Epi-microRNA mediated metabolic reprogramming ensures affinity maturation.

To increase antibody affinity against pathogens, positively selected GC-B cells initiate cell division in the light zone (LZ) of germinal centres (GCs). Among those, higher-affinity clones migrate to the dark zone (DZ) and vigorously proliferate by relying on oxidative phosphorylation (OXPHOS). However, it remains unknown how positively selected GC-B cells adapt their metabolism for cell division in the glycolysis-dominant, cell cycle arrest-inducing, hypoxic LZ microenvironment. Here, we show that microRNA (miR)-155 mediates metabolic reprogramming during positive selection to protect high-affinity clones. Transcriptome examination and mass spectrometry analysis revealed that miR-155 regulates H3K36me2 levels by directly repressing hypoxia-induced histone lysine demethylase, Kdm2a. This is indispensable for enhancing OXPHOS through optimizing the expression of vital nuclear mitochondrial genes under hypoxia. The miR-155-Kdm2a interaction is crucial to prevent excessive production of reactive oxygen species and apoptosis. Thus, miR-155-mediated epigenetic regulation promotes mitochondrial fitness in high-affinity clones, ensuring their expansion and consequently affinity maturation.

A CpG island-encoded mechanism protects genes from premature transcription termination.

Transcription must be tightly controlled to regulate gene expression and development. However, our understanding of the molecular mechanisms that influence transcription and how these are coordinated in cells to ensure normal gene expression remains rudimentary. Here, by dissecting the function of the SET1 chromatin-modifying complexes that bind to CpG island-associated gene promoters, we discover that they play a specific and essential role in enabling the expression of low to moderately transcribed genes. Counterintuitively, this effect can occur independently of SET1 complex histone-modifying activity and instead relies on an interaction with the RNA Polymerase II-binding protein WDR82. Unexpectedly, we discover that SET1 complexes enable gene expression by antagonising premature transcription termination by the ZC3H4/WDR82 complex at CpG island-associated genes. In contrast, at extragenic sites of transcription, which typically lack CpG islands and SET1 complex occupancy, we show that the activity of ZC3H4/WDR82 is unopposed. Therefore, we reveal a gene regulatory mechanism whereby CpG islands are bound by a protein complex that specifically protects genic transcripts from premature termination, effectively distinguishing genic from extragenic transcription and enabling normal gene expression.

Variant PCGF1-PRC1 links PRC2 recruitment with differentiation-associated transcriptional inactivation at target genes.

Polycomb repressive complexes-1 and -2 (PRC1 and 2) silence developmental genes in a spatiotemporal manner during embryogenesis. How Polycomb group (PcG) proteins orchestrate down-regulation of target genes upon differentiation, however, remains elusive. Here, by differentiating embryonic stem cells into embryoid bodies, we reveal a crucial role for the PCGF1-containing variant PRC1 complex (PCGF1-PRC1) to mediate differentiation-associated down-regulation of a group of genes. Upon differentiation cues, transcription is down-regulated at these genes, in association with PCGF1-PRC1-mediated deposition of histone H2AK119 mono-ubiquitination (H2AK119ub1) and PRC2 recruitment. In the absence of PCGF1-PRC1, both H2AK119ub1 deposition and PRC2 recruitment are disrupted, leading to aberrant expression of target genes. PCGF1-PRC1 is, therefore, required for initiation and consolidation of PcG-mediated gene repression during differentiation.

The molecular principles of gene regulation by Polycomb repressive complexes.

Precise control of gene expression is fundamental to cell function and development. Although ultimately gene expression relies on DNA-binding transcription factors to guide the activity of the transcription machinery to genes, it has also become clear that chromatin and histone post-translational modification have fundamental roles in gene regulation. Polycomb repressive complexes represent a paradigm of chromatin-based gene regulation in animals. The Polycomb repressive system comprises two central protein complexes, Polycomb repressive complex 1 (PRC1) and PRC2, which are essential for normal gene regulation and development. Our early understanding of Polycomb function relied on studies in simple model organisms, but more recently it has become apparent that this system has expanded and diverged in mammals. Detailed studies are now uncovering the molecular mechanisms that enable mammalian PRC1 and PRC2 to identify their target sites in the genome, communicate through feedback mechanisms to create Polycomb chromatin domains and control transcription to regulate gene expression. In this Review, we discuss and contextualize the emerging principles that define how this fascinating chromatin-based system regulates gene expression in mammals.

BAP1 constrains pervasive H2AK119ub1 to control the transcriptional potential of the genome.

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.

Getting under the skin of Polycomb-dependent gene regulation.

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.

Cohesin Disrupts Polycomb-Dependent Chromosome Interactions in Embryonic Stem Cells.

How chromosome organization is related to genome function remains poorly understood. Cohesin, loop extrusion, and CCCTC-binding factor (CTCF) have been proposed to create topologically associating domains (TADs) to regulate gene expression. Here, we examine chromosome conformation in embryonic stem cells lacking cohesin and find, as in other cell types, that cohesin is required to create TADs and regulate A/B compartmentalization. However, in the absence of cohesin, we identify a series of long-range chromosomal interactions that persist. These correspond to regions of the genome occupied by the polycomb repressive system and are dependent on PRC1. Importantly, we discover that cohesin counteracts these polycomb-dependent interactions, but not interactions between super-enhancers. This disruptive activity is independent of CTCF and insulation and appears to modulate gene repression by the polycomb system. Therefore, we discover that cohesin disrupts polycomb-dependent chromosome interactions to modulate gene expression in embryonic stem cells.

PRC1 Catalytic Activity Is Central to Polycomb System Function.

The Polycomb repressive system is an essential chromatin-based regulator of gene expression. Despite being extensively studied, how the Polycomb system selects its target genes is poorly understood, and whether its histone-modifying activities are required for transcriptional repression remains controversial. Here, we directly test the requirement for PRC1 catalytic activity in Polycomb system function. To achieve this, we develop a conditional mutation system in embryonic stem cells that completely removes PRC1 catalytic activity. Using this system, we demonstrate that catalysis by PRC1 drives Polycomb chromatin domain formation and long-range chromatin interactions. Furthermore, we show that variant PRC1 complexes with DNA-binding activities occupy target sites independently of PRC1 catalytic activity, providing a putative mechanism for Polycomb target site selection. Finally, we discover that Polycomb-mediated gene repression requires PRC1 catalytic activity. Together these discoveries provide compelling evidence that PRC1 catalysis is central to Polycomb system function and gene regulation.

Synergy between Variant PRC1 Complexes Defines Polycomb-Mediated Gene Repression.

The Polycomb system modifies chromatin and plays an essential role in repressing gene expression to control normal mammalian development. However, the components and mechanisms that define how Polycomb protein complexes achieve this remain enigmatic. Here, we use combinatorial genetic perturbation coupled with quantitative genomics to discover the central determinants of Polycomb-mediated gene repression in mouse embryonic stem cells. We demonstrate that canonical Polycomb repressive complex 1 (PRC1), which mediates higher-order chromatin structures, contributes little to gene repression. Instead, we uncover an unexpectedly high degree of synergy between variant PRC1 complexes, which is fundamental to gene repression. We further demonstrate that variant PRC1 complexes are responsible for distinct pools of H2A monoubiquitylation that are associated with repression of Polycomb target genes and silencing during X chromosome inactivation. Together, these discoveries reveal a new variant PRC1-dependent logic for Polycomb-mediated gene repression.

Polycomb repressive complex 1 shapes the nucleosome landscape but not accessibility at target genes.

Polycomb group (PcG) proteins are transcriptional repressors that play important roles in regulating gene expression during animal development. In vitro experiments have shown that PcG protein complexes can compact chromatin to limit the activity of chromatin remodeling enzymes and access of the transcriptional machinery to DNA. In fitting with these ideas, gene promoters associated with PcG proteins have been reported to be less accessible than other gene promoters. However, it remains largely untested in vivo whether PcG proteins define chromatin accessibility or other chromatin features. To address this important question, we examine the chromatin accessibility and nucleosome landscape at PcG protein-bound promoters in mouse embryonic stem cells using the assay for transposase accessible chromatin (ATAC)-seq. Combined with genetic ablation strategies, we unexpectedly discover that although PcG protein-occupied gene promoters exhibit reduced accessibility, this does not rely on PcG proteins. Instead, the Polycomb repressive complex 1 (PRC1) appears to play a unique role in driving elevated nucleosome occupancy and decreased nucleosomal spacing in Polycomb chromatin domains. Our new genome-scale observations argue, in contrast to the prevailing view, that PcG proteins do not significantly affect chromatin accessibility and highlight an underappreciated complexity in the relationship between chromatin accessibility, the nucleosome landscape, and PcG-mediated transcriptional repression.

Biochemical Identification of Nonmethylated DNA by BioCAP-Seq.

CpG islands are regions of vertebrate genomes that often function as gene regulatory elements and are associated with most gene promoters. CpG island elements usually contain nonmethylated CpG dinucleotides, while the remainder of the genome is pervasively methylated. We developed a biochemical approach called biotinylated CxxC affinity purification (BioCAP) to unbiasedly isolate regions of the genome that contain nonmethylated CpG dinucleotides. The resulting highly pure nonmethylated DNA is easily analyzed by quantitative PCR to interrogate specific loci or via massively parallel sequencing to yield genome-wide profiles.

The SET1 Complex Selects Actively Transcribed Target Genes via Multivalent Interaction with CpG Island Chromatin.

Chromatin modifications and the promoter-associated epigenome are important for the regulation of gene expression. However, the mechanisms by which chromatin-modifying complexes are targeted to the appropriate gene promoters in vertebrates and how they influence gene expression have remained poorly defined. Here, using a combination of live-cell imaging and functional genomics, we discover that the vertebrate SET1 complex is targeted to actively transcribed gene promoters through CFP1, which engages in a form of multivalent chromatin reading that involves recognition of non-methylated DNA and histone H3 lysine 4 trimethylation (H3K4me3). CFP1 defines SET1 complex occupancy on chromatin, and its multivalent interactions are required for the SET1 complex to place H3K4me3. In the absence of CFP1, gene expression is perturbed, suggesting that normal targeting and function of the SET1 complex are central to creating an appropriately functioning vertebrate promoter-associated epigenome.

MLL-AF4 Spreading Identifies Binding Sites that Are Distinct from Super-Enhancers and that Govern Sensitivity to DOT1L Inhibition in Leukemia.

Understanding the underlying molecular mechanisms of defined cancers is crucial for effective personalized therapies. Translocations of the mixed-lineage leukemia (MLL) gene produce fusion proteins such as MLL-AF4 that disrupt epigenetic pathways and cause poor-prognosis leukemias. Here, we find that at a subset of gene targets, MLL-AF4 binding spreads into the gene body and is associated with the spreading of Menin binding, increased transcription, increased H3K79 methylation (H3K79me2/3), a disruption of normal H3K36me3 patterns, and unmethylated CpG regions in the gene body. Compared to other H3K79me2/3 marked genes, MLL-AF4 spreading gene expression is downregulated by inhibitors of the H3K79 methyltransferase DOT1L. This sensitivity mediates synergistic interactions with additional targeted drug treatments. Therefore, epigenetic spreading and enhanced susceptibility to epidrugs provides a potential marker for better understanding combination therapies in humans.

RYBP stimulates PRC1 to shape chromatin-based communication between Polycomb repressive complexes.

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.

Targeting Polycomb systems to regulate gene expression: modifications to a complex story.

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.

Targeting polycomb to pericentric heterochromatin in embryonic stem cells reveals a role for H2AK119u1 in PRC2 recruitment.

The mechanisms by which the major Polycomb group (PcG) complexes PRC1 and PRC2 are recruited to target sites in vertebrate cells are not well understood. Building on recent studies that determined a reciprocal relationship between DNA methylation and Polycomb activity, we demonstrate that, in methylation-deficient embryonic stem cells (ESCs), CpG density combined with antagonistic effects of H3K9me3 and H3K36me3 redirects PcG complexes to pericentric heterochromatin and gene-rich domains. Surprisingly, we find that PRC1-linked H2A monoubiquitylation is sufficient to recruit PRC2 to chromatin in vivo, suggesting a mechanism through which recognition of unmethylated CpG determines the localization of both PRC1 and PRC2 at canonical and atypical target sites. We discuss our data in light of emerging evidence suggesting that PcG recruitment is a default state at licensed chromatin sites, mediated by interplay between CpG hypomethylation and counteracting H3 tail modifications.

Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation.

Chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development. However, the mechanisms by which these complexes recognize their target sites and function together to form repressive chromatin domains remain poorly understood. Recruitment of PRC1 to target sites has been proposed to occur through a hierarchical process, dependent on prior nucleation of PRC2 and placement of H3K27me3. Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain. This activity is restricted to variant PRC1 complexes, and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for normal polycomb domain formation and mouse development. These observations provide a surprising PRC1-dependent logic for PRC2 occupancy at target sites in vivo.

CpG island chromatin is shaped by recruitment of ZF-CxxC proteins.

Most mammalian gene promoters are embedded within genomic regions called CpG islands, characterized by elevated levels of nonmethylated CpG dinucleotides. Here, we describe recent work demonstrating that CpG islands act as specific nucleation sites for the zinc finger CxxC domain-containing proteins CFP1 and KDM2A. Importantly, both CFP1 and KDM2A are associated with enzymatic activities that modulate specific histone lysine methylation marks. The action of these zinc finger CxxC domain proteins therefore imposes a defined chromatin architecture on CpG islands that distinguishes these important regulatory elements from the surrounding genome. The functional consequence of this CpG island-directed chromatin environment is discussed.

ZF-CxxC domain-containing proteins, CpG islands and the chromatin connection.

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.