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The Polycomb repressive system functions through chromatin to regulate gene expression and development. In this issue of Genes & Development, Cohen and colleagues (pp. 354–366) use the developing mouse epidermis as a model system to show that the two central Polycomb repressive complexes, PRC1 and PRC2, have autonomous yet overlapping functions in repressing Polycomb target genes. They show that this cooperation enables the stable repression of nonepidermal transcription factors that would otherwise compromise epidermal cell identity and disrupt normal skin development.

Histone-modifying systems play fundamental roles in gene regulation and the development of multicellular organisms. Histone modifications that are enriched at gene regulatory elements have been heavily studied, but the function of modifications found more broadly throughout the genome remains poorly understood. This is exemplified by histone H2A monoubiquitylation (H2AK119ub1), which is enriched at Polycomb-repressed gene promoters but also covers the genome at lower levels. Here, using inducible genetic perturbations and quantitative genomics, we found that the BAP1 deubiquitylase plays an essential role in constraining H2AK119ub1 throughout the genome. Removal of BAP1 leads to pervasive genome-wide accumulation of H2AK119ub1, which causes widespread reductions in gene expression. We show that elevated H2AK119ub1 preferentially counteracts Ser5 phosphorylation on the C-terminal domain of RNA polymerase II at gene regulatory elements and causes reductions in transcription and transcription-associated histone modifications. Furthermore, failure to constrain pervasive H2AK119ub1 compromises Polycomb complex occupancy at a subset of Polycomb target genes, which leads to their derepression, providing a potential molecular rationale for why the BAP1 ortholog in Drosophila has been characterized as a Polycomb group gene. Together, these observations reveal that the transcriptional potential of the genome can be modulated by regulating the levels of a pervasive histone modification.

The precise regulation of gene transcription is required to establish and maintain cell type-specific gene expression programs during multicellular development. In addition to transcription factors, chromatin, and its chemical modification, play a central role in regulating gene expression. In vertebrates, DNA is pervasively methylated at CG dinucleotides, a modification that is repressive to transcription. However, approximately 70% of vertebrate gene promoters are associated with DNA elements called CpG islands (CGIs) that are refractory to DNA methylation. CGIs integrate the activity of a range of chromatin-regulating factors that can post-translationally modify histones and modulate gene expression. This is exemplified by the trimethylation of histone H3 at lysine 4 (H3K4me3), which is enriched at CGI-associated gene promoters and correlates with transcriptional activity. Through studying H3K4me3 at CGIs it has become clear that CGIs shape the distribution of H3K4me3 and, in turn, H3K4me3 influences the chromatin landscape at CGIs. Here we will discuss our understanding of the emerging relationship between CGIs, H3K4me3, and gene expression., Highlights • The deposition of H3K4me3 is intrinsically linked to CpG islands via ZF-CxxC domain-containing proteins. • H3K4me3 levels at CpG islands are regulated by transcription and other chromatin features. • H3K4me3 influences chromatin architecture at CpG islands and has been proposed to regulate gene expression. • H3K4me3 may contribute to bistable chromatin states at CpG islands.

Epigenetic modifications on chromatin play important roles in regulating gene expression. While chromatin states are often governed by multi-layered structure, how individual pathways contribute to gene expression remains poorly understood. For example, DNA methylation is known to regulate transcription factor binding but also to recruit methyl-CpG binding proteins that affect chromatin structure through the activity of histone deacetylase complexes (HDACs). Both of these mechanisms can potentially affect gene expression, but the importance of each, and whether these activities are integrated to achieve appropriate gene regulation, remains largely unknown. To address this important question, we measured gene expression, chromatin accessibility, and transcription factor occupancy in wild-type or DNA methylation-deficient mouse embryonic stem cells following HDAC inhibition. Interestingly, we observe widespread increases in chromatin accessibility at repeat elements when HDACs are inhibited, and this is magnified when cells also lack DNA methylation. A subset of these elements have elevated binding of the YY1 and GABPA transcription factors and increased expression. The pronounced additive effect of HDAC inhibition in DNA methylation deficient cells demonstrate that DNA methylation and histone deacetylation act largely independently to suppress transcription factor binding and gene expression.

Pioneer transcription factors recognise and bind their target sequences in inaccessible chromatin to establish new transcriptional networks throughout development and cellular reprogramming. During this process, pioneer factors establish an accessible chromatin state to facilitate additional transcription factor binding, yet it remains unclear how different pioneer factors achieve this. Here, we discover that the pluripotency-associated pioneer factor OCT4 binds chromatin to shape accessibility, transcription factor co-binding, and regulatory element function in mouse embryonic stem cells. Chromatin accessibility at OCT4-bound sites requires the chromatin remodeller BRG1, which is recruited to these sites by OCT4 to support additional transcription factor binding and expression of the pluripotency-associated transcriptome. Furthermore, the requirement for BRG1 in shaping OCT4 binding reflects how these target sites are used during cellular reprogramming and early mouse development. Together this reveals a distinct requirement for a chromatin remodeller in promoting the activity of the pioneer factor OCT4 and regulating the pluripotency network. DOI: http://dx.doi.org/10.7554/eLife.22631.001, eLife digest All cells in your body contain the same genetic information in the form of genes encoded within DNA. Yet, cells use this information in different ways so that the activities of individual genes within that DNA can vary from cell to cell. This allows identical cells to become different to each other and to adapt to changing circumstances. A group of proteins called transcription factors control the activity of certain genes by binding to specific sites on DNA. However, this isn’t a straightforward process because DNA in human and other animal cells is usually associated with structures called nucleosomes that can block access to the DNA. Pioneer transcription factors, such as OCT4, are a specific group of transcription factors that can attach to DNA in spite of the nucleosomes, but it’s not clear how this is possible. Once pioneer transcription factors attach to DNA they can help other transcription factors to bind alongside them. King et al. studied OCT4 in stem cells from mouse embryos to investigate how it is able to act as a pioneer transcription factor and control gene activity. The experiments show that several other transcription factors lose the ability to bind to DNA when OCT4 is absent. This leads to widespread changes in gene activity in the cells, which seems to be due to other transcription factors being unable to get past the nucleosomes to attach to the DNA. Further experiments showed that OCT4 needs a protein called BRG1 in order to act as a pioneer transcription factor. BRG1 is an enzyme that is able to move and remove (remodel) nucleosomes attached to DNA, suggesting that normal transcription factor binding requires this activity. The next challenge is to investigate whether BRG1, or similar enzymes, are also needed by other pioneer transcription factors that are required for normal gene activity and cell identity. This will be important because many enzymes that remodel nucleosomes are disrupted in human diseases like cancer where cells lose their normal identity. DOI: http://dx.doi.org/10.7554/eLife.22631.002

Polycomb group (PcG) proteins function as chromatin-based transcriptional repressors that are essential for normal gene regulation during development. However, how these systems function to achieve transcriptional regulation remains very poorly understood. Here, we discover that the histone H2AK119 E3 ubiquitin ligase activity of Polycomb repressive complex 1 (PRC1) is defined by the composition of its catalytic subunits and is highly regulated by RYBP/YAF2-dependent stimulation. In mouse embryonic stem cells, RYBP plays a central role in shaping H2AK119 mono-ubiquitylation at PcG targets and underpins an activity-based communication between PRC1 and Polycomb repressive complex 2 (PRC2) which is required for normal histone H3 lysine 27 trimethylation (H3K27me3). Without normal histone modification-dependent communication between PRC1 and PRC2, repressive Polycomb chromatin domains can erode, rendering target genes susceptible to inappropriate gene expression signals. This suggests that activity-based communication and histone modification-dependent thresholds create a localized form of epigenetic memory required for normal PcG chromatin domain function in gene regulation. DOI: http://dx.doi.org/10.7554/eLife.18591.001

Most human aneuploidies originate maternally, due in part to the presence of highly stringent checkpoints during male meiosis. Indeed, male sterility is common among aneuploid mice used to study chromosomal abnormalities, and male germline transmission of exogenous DNA has been rarely reported. Here we show that, despite aberrant testis architecture, males of the aneuploid Tc1 mouse strain produce viable sperm and transmit human chromosome 21 to create aneuploid offspring. In these offspring, we mapped transcription, transcriptional initiation, enhancer activity, non-methylated DNA, and transcription factor binding in adult tissues. Remarkably, when compared with mice derived from female passage of human chromosome 21, the chromatin condensation during spermatogenesis and the extensive epigenetic reprogramming specific to male germline transmission resulted in almost indistinguishable patterns of transcriptional deployment. Our results reveal an unexpected tolerance of aneuploidy during mammalian spermatogenesis, and the surprisingly robust ability of mouse developmental machinery to accurately deploy an exogenous chromosome, regardless of germline transmission. DOI: http://dx.doi.org/10.7554/eLife.20235.001

Mutations of human PHF8 cluster within its JmjC encoding exons and are linked to mental retardation (MR) and a cleft lip/palate phenotype. Sequence comparisons, employing structural insights, suggest that PHF8 contains the double stranded beta-helix fold and ferrous iron binding residues that are present in 2-oxoglutarate-dependent oxygenases. We report that recombinant PHF8 is an Fe(II) and 2-oxoglutarate-dependent N(epsilon)-methyl lysine demethylase, which acts on histone substrates. PHF8 is selective in vitro for N(epsilon)-di- and mono-methylated lysine residues and does not accept trimethyl substrates. Clinically observed mutations to the PHF8 gene cluster in exons encoding for the double stranded beta-helix fold and will therefore disrupt catalytic activity. The PHF8 missense mutation c.836C>T is associated with mild MR, mild dysmorphic features, and either unilateral or bilateral cleft lip and cleft palate in two male siblings. This mutant encodes a F279S variant of PHF8 that modifies a conserved hydrophobic region; assays with both peptides and intact histones reveal this variant to be catalytically inactive. The dependence of PHF8 activity on oxygen availability is interesting because the occurrence of fetal cleft lip has been demonstrated to increase with maternal hypoxia in mouse studies. Cleft lip and other congenital anomalies are also linked indirectly to maternal hypoxia in humans, including from maternal smoking and maternal anti-hypertensive treatment. Our results will enable further studies aimed at defining the molecular links between developmental changes in histone methylation status, congenital disorders and MR.

Methylation of DNA at position five of the cytosine ring occurs at most CpG dinucleotides in the mammalian genome and is essential for embryonic viability. With several of the key proteins now known, it has become possible to approach the biological significance of this epigenetic system through both biochemistry and genetics. As a result, advances have been made in our understanding of the mechanisms by which DNA methylation is targeted to specific regions of the genome and interpreted by methyl-CpG-binding proteins. Recent studies have illuminated the role of DNA methylation in controlling gene expression and have strengthened its links with histone modification and chromatin remodelling.

Histone methylation has important roles in regulating gene expression and forms part of the epigenetic memory system that regulates cell fate and identity. Enzymes that directly remove methyl marks from histones have recently been identified, revealing a new level of plasticity within this epigenetic modification system. Here we analyse the evolutionary relationship between Jumonji C (JmjC)-domain-containing proteins and discuss their cellular functions in relation to their potential enzymatic activities.

Post-translational modification of chromatin is emerging as an increasingly important regulator of chromosomal processes. In particular, histone lysine and arginine methylation play important roles in regulating transcription, maintaining genomic integrity, and contributing to epigenetic memory. Recently, the use of new approaches to analyse histone methylation, the generation of genetic model systems, and the ability to interrogate genome wide histone modification profiles has aided in defining how histone methylation contributes to these processes. Here we focus on the recent advances in our understanding of the histone methylation system and examine how dynamic histone methylation contributes to normal cellular function in mammals.

Vertebrate DNA can be chemically modified by methylation of the 5 position of the cytosine base in the context of CpG dinucleotides. This modification creates a binding site for MBD (methyl-CpG-binding domain) proteins which target chromatin-modifying activities that are thought to contribute to transcriptional repression and maintain heterochromatic regions of the genome. In contrast with DNA methylation, which is found broadly across vertebrate genomes, non-methylated DNA is concentrated in regions known as CGIs (CpG islands). Recently, a family of proteins which encode a ZF-CxxC (zinc finger-CxxC) domain have been shown to specifically recognize non-methylated DNA and recruit chromatin-modifying activities to CGI elements. For example, CFP1 (CxxC finger protein 1), MLL (mixed lineage leukaemia protein), KDM (lysine demethylase) 2A and KDM2B regulate lysine methylation on histone tails, whereas TET (ten-eleven translocation) 1 and TET3 hydroxylate methylated cytosine bases. In the present review, we discuss the most recent advances in our understanding of how ZF-CxxC domain-containing proteins recognize non-methylated DNA and describe their role in chromatin modification at CGIs. © 2013 The Author(s).

Summary In higher eukaryotes, up to 70% of genes have high levels of nonmethylated cytosine/guanine base pairs (CpGs) surrounding promoters and gene regulatory units. These features, called CpG islands, were identified over 20 years ago, but there remains little mechanistic evidence to suggest how these enigmatic elements contribute to promoter function, except that they are refractory to epigenetic silencing by DNA methylation. Here we show that CpG islands directly recruit the H3K36-specific lysine demethylase enzyme KDM2A. Nucleation of KDM2A at these elements results in removal of H3K36 methylation, creating CpG island chromatin that is uniquely depleted of this modification. KDM2A utilizes a zinc finger CxxC (ZF-CxxC) domain that preferentially recognizes nonmethylated CpG DNA, and binding is blocked when the CpG DNA is methylated, thus constraining KDM2A to nonmethylated CpG islands. These data expose a straightforward mechanism through which KDM2A delineates a unique architecture that differentiates CpG island chromatin from bulk chromatin., Highlights ► A ZF-CxxC domain in KDM2A specifically binds nonmethylated CpG dinucleotides ► KDM2A is targeted to nonmethylated CpG islands genome-wide ► KDM2A actively removes histone H3 lysine 36 dimethylation from CpG islands ► This unique chromatin architecture distinguishes CpG islands from bulk chromatin

Polycomb group proteins are transcriptional repressors that are essential for normal gene regulation during development. Recent studies suggest that Polycomb repressive complexes (PRCs) recognize and are recruited to their genomic target sites through a range of different mechanisms, which involve transcription factors, CpG island elements and non-coding RNAs. Together with the realization that the interplay between PRC1 and PRC2 is more intricate than was previously appreciated, this has increased our understanding of the vertebrate Polycomb system at the molecular level.

Two-thirds of gene promoters in mammals are associated with regions of non-methylated DNA, called CpG islands (CGIs), which counteract the repressive effects of DNA methylation on chromatin. In cold-blooded vertebrates, computational CGI predictions often reside away from gene promoters, suggesting a major divergence in gene promoter architecture across vertebrates. By experimentally identifying non-methylated DNA in the genomes of seven diverse vertebrates, we instead reveal that non-methylated islands (NMIs) of DNA are a central feature of vertebrate gene promoters. Furthermore, NMIs are present at orthologous genes across vast evolutionary distances, revealing a surprising level of conservation in this epigenetic feature. By profiling NMIs in different tissues and developmental stages we uncover a unifying set of features that are central to the function of NMIs in vertebrates. Together these findings demonstrate an ancient logic for NMI usage at gene promoters and reveal an unprecedented level of epigenetic conservation across vertebrate evolution. DOI: http://dx.doi.org/10.7554/eLife.00348.001, eLife digest DNA methylation—the addition of a methyl group to cytosine, one of the four bases found in DNA—is a central process in genetics. By preventing genes from being expressed as proteins, DNA methylation is one of a number of epigenetic mechanisms that can determine which proteins are made in different cell types without changing the underlying DNA sequence. In warm-blooded vertebrates such as mammals most of the genome is methylated, however short regions of non-methylated DNA are known to be associated with gene promoters (regions of DNA that act as binding sites for the enzymes and transcription factors that transcribe the DNA in the gene into RNA). Much of our current understanding of the role of these islands of non-methylated DNA is based on computational predictions rather than experimental data. In cold-blooded vertebrates, for example, computer models often predict that non-methylated islands are not associated with gene promoters, which potentially suggests an evolutionary divergence in the role of methylation amongst vertebrates. However, this idea has not been confirmed by experimental data. Long et al. have performed experiments to compare the location of non-methylated islands in seven different vertebrate species. In general they find that computational models are not a reliable method for identifying non-methylated islands. Moreover they find that non-methylated islands are a central epigenetic feature of gene promoters in all vertebrates analysed–including three mammals, a bird, a lizard, a frog and a fish—and not just in warm-blooded vertebrates as suggested by computational models. This shows that the epigenetic function of these non-methylated islands has been conserved over more than 450 million years of evolution. In addition to the non-methylated islands associated with gene promoters, Long et al. identify two other types: intergenic non-methylated islands that are found away from gene promoters and are said to be ‘plastic’ because the DNA in these islands can acquire methyl groups, and ‘broad’ non-methylated islands that span many of the genes that are involved in embryonic development. By showing that the epigenetic role of non-methylated islands has been conserved over time, and identifying three specific types of island, the work of Long et al. marks an important change in our understanding of epigenetics in vertebrates. DOI: http://dx.doi.org/10.7554/eLife.00348.002

CpG islands (CGIs) are associated with most mammalian gene promoters. A subset of CGIs act as polycomb response elements (PREs) and are recognized by the polycomb silencing systems to regulate expression of genes involved in early development. How CGIs function mechanistically as nucleation sites for polycomb repressive complexes remains unknown. Here we discover that KDM2B (FBXL10) specifically recognizes non-methylated DNA in CGIs and recruits the polycomb repressive complex 1 (PRC1). This contributes to histone H2A lysine 119 ubiquitylation (H2AK119ub1) and gene repression. Unexpectedly, we also find that CGIs are occupied by low levels of PRC1 throughout the genome, suggesting that the KDM2B-PRC1 complex may sample CGI-associated genes for susceptibility to polycomb-mediated silencing. These observations demonstrate an unexpected and direct link between recognition of CGIs by KDM2B and targeting of the polycomb repressive system. This provides the basis for a new model describing the functionality of CGIs as mammalian PREs. DOI: http://dx.doi.org/10.7554/eLife.00205.001, eLife digest Gene expression in eukaryotic cells can be controlled in a number of different ways, including various epigenetic mechanisms that do not involve making changes to DNA sequences that define the genes themselves. A well-known epigenetic mechanism for silencing genes in vertebrates is DNA methylation—the addition of a methyl group (CH3) to cytosine, which is one of the four bases found in the DNA. Methylation is thought to silence genes by preventing transcription factors from binding to the DNA, and also by recruiting proteins that inhibit the transcription of DNA. DNA methylation occurs naturally throughout the genome, mostly at positions where cytosine is bonded to guanine to form a CpG dinucleotide. While the cytosine bases in most CpG dinucleotides are methylated, there are short stretches of DNA known as CpG islands that contain a high proportion of unmethylated CpG dinucleotides. These islands contain a large number of cytosine and guanine bases, and they are often found at or near transcription start sites. The lack of methylation at CpG islands has long been assumed to have a passive role in gene expression, leaving the DNA easily accessible and available for transcription factors to bind and initiate transcription. However, recent work suggests that CpG islands may have a more active role. In particular, it has been shown that specific proteins bind to CpG islands to create chromatin environments that are more favourable for the initiation of gene expression. Moreover, a subset of CpG islands can also bind polycomb-group proteins, including the polycomb repressive complex 1 (PRC1) that silence gene expression. These complexes have an important role in the regulation of genes during early development in animals, but the mechanism by which PRC1 recognizes CpG islands in mammals has remained enigmatic. Farcas et al. now reveal that a protein, KDM2B (FBXL10), can recognize CpG islands and recruit PRC1 to them. To achieve this, KDM2B encodes a DNA binding domain that specifically recognizes non-methylated CpG dinucleotides. By interacting biochemically with a variant PRC1 complex, KDM2B then nucleates PRC1 at CpG islands, and PRC1 activity silences certain polycomb target genes in embryonic stem cells. Surprisingly, Farcas et al. also find low but appreciable levels of PRC1 at most CpG islands genome-wide, in addition to the high levels of PRC1 at selected islands: this suggests that KDM2B may sample the whole genome to find CpG islands where PRC1 can establish silencing. An improved understanding of the polycomb repressive system, and the role of CpG islands within it, could lead to new insights into the role of epigenetic mechanisms in mammalian development. DOI: http://dx.doi.org/10.7554/eLife.00205.002

Across vertebrate genomes methylation of cytosine residues within the context of CpG dinucleotides is a pervasive epigenetic mark that can impact gene expression and has been implicated in various developmental and disease-associated processes. Several biochemical approaches exist to profile DNA methylation, but recently an alternative approach based on profiling non-methylated CpGs was developed. This technique, called CxxC affinity purification (CAP), uses a ZF-CxxC (CxxC) domain to specifically capture DNA containing clusters of non-methylated CpGs. Here we describe a new CAP approach, called biotinylated CAP (Bio-CAP), which eliminates the requirement for specialized equipment while dramatically improving and simplifying the CxxC-based DNA affinity purification. Importantly, this approach isolates non-methylated DNA in a manner that is directly proportional to the density of non-methylated CpGs, and discriminates non-methylated CpGs from both methylated and hydroxymethylated CpGs. Unlike conventional CAP, Bio-CAP can be applied to nanogram quantities of genomic DNA and in a magnetic format is amenable to efficient parallel processing of samples. Furthermore, Bio-CAP can be applied to genome-wide profiling of non-methylated DNA with relatively small amounts of input material. Therefore, Bio-CAP is a simple and streamlined approach for characterizing regions of the non-methylated DNA, whether at specific target regions or genome wide. © 2012 The Author(s).

Histone lysine methylation regulates a variety of nuclear processes including transcription and maintenance of genome integrity. Many attributes of the histone lysine methylation system suggest that it might also contribute to epigenetic memory. Here, we examine the recent advances in our understanding of three intensely-studied histone lysine methylation marks, focusing on the potential mechanisms by which these marks may be maintained during cell proliferation and adhere to the principles of epigenetics.

Mutations in isocitrate dehydrogenases (IDHs) have a gain-of-function effect leading to R(-)-2-hydroxyglutarate (R-2HG) accumulation. By using biochemical, structural and cellular assays, we show that either or both R- and S-2HG inhibit 2-oxoglutarate (2OG)-dependent oxygenases with varying potencies. Half-maximal inhibitory concentration (IC(50)) values for the R-form of 2HG varied from approximately 25 μM for the histone N(ɛ)-lysine demethylase JMJD2A to more than 5 mM for the hypoxia-inducible factor (HIF) prolyl hydroxylase. The results indicate that candidate oncogenic pathways in IDH-associated malignancy should include those that are regulated by other 2OG oxygenases than HIF hydroxylases, in particular those involving the regulation of histone methylation.

Methylation of CpG islands in gene promoters results in silencing of those genes. Mutation of a methyl-CpG binding domain protein called MeCP2 that contributes to the maintenance of methylation-mediated gene silencing is associated with a severe neurological disease called Rett syndrome. As Klose and Bird report in their Perspective, new work published here ( Chen et al ., Martinowich et al .) and elsewhere demonstrates that MeCP2 normally represses the expression of two genes- BDNF in rat and Hairy2a in frog-that are crucial for normal development of the nervous system.

CpG islands (CGIs) are associated with the majority of mammalian gene promoters and function to recruit chromatin modifying enzymes. It has therefore been proposed that CGIs regulate gene expression through chromatin-based mechanisms, however in most cases this has not been directly tested. Here, we reveal that the histone H3 lysine 36 (H3K36) demethylase activity of the CGI-binding KDM2 proteins contributes only modestly to the H3K36me2-depleted state at CGI-associated gene promoters and is dispensable for normal gene expression. Instead, we discover that KDM2 proteins play a widespread and demethylase-independent role in constraining gene expression from CGI-associated gene promoters. We further show that KDM2 proteins shape RNA Polymerase II occupancy but not chromatin accessibility at CGI-associated promoters. Together this reveals a demethylase-independent role for KDM2 proteins in transcriptional repression and uncovers a new function for CGIs in constraining gene expression.