and H3K27me3 coincidence is rare, and in fact, mC antagonizes H3K27me3 in both embryonic stem cells and primary mouse fibroblasts, indicating this antagonism is shared among primary cells. However, upon immortalization or tumorigenic transformation of mouse fibroblasts, mC is required for complete H3K27me3 placement. Importantly, in human promyelocytic cells, H3K27me3 is also dependent on mC. Because aberrant placement of gene silencing marks at tumor suppressor genes contributes to tumor progression, the improper dependency of H3K27me3 by mC in immortalized cells is likely to be fundamental to cancer. Their platform can enable other studies involving coordination of epigenetic marks and leverage efforts to discover disease biomarkers and epigenome-modifying drugs.
Murphy et al. (2013) PNAS. doi:10.1073/pnas.1218495110

Abstract.

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Inhibition of PRC2 Activity by a Gain-of-Function H3 Mutation Found in Pediatric Glioblastoma
Sequencing of pediatric gliomas has identified missense mutations Lys27Met (K27M) and Gly34Arg/Val (G34R/V) in genes encoding histone H3.3 (H3F3A) and H3.1 (HIST3H1B). Researchers report that human diffuse intrinsic pontine gliomas (DIPGs) containing the K27M mutation display significantly lower overall H3K27me3 levels, and that histone H3K27M transgenes are sufficient to reduce H3K27me3 levels in vitro and in vivo. They find that H3K27M inhibits the enzymatic activity of the Polycomb repressive complex 2 (PRC2) through interaction with the EZH2 subunit. Additionally, transgenes containing lysine-to-methionine substitutions at other known methylated lysines (H3K9 and H3K36) are sufficient to cause specific reduction in methylation levels through inhibition of SET-domain enzymes. They propose that K-to-M substitutions may represent a mechanism to alter epigenetic states in a variety of pathologies.
Lewis et al. (2013) Science. doi:10.1126/science.1232245
Abstract.
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The Histone H3.3K27M Mutation in Pediatric Glioma Reprograms H3K27 Methylation and Gene Expression
Recent studies have identified a Lys 27-to-methionine (K27M) mutation at one allele of H3F3A, one of the two genes encoding histone H3 variant H3.3, in 60% of high-grade pediatric glioma cases. The median survival of this group of patients after diagnosis is ∼1 yr. Here, researchers show that the levels of H3K27 di- and trimethylation (H3K27me2 and H3K27me3) are reduced globally in H3.3K27M patient samples due to the expression of the H3.3K27M mutant allele. Remarkably, they also observed that H3K27me3 and Ezh2 (the catalytic subunit of H3K27 methyltransferase) at chromatin are dramatically increased locally at hundreds of gene loci in H3.3K27M patient cells. Moreover, the gain of H3K27me3 and EZH2 at gene promoters alters the expression of genes that are associated with various cancer pathways. These results indicate that H3.3K27M mutation reprograms epigenetic landscape and gene expression, which may drive tumorigenesis.
Chan et al. (2013) Genes & Dev. doi:10.1101/gad.217778.113
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Epigenetic Protein Families: A New Frontier for Drug Discovery
Epigenetic regulation of gene expression is a dynamic and reversible process that establishes normal cellular phenotypes but also contributes to human diseases. At the molecular level, epigenetic regulation involves hierarchical covalent modification of DNA and the proteins that package DNA, such as histones. Here, researchers review the key protein families that mediate epigenetic signaling through the acetylation and methylation of histones, including histone deacetylases, protein methyltransferases, lysine demethylases, bromodomain-containing proteins and proteins that bind to methylated histones. These protein families are emerging as druggable classes of enzymes and druggable classes of protein–protein interaction domains. In this article, they discuss the known links with disease, basic molecular mechanisms of action and recent progress in the pharmacological modulation of each class of proteins.
Arrowsmith et al. (2012) Nature Reviews Drug Discovery. doi:10.1038/nrd3674

Abstract.
Time magazine article can be found here.

Phospho Switch Triggers Brd4 Chromatin Binding and Activator Recruitment for Gene-Specific Targeting
Bromodomain-containing protein 4 (Brd4) is an epigenetic reader and transcriptional regulator recently identified as a cancer therapeutic target for acute myeloid leukemia, multiple myeloma, and Burkitt’s lymphoma. Although chromatin targeting is a crucial function of Brd4, there is little understanding of how bromodomains that bind acetylated histones are regulated, nor how the gene-specific activity of Brd4 is determined. Via interaction screen and domain mapping, researchers identified p53 as a functional partner of Brd4. Interestingly, Brd4 association with p53 is modulated by casein kinase II (CK2)-mediated phosphorylation of a conserved acidic region in Brd4 that selectively contacts either a juxtaposed bromodomain or an adjacent basic region to dictate the ability of Brd4 binding to chromatin and also the recruitment of p53 to regulated promoters. The unmasking of bromodomains and activator recruitment, concurrently triggered by the CK2 phospho switch, provide an intriguing mechanism for gene-specific targeting by a universal epigenetic reader.
Wu et al. (2013) Molecular Cell. doi:10.1016/j.molcel.2012.12.006.
Abstract.
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Targeting MYCN in Neuroblastoma by BET Bromodomain Inhibition
Bromodomain inhibition comprises a promising therapeutic strategy in cancer, particularly for hematologic malignancies. To date, however, genomic biomarkers to direct clinical translation have been lacking. Researchers conducted a cell-based screen of genetically defined cancer cell lines using a prototypical inhibitor of BET bromodomains. Integration of genetic features with chemosensitivity data revealed a robust correlation between MYCN amplification and sensitivity to bromodomain inhibition. They characterized the mechanistic and translational significance of this finding in neuroblastoma, a childhood cancer with frequent amplification of MYCN. Genome-wide expression analysis showed down regulation of the MYCN transcriptional program accompanied by suppression of MYCN transcription. Functionally, bromodomain-mediated inhibition of MYCN impaired growth and induced apoptosis in neuroblastoma. BRD4 knockdown phenocopied these effects, establishing BET bromodomains as transcriptional regulators of MYCN. BET inhibition conferred a significant survival advantage in 3 in vivo neuroblastoma models, providing a compelling rationale for developing BET bromodomain inhibitors in patients with neuroblastoma.
Puissant et al. (2013) Cancer Discovery. doi: 10.1158/2159-8290.CD-12-0418.
Abstract.
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Epigenetic Regulation by Long Noncoding RNAs
Recent studies show that transcription of the mammalian genome is not only pervasive but also enormously complex. It is estimated that an average of 10 transcription units, the vast majority of which make long noncoding RNAs (lncRNAs), may overlap each traditional coding gene. These lncRNAs include not only antisense, intronic, and intergenic transcripts but also pseudogenes and retrotransposons. Do they universally have function, or are they merely transcriptional by-products of conventional coding genes? A glimpse into the molecular biology of multiple emerging lncRNA systems reveals the “Wild West” landscape of their functions and mechanisms and the key problems to solve in the years ahead toward understanding these intriguing macromolecules.
Lee et al. (2012) Science. doi:10.1126/science.1231776

Abstract.
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Activating RNAs Associate with Mediator to Enhance Chromatin Architecture and Transcription
Recent advances in genomic research have revealed the existence of a large number of transcripts devoid of protein-coding potential in multiple organisms. Although the functional role for long non-coding RNAs (lncRNAs) has been best defined in epigenetic phenomena such as X-chromosome inactivation and imprinting, different classes of lncRNAs may have varied biological functions. Researchers have identified a class of lncRNAs, termed ncRNA-activating (ncRNA-a), that function to activate their neighbouring genes using a cis-mediated mechanism. To define the precise mode by which such enhancer-like RNAs function, they depleted factors with known roles in transcriptional activation and assessed their role in RNA-dependent activation. Here we report that depletion of the components of the co-activator complex, Mediator, specifically and potently diminished the ncRNA-induced activation of transcription in a heterologous reporter assay using human HEK293 cells. In vivo, Mediator is recruited to ncRNA-a target genes and regulates their expression. They show that ncRNA-a interact with Mediator to regulate its chromatin localization and kinase activity towards histone H3 serine 10. The Mediator complex harbouring disease displays diminished ability to associate with activating ncRNAs. Chromosome conformation capture confirmed the presence of DNA looping between the ncRNA-a loci and its targets. Importantly, depletion of Mediator subunits or ncRNA-a reduced the chromatin looping between the two loci. Their results identify the human Mediator complex as the transducer of activating ncRNAs and highlight the importance of Mediator and activating ncRNA association in human disease.
Lai et al. (2012) Nature. doi:10.1038/nature11884.
Abstract.
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The NeST Long ncRNA Controls Microbial Susceptibility and Epigenetic Activation of the Interferon-γ Locus
Long noncoding RNAs (lncRNAs) are increasingly appreciated as regulators of cell-specific gene expression. Here, an enhancer-like lncRNA termed NeST is shown to be causal for all phenotypes conferred by murine viral susceptibility locus Tmevp3. This locus was defined by crosses between SJL/J and B10.S mice and contains several candidate genes, including NeST. The SJL/J-derived locus confers higher lncRNA expression, increased interferon-γ (IFN-γ) abundance in activated CD8+ T cells, increased Theiler’s virus persistence, and decreased Salmonella enterica pathogenesis. Transgenic expression of NeST lncRNA alone was sufficient to confer all phenotypes of the SJL/J locus. NeST RNA was found to bind WDR5, a component of the histone H3 lysine 4 methyltransferase complex, and to alter histone 3 methylation at the IFN-γ locus. Thus, this lncRNA regulates epigenetic marking of IFN-γ-encoding chromatin, expression of IFN-γ, and susceptibility to a viral and a bacterial pathogen.
Antonio Gomez et al. (2012) Cell. doi:10.1016/j.cell.2013.01.015.
Abstract.
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Germline DNA Demethylation Dynamics and Imprint Erasure Through 5-Hydroxymethylcytosine
Epigenetic reprogramming, characterized by the loss of CpG methylation and histone modifications, occurs in a sequential manner in primordial germ cells (PGCs) to generate totipotency. As the dynamics and underlying mechanism of this process is poorly characterized, the authors performed a comprehensive analysis of PGCs to address the mechanistic basis of epigenetic reprogramming. They not only demonstrate that the depletion of cytosine methylation (5-mC), including imprint erasure, occurs via the conversion to 5-hydroxymethylcytosine (5-hmC), but also that this process is driven by TET1 and TET2 in mouse PGCs. The rate of progressive decline in 5-hmC was shown to be consistent with a replication-dependent mechanism towards a completely unmodified state. In essence, reprograming in PGCs involves multiple redundant mechanisms to reset the genome for totipotency, emphasizing the comprehensive nature of DNA demethylation in PGCs.
Hacket et al. (2012) Science Published online Dec. 6. Abstract.
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Epigenome-wide Association Data Implicate DNA Methylation as an Intermediary of Genetic Risk in Rheumatoid Arthritis
Epigenetic mechanisms integrate genetic and environmental causes of disease, but comprehensive genome-wide analyses of epigenetic modifications have not yet demonstrated robust association with common diseases. Using Illumina HumanMethylation450 arrays on 354 anti-citrullinated protein antibody–associated rheumatoid arthritis cases and 337 controls, researchers identified two clusters within the major histocompatibility complex (MHC) region whose differential methylation potentially mediates genetic risk for rheumatoid arthritis. To reduce confounding factors that have hampered previous epigenome-wide studies, they corrected for cellular heterogeneity by estimating and adjusting for cell-type proportions in their blood-derived DNA samples and used mediation analysis to filter out associations likely to be a consequence of disease. Four CpGs also showed an association between genotype and variance of methylation. The associations for both clusters replicated at least one CpG (P < 0.01), with the rest showing suggestive association, in monocyte cell fractions in an independent cohort of 12 cases and 12 controls. Thus, DNA methylation is a potential mediator of genetic risk.
Liu et al. (2012) Nature Biotechnology. 26(23):2604-2620.
Abstract.
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Mapping Recently Identified Nucleotide Variants in the Genome and Transcriptome
Nucleotide variants, especially those related to epigenetic functions, provide critical regulatory information beyond simple genomic sequence, and they define cell status in higher organisms. 5-methylcytosine, which is found in DNA, was until recently the only nucleotide variant studied in terms of epigenetics in eukaryotes. However, 5-methylcytosine has turned out to be just one component of a dynamic DNA epigenetic regulatory network that also includes 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. Recently, reversible methylation of N6-methyladenosine in RNA has also been demonstrated. The discovery of these new nucleotide variants triggered an explosion of new information in the epigenetics field. This rapid research progress has benefited significantly from timely developments of new technologies that specifically recognize, enrich and sequence nucleotide modifications, as evidenced by the wide application of the bisulfite sequencing of 5-methylcytosine and very recent modifications of bisulfite sequencing to resolve 5-hydroxymethylcytosine from 5-methylcytosine with base-resolution information.
Song et al. (2012) Nature Biotechnology. 30:1107-1116.
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Carm1 Regulates Pax7 Transcriptional Activity Through MLL1/2 Recruitment During Asymmetric Satellite Stem Cell Divisions
In skeletal muscle, asymmetrically dividing satellite stem cells give rise to committed satellite cells that transcribe the myogenic determination factor Myf5, a Pax7-target gene. We identified the arginine methyltransferase Carm1 as a Pax7 interacting protein and found that Carm1 specifically methylates multiple arginines in the N terminus of Pax7. Methylated Pax7 directly binds the C-terminal cleavage forms of the trithorax proteins MLL1/2 resulting in the recruitment of the ASH2L:MLL1/2:WDR5:RBBP5 histone H3K4 methyltransferase complex to regulatory enhancers and the proximal promoter of Myf5. Finally, Carm1 is required for the induction of de novo Myf5 transcription following asymmetric satellite stem cell divisions. We defined the C-terminal MLL region as a reader domain for the recognition of arginine methylated proteins such as Pax7. Thus, arginine methylation of Pax7 by Carm1 functions as a molecular switch controlling the epigenetic induction of Myf5 during satellite stem cell asymmetric division and entry into the myogenic program.
Kawabe et al. (2012) Cell Stem Cell. 11(3):333-345.
Abstract.
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Enhancer-associated H3K4 Monomethylation by Trithorax-related, the Drosophila Homolog of Mammalian Mll3/Mll4
Monomethylation of histone H3 on Lys 4 (H3K4me1) and acetylation of histone H3 on Lys 27 (H3K27ac) are histone modifications that are highly enriched over the body of actively transcribed genes and on enhancers. Although in yeast all H3K4 methylation patterns, including H3K4me1, are implemented by Set1/COMPASS (complex of proteins associated with Set1), there are three classes of COMPASS-like complexes in Drosophila that could carry out H3K4me1 on enhancers: dSet1, Trithorax, and Trithorax-related (Trr). Here, we report that Trr, the Drosophila homolog of the mammalian Mll3/4 COMPASS-like complexes, can function as a major H3K4 monomethyltransferase on enhancers in vivo. Loss of Trr results in a global decrease of H3K4me1 and H3K27ac levels in various tissues. Assays with the cut wing margin enhancer implied a functional role for Trr in enhancer-mediated processes. A genome-wide analysis demonstrated that Trr is required to maintain the H3K4me1 and H3K27ac chromatin signature that resembles the histone modification patterns described for enhancers. Furthermore, studies in the mammalian system suggested a role for the Trr homolog Mll3 in similar processes. Since Trr and mammalian Mll3/4 complexes are distinguished by bearing a unique subunit, the H3K27 demethylase UTX, we propose a model in which the H3K4 monomethyltransferases Trr/Mll3/Mll4 and the H3K27 demethylase UTX cooperate to regulate the transition from inactive/poised to active enhancers.
Herz et al. (2012) Genes & Development. 26(23):2604-2620.
Abstract.
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The COMPASS Subunit Spp1 Links Histone Methylation to Initiation of Meiotic Recombination
Novel combinatorial associations of genetic traits arise from homologous recombination between parental chromosomes during meiosis. Histone H3 lysine 4 trimethylation marks meiotic recombination hotspots in yeast and mammals, but how this ubiquitous chromatin modification relates to initiation of Spo11-dependent double-strand breaks (DSB) remains unknown. Here, we show that the tethering of the PHD-containing protein, Spp1, a component of the COMPASS complex to recombinationally cold regions is sufficient to induce DSB formation. Furthermore, we found that Spp1 physically interacts with Mer2, a key protein of the differentiated chromosomal axis required for DSB formation. Thus, Spp1, by interacting with H3K4me3 and Mer2, promotes recruitment of potential meiotic DSB sites to the chromosomal axis, allowing Spo11 cleavage at nearby nucleosome-depleted regions.
Acquaviva et al. (2012) Science November 15, E-pub ahead of print.
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Genome-Wide Mapping of Nucleosome Positioning and DNA Methylation within Individual DNA Molecules
DNA methylation and nucleosome positioning work together to generate chromatin structures that regulate gene expression. Nucleosomes are typically mapped using nuclease digestion requiring significant amounts of material and varying enzyme concentrations. Researchers at the University of Southern California have developed a method (NOMe-Seq) that uses a GpC methyltransferase (M.CviPI) and next generation sequencing to generate a high resolution footprint of nucleosome positioning genome-wide using less than 1 million cells while retaining endogenous DNA methylation information from the same DNA strand. Using a novel bioinformatics pipeline, they show a striking anti-correlation between nucleosome occupancy and DNA methylation at CTCF regions that is not present at promoters. They further show that the extent of nucleosome depletion at promoters is directly correlated to expression level and can accommodate multiple nucleosomes and provide genome-wide evidence that expressed non-CpG island promoters are nucleosome-depleted. Importantly, NOMe-Seq obtains DNA methylation and nucleosome positioning information from the same DNA molecule, giving the first genome-wide DNA methylation and nucleosome positioning correlation at the single molecule, and thus, single cell level, that can be used to monitor disease progression and response to therapy.
Kelly et al. (2012) Genome Res. doi:10.1101/gr.143008.112.
Abstract.
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Genome-Wide Nucleosome Positioning During Embryonic Stem Cell Development
Researchers at Deutsches Krebsforschungszentrum determined genome-wide nucleosome occupancies in mouse embryonic stem cells and their neural progenitor and embryonic fibroblast counterparts to assess features associated with nucleosome positioning during lineage commitment. Cell-type- and protein-specific binding preferences of transcription factors to sites with either low (Myc, Klf4 and Zfx) or high (Nanog, Oct4 and Sox2) nucleosome occupancy as well as complex patterns for CTCF were identified. Nucleosome-depleted regions around transcription start and transcription termination sites were broad and more pronounced for active genes, with distinct patterns for promoters classified according to CpG content or histone methylation marks. Throughout the genome, nucleosome occupancy was correlated with certain histone methylation or acetylation modifications. In addition, the average nucleosome repeat length increased during differentiation by 5–7 base pairs, with local variations for specific regions. Their results reveal regulatory mechanisms of cell differentiation that involve nucleosome repositioning.
Teif et al. (2012) Nature Structural & Molecular Biology. doi:10.1038/nsmb.2419.
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Asymmetrically Modified Nucleosomes
Mononucleosomes, the basic building blocks of chromatin, contain two copies of each core histone. The associated post-translational modifications regulate essential chromatin-dependent processes, yet whether each histone copy is identically modified in vivo is unclear. Researchers at New York University School of Medicine demonstrate that nucleosomes in embryonic stem cells, fibroblasts, and cancer cells exist in both symmetrically and asymmetrically modified populations for histone H3 lysine 27 di/trimethylation (H3K27me2/3) and H4K20me1. Further, they obtained direct physical evidence for bivalent nucleosomes carrying H3K4me3 or H3K36me3 along with H3K27me3, albeit on opposite H3 tails. Bivalency at target genes was resolved upon differentiation of ES cells. Polycomb repressive complex 2-mediated methylation of H3K27 was inhibited when nucleosomes contain symmetrically, but not asymmetrically, placed H3K4me3 or H3K36me3. These findings uncover a potential mechanism for the incorporation of bivalent features into nucleosomes and demonstrate how asymmetry might set the stage to diversify functional nucleosome states.
Voigt et al. (2012) Cell. doi:10.1016/j.cell.2012.09.002.
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