Modularity of PRC1 composition and chromatin interaction define condensate properties.

Polycomb repressive complexes (PRCs) play a key role in gene repression and are indispensable for proper development. Canonical PRC1 forms condensates in vitro and in cells that are proposed to contribute to the maintenance of repression. However, how chromatin and the various subunits of PRC1 contribute to condensation is largely unexplored. Using a reconstitution approach and single-molecule imaging, we demonstrate that nucleosomal arrays and PRC1 act synergistically, reducing the critical concentration required for condensation by more than 20-fold. We find that the exact combination of PHC and CBX subunits determines condensate initiation, morphology, stability, and dynamics. Particularly, PHC2's polymerization activity influences condensate dynamics by promoting the formation of distinct domains that adhere to each other but do not coalesce. Live-cell imaging confirms CBX's role in condensate initiation and highlights PHC's importance for condensate stability. We propose that PRC1 composition can modulate condensate properties, providing crucial regulatory flexibility across developmental stages.

Cell type-specific role of CBX2 and its disordered region in spermatogenesis.

Polycomb group (PcG) proteins maintain the repressed state of lineage-inappropriate genes and are therefore essential for embryonic development and adult tissue homeostasis. One critical function of PcG complexes is modulating chromatin structure. Canonical Polycomb repressive complex 1 (cPRC1), particularly its component CBX2, can compact chromatin and phase-separate in vitro. These activities are hypothesized to be critical for forming a repressed physical environment in cells. While much has been learned by studying these PcG activities in cell culture models, it is largely unexplored how cPRC1 regulates adult stem cells and their subsequent differentiation in living animals. Here, we show in vivo evidence of a critical nonenzymatic repressive function of cPRC1 component CBX2 in the male germline. CBX2 is up-regulated as spermatogonial stem cells differentiate and is required to repress genes that were active in stem cells. CBX2 forms condensates (similar to previously described Polycomb bodies) that colocalize with target genes bound by CBX2 in differentiating spermatogonia. Single-cell analyses of mosaic Cbx2 mutant testes show that CBX2 is specifically required to produce differentiating A1 spermatogonia. Furthermore, the region of CBX2 responsible for compaction and phase separation is needed for the long-term maintenance of male germ cells in the animal. These results emphasize that the regulation of chromatin structure by CBX2 at a specific stage of spermatogenesis is critical, which distinguishes this from a mechanism that is reliant on histone modification.

TRACE generates fluorescent human reporter cell lines to characterize epigenetic pathways.

Genetically encoded biosensors are powerful tools to monitor cellular behavior, but the difficulty in generating appropriate reporters for chromatin factors hampers our ability to dissect epigenetic pathways. Here, we present TRACE (transgene reporters across chromatin environments), a high-throughput, genome-wide technique to generate fluorescent human reporter cell lines responsive to manipulation of epigenetic factors. By profiling GFP expression from a large pool of individually barcoded lentiviral integrants in the presence and absence of a perturbation, we identify reporters responsive to pharmacological inhibition of the histone lysine demethylase LSD1 and genetic ablation of the PRC2 subunit SUZ12. Furthermore, by manipulating the HIV-1 host factor LEDGF through targeted deletion or fusion to chromatin reader domains, we alter lentiviral integration site preferences, thus broadening the types of chromatin examined by TRACE. The phenotypic reporters generated through TRACE will allow the genetic interrogation of a broad range of epigenetic pathways, furthering our mechanistic understanding of chromatin biology.

A chromatin-dependent role of the fragile X mental retardation protein FMRP in the DNA damage response.

Fragile X syndrome, a common form of inherited intellectual disability, is caused by loss of the fragile X mental retardation protein FMRP. FMRP is present predominantly in the cytoplasm, where it regulates translation of proteins that are important for synaptic function. We identify FMRP as a chromatin-binding protein that functions in the DNA damage response (DDR). Specifically, we show that FMRP binds chromatin through its tandem Tudor (Agenet) domain in vitro and associates with chromatin in vivo. We also demonstrate that FMRP participates in the DDR in a chromatin-binding-dependent manner. The DDR machinery is known to play important roles in developmental processes such as gametogenesis. We show that FMRP occupies meiotic chromosomes and regulates the dynamics of the DDR machinery during mouse spermatogenesis. These findings suggest that nuclear FMRP regulates genomic stability at the chromatin interface and may impact gametogenesis and some developmental aspects of fragile X syndrome.

A Polycomb domain found in committed cells impairs differentiation when introduced into PRC1 in pluripotent cells.

The CBX family of proteins is central to proper mammalian development via key roles in Polycomb-mediated maintenance of repression. CBX proteins in differentiated lineages have chromatin compaction and phase separation activities that might contribute to maintaining repressed chromatin. The predominant CBX protein in pluripotent cells, CBX7, lacks the domain required for these activities. We inserted this functional domain into CBX7 in embryonic stem cells (ESCs) to test the hypothesis that it contributes a key epigenetic function. ESCs expressing this chimeric CBX7 were impaired in their ability to properly form embryoid bodies and neural progenitor cells and showed reduced activation of lineage-specific genes across differentiation. Neural progenitors exhibited a corresponding inappropriate maintenance of Polycomb binding at neural-specific loci over the course of differentiation. We propose that a switch in the ability to compact and phase separate is a central aspect of Polycomb group function during the transition from pluripotency to differentiated lineages.

Context-specific Polycomb mechanisms in development.

Polycomb group (PcG) proteins are crucial chromatin regulators that maintain repression of lineage-inappropriate genes and are therefore required for stable cell fate. Recent advances show that PcG proteins form distinct multi-protein complexes in various cellular environments, such as in early development, adult tissue maintenance and cancer. This surprising compositional diversity provides the basis for mechanistic diversity. Understanding this complexity deepens and refines the principles of PcG complex recruitment, target-gene repression and inheritance of memory. We review how the core molecular mechanism of Polycomb complexes operates in diverse developmental settings and propose that context-dependent changes in composition and mechanism are essential for proper epigenetic regulation in development.

Phase separation of Polycomb-repressive complex 1 is governed by a charged disordered region of CBX2.

Mammalian development requires effective mechanisms to repress genes whose expression would generate inappropriately specified cells. The Polycomb-repressive complex 1 (PRC1) family complexes are central to maintaining this repression. These include a set of canonical PRC1 complexes, each of which contains four core proteins, including one from the CBX family. These complexes have been shown previously to reside in membraneless organelles called Polycomb bodies, leading to speculation that canonical PRC1 might be found in a separate phase from the rest of the nucleus. We show here that reconstituted PRC1 readily phase-separates into droplets in vitro at low concentrations and physiological salt conditions. This behavior is driven by the CBX2 subunit. Point mutations in an internal domain of Cbx2 eliminate phase separation. These same point mutations eliminate the formation of puncta in cells and have been shown previously to eliminate nucleosome compaction in vitro and generate axial patterning defects in mice. Thus, the domain of CBX2 that is important for phase separation is the same domain shown previously to be important for chromatin compaction and proper development, raising the possibility of a mechanistic or evolutionary link between these activities.

Beyond the Histone Code: A Physical Map of Chromatin States.

Post-translational modifications of histones are widely used to discriminate between different types of chromatin. In a recent issue of Molecular Cell, Becker et al. (2017) delineate chromatin states based on physical properties, thereby expanding our understanding of chromatin function.

PGE2 alters chromatin through H2A.Z-variant enhancer nucleosome modification to promote hematopoietic stem cell fate.

Prostaglandin E2 (PGE2) and 16,16-dimethyl-PGE2 (dmPGE2) are important regulators of hematopoietic stem and progenitor cell (HSPC) fate and offer potential to enhance stem cell therapies [C. Cutler et al. Blood 122, 3074-3081(2013); W. Goessling et al. Cell Stem Cell 8, 445-458 (2011); W. Goessling et al. Cell 136, 1136-1147 (2009)]. Here, we report that PGE2-induced changes in chromatin at enhancer regions through histone-variant H2A.Z permit acute inflammatory gene induction to promote HSPC fate. We found that dmPGE2-inducible enhancers retain MNase-accessible, H2A.Z-variant nucleosomes permissive of CREB transcription factor (TF) binding. CREB binding to enhancer nucleosomes following dmPGE2 stimulation is concomitant with deposition of histone acetyltransferases p300 and Tip60 on chromatin. Subsequent H2A.Z acetylation improves chromatin accessibility at stimuli-responsive enhancers. Our findings support a model where histone-variant nucleosomes retained within inducible enhancers facilitate TF binding. Histone-variant acetylation by TF-associated nucleosome remodelers creates the accessible nucleosome landscape required for immediate enhancer activation and gene induction. Our work provides a mechanism through which inflammatory mediators, such as dmPGE2, lead to acute transcriptional changes and modify HSPC behavior to improve stem cell transplantation.

A region of the human HOXD cluster that confers polycomb-group responsiveness.

Polycomb group (PcG) proteins are essential for accurate axial body patterning during embryonic development. PcG-mediated repression is conserved in metazoans and is targeted in Drosophila by Polycomb response elements (PREs). However, targeting sequences in humans have not been described. While analyzing chromatin architecture in the context of human embryonic stem cell (hESC) differentiation, we discovered a 1.8kb region between HOXD11 and HOXD12 (D11.12) that is associated with PcG proteins, becomes nuclease hypersensitive, and then shows alteration in nuclease sensitivity as hESCs differentiate. The D11.12 element repressed luciferase expression from a reporter construct and full repression required a highly conserved region and YY1 binding sites. Furthermore, repression was dependent on the PcG proteins BMI1 and EED and a YY1-interacting partner, RYBP. We conclude that D11.12 is a Polycomb-dependent regulatory region with similarities to Drosophila PREs, indicating conservation in the mechanisms that target PcG function in mammals and flies.

Polycomb Repressive Complex 1 Generates Discrete Compacted Domains that Change during Differentiation.

Master regulatory genes require stable silencing by the polycomb group (PcG) to prevent misexpression during differentiation and development. Some PcG proteins covalently modify histones, which contributes to heritable repression. The role for other effects on chromatin structure is less understood. We characterized the organization of PcG target genes in ESCs and neural progenitors using 5C and super-resolution microscopy. The genomic loci of repressed PcG targets formed discrete, small (20-140 Kb) domains of tight interaction that corresponded to locations bound by canonical polycomb repressive complex 1 (PRC1). These domains changed during differentiation as PRC1 binding changed. Their formation depended upon the Polyhomeotic component of canonical PRC1 and occurred independently of PRC1-catalyzed ubiquitylation. PRC1 domains differ from topologically associating domains in size and boundary characteristics. These domains have the potential to play a key role in transmitting epigenetic silencing of PcG targets by linking PRC1 to formation of a repressive higher-order structure.

Polycomb Repressive Complex 2 Methylates Elongin A to Regulate Transcription.

Polycomb repressive complex 2 (PRC2-EZH2) methylates histone H3 at lysine 27 (H3K27) and is required to maintain gene repression during development. Misregulation of PRC2 is linked to a range of neoplastic malignancies, which is believed to involve methylation of H3K27. However, the full spectrum of non-histone substrates of PRC2 that might also contribute to PRC2 function is not known. We characterized the target recognition specificity of the PRC2 active site and used the resultant data to screen for uncharacterized potential targets. The RNA polymerase II (Pol II) transcription elongation factor, Elongin A (EloA), is methylated by PRC2 in vivo. Mutation of the methylated EloA residue decreased repression of a subset of PRC2 target genes as measured by both steady-state and nascent RNA levels and perturbed embryonic stem cell differentiation. We propose that PRC2 modulates transcription of a subset of low expression target genes in part via methylation of EloA.

Multitasking by Polycomb response elements.

Development requires the expression of master regulatory genes necessary to specify a cell lineage. Equally significant is the stable and heritable silencing of master regulators that would specify alternative lineages. This regulated gene silencing is carried out by Polycomb group (PcG) proteins, which must be correctly recruited only to the subset of their target loci that requires lineage-specific silencing. A recent study by Erceg and colleagues (pp. 590-602) expands on a key aspect of that targeting: The same DNA elements that recruit PcG complexes to a repressed locus also encode transcriptional enhancers that function in different lineages where that locus must be expressed. Thus, PcG targeting elements overlap with enhancers.

Widespread changes in nucleosome accessibility without changes in nucleosome occupancy during a rapid transcriptional induction.

Activation of transcription requires alteration of chromatin by complexes that increase the accessibility of nucleosomal DNA. Removing nucleosomes from regulatory sequences has been proposed to play a significant role in activation. We tested whether changes in nucleosome occupancy occurred on the set of genes that is activated by the unfolded protein response (UPR). We observed no decrease in occupancy on most promoters, gene bodies, and enhancers. Instead, there was an increase in the accessibility of nucleosomes, as measured by micrococcal nuclease (MNase) digestion and ATAC-seq (assay for transposase-accessible chromatin [ATAC] using sequencing), that did not result from removal of the nucleosome. Thus, changes in nucleosome accessibility predominate over changes in nucleosome occupancy during rapid transcriptional induction during the UPR.

Full methylation of H3K27 by PRC2 is dispensable for initial embryoid body formation but required to maintain differentiated cell identity.

Polycomb repressive complex 2 (PRC2) catalyzes methylation of histone H3 on lysine 27 and is required for normal development of complex eukaryotes. The nature of that requirement is not clear. H3K27me3 is associated with repressed genes, but the modification is not sufficient to induce repression and, in some instances, is not required. We blocked full methylation of H3K27 with both a small molecule inhibitor, GSK343, and by introducing a point mutation into EZH2, the catalytic subunit of PRC2, in the mouse CJ7 cell line. Cells with substantively decreased H3K27 methylation differentiate into embryoid bodies, which contrasts with EZH2 null cells. PRC2 targets had varied requirements for H3K27me3, with a subset that maintained normal levels of repression in the absence of methylation. The primary cellular phenotype of blocked H3K27 methylation was an inability of altered cells to maintain a differentiated state when challenged. This phenotype was determined by H3K27 methylation in embryonic stem cells through the first 4 days of differentiation. Full H3K27 methylation therefore was not necessary for formation of differentiated cell states during embryoid body formation but was required to maintain a stable differentiated state.

Purification of proteins associated with specific genomic Loci.

Eukaryotic DNA is bound and interpreted by numerous protein complexes in the context of chromatin. A description of the full set of proteins that regulate specific loci is critical to understanding regulation. Here, we describe a protocol called proteomics of isolated chromatin segments (PICh) that addresses this issue. PICh uses a specific nucleic acid probe to isolate genomic DNA with its associated proteins in sufficient quantity and purity to allow identification of the bound proteins. Purification of human telomeric chromatin using PICh identified the majority of known telomeric factors and uncovered a large number of novel associations. We compared proteins found at telomeres maintained by the alternative lengthening of telomeres (ALT) pathway to proteins bound at telomeres maintained by telomerase. We identified and validated several proteins, including orphan nuclear receptors, that specifically bind to ALT telomeres, establishing PICh as a useful tool for characterizing chromatin composition.

De Novo Variants in the ATPase Module of MORC2 Cause a Neurodevelopmental Disorder with Growth Retardation and Variable Craniofacial Dysmorphism.

MORC2 encodes an ATPase that plays a role in chromatin remodeling, DNA repair, and transcriptional regulation. Heterozygous variants in MORC2 have been reported in individuals with autosomal-dominant Charcot-Marie-Tooth disease type 2Z and spinal muscular atrophy, and the onset of symptoms ranges from infancy to the second decade of life. Here, we present a cohort of 20 individuals referred for exome sequencing who harbor pathogenic variants in the ATPase module of MORC2. Individuals presented with a similar phenotype consisting of developmental delay, intellectual disability, growth retardation, microcephaly, and variable craniofacial dysmorphism. Weakness, hyporeflexia, and electrophysiologic abnormalities suggestive of neuropathy were frequently observed but were not the predominant feature. Five of 18 individuals for whom brain imaging was available had lesions reminiscent of those observed in Leigh syndrome, and five of six individuals who had dilated eye exams had retinal pigmentary abnormalities. Functional assays revealed that these MORC2 variants result in hyperactivation of epigenetic silencing by the HUSH complex, supporting their pathogenicity. The described set of morphological, growth, developmental, and neurological findings and medical concerns expands the spectrum of genetic disorders resulting from pathogenic variants in MORC2.

Elongin A regulates transcription in vivo through enhanced RNA polymerase processivity.

Elongin is an RNA polymerase II (RNAPII)-associated factor that has been shown to stimulate transcriptional elongation in vitro. The Elongin complex is thought to be required for transcriptional induction in response to cellular stimuli and to ubiquitinate RNAPII in response to DNA damage. Yet, the impact of the Elongin complex on transcription in vivo has not been well studied. Here, we performed comprehensive studies of the role of Elongin A, the largest subunit of the Elongin complex, on RNAPII transcription genome-wide. Our results suggest that Elongin A localizes to actively transcribed regions and potential enhancers, and the level of recruitment correlated with transcription levels. We also identified a large group of factors involved in transcription as Elongin A-associated factors. In addition, we found that loss of Elongin A leads to dramatically reduced levels of serine2-phosphorylated, but not total, RNAPII, and cells depleted of Elongin A show stronger promoter RNAPII pausing, suggesting that Elongin A may be involved in the release of paused RNAPII. Our RNA-seq studies suggest that loss of Elongin A did not alter global transcription, and unlike prior in vitro studies, we did not observe a dramatic impact on RNAPII elongation rates in our cell-based nascent RNA-seq experiments upon Elongin A depletion. Taken together, our studies provide the first comprehensive analysis of the role of Elongin A in regulating transcription in vivo. Our studies also revealed that unlike prior in vitro findings, depletion of Elongin A has little impact on global transcription profiles and transcription elongation in vivo.

Elongin A associates with actively transcribed genes and modulates enhancer RNA levels with limited impact on transcription elongation rate in vivo.

Elongin A (EloA) is an essential transcription factor that stimulates the rate of RNA polymerase II (Pol II) transcription elongation in vitro. However, its role as a transcription factor in vivo has remained underexplored. Here we show that in mouse embryonic stem cells, EloA localizes to both thousands of Pol II transcribed genes with preference for transcription start site and promoter regions and a large number of active enhancers across the genome. EloA deletion results in accumulation of transcripts from a subset of enhancers and their adjacent genes. Notably, EloA does not substantially enhance the elongation rate of Pol II in vivo. We also show that EloA localizes to the nucleoli and associates with RNA polymerase I transcribed ribosomal RNA gene, Rn45s. EloA is a highly disordered protein, which we demonstrate forms phase-separated condensates in vitro, and truncation mutations in the intrinsically disordered regions (IDR) of EloA interfere with its targeting and localization to the nucleoli. We conclude that EloA broadly associates with transcribed regions, tunes RNA Pol II transcription levels via impacts on enhancer RNA synthesis, and interacts with the rRNA producing/processing machinery in the nucleolus. Our work opens new avenues for further investigation of the role of this functionally multifaceted transcription factor in enhancer and ribosomal RNA biology.

Dynamics of activating and repressive histone modifications in Drosophila neural stem cell lineages and brain tumors.

During central nervous system development, spatiotemporal gene expression programs mediate specific lineage decisions to generate neuronal and glial cell types from neural stem cells (NSCs). However, little is known about the epigenetic landscape underlying these highly complex developmental events. Here, we perform ChIP-seq on distinct subtypes of Drosophila FACS-purified NSCs and their differentiated progeny to dissect the epigenetic changes accompanying the major lineage decisions in vivo By analyzing active and repressive histone modifications, we show that stem cell identity genes are silenced during differentiation by loss of their activating marks and not via repressive histone modifications. Our analysis also uncovers a new set of genes specifically required for altering lineage patterns in type II neuroblasts (NBs), one of the two main Drosophila NSC identities. Finally, we demonstrate that this subtype specification in NBs, unlike NSC differentiation, requires Polycomb-group-mediated repression.