Region Capture Micro-C reveals coalescence of enhancers and promoters into nested microcompartments.

Although enhancers are central regulators of mammalian gene expression, the mechanisms underlying enhancer-promoter (E-P) interactions remain unclear. Chromosome conformation capture (3C) methods effectively capture large-scale three-dimensional (3D) genome structure but struggle to achieve the depth necessary to resolve fine-scale E-P interactions. Here, we develop Region Capture Micro-C (RCMC) by combining micrococcal nuclease (MNase)-based 3C with a tiling region-capture approach and generate the deepest 3D genome maps reported with only modest sequencing. By applying RCMC in mouse embryonic stem cells and reaching the genome-wide equivalent of ~317 billion unique contacts, RCMC reveals previously unresolvable patterns of highly nested and focal 3D interactions, which we term microcompartments. Microcompartments frequently connect enhancers and promoters, and although loss of loop extrusion and inhibition of transcription disrupts some microcompartments, most are largely unaffected. We therefore propose that many E-P interactions form through a compartmentalization mechanism, which may partially explain why acute cohesin depletion only modestly affects global gene expression.

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.

Live-cell single particle tracking of PRC1 reveals a highly dynamic system with low target site occupancy.

Polycomb repressive complex 1 (PRC1) is an essential chromatin-based repressor of gene transcription. How PRC1 engages with chromatin to identify its target genes and achieve gene repression remains poorly defined, representing a major hurdle to our understanding of Polycomb system function. Here, we use genome engineering and single particle tracking to dissect how PRC1 binds to chromatin in live mouse embryonic stem cells. We observe that PRC1 is highly dynamic, with only a small fraction stably interacting with chromatin. By integrating subunit-specific dynamics, chromatin binding, and abundance measurements, we discover that PRC1 exhibits low occupancy at target sites. Furthermore, we employ perturbation approaches to uncover how specific components of PRC1 define its kinetics and chromatin binding. Together, these discoveries provide a quantitative understanding of chromatin binding by PRC1 in live cells, suggesting that chromatin modification, as opposed to PRC1 complex occupancy, is central to gene repression.

Identifying proteins bound to native mitotic ESC chromosomes reveals chromatin repressors are important for compaction.

Epigenetic information is transmitted from mother to daughter cells through mitosis. Here, to identify factors that might play a role in conveying epigenetic memory through cell division, we report on the isolation of unfixed, native chromosomes from metaphase-arrested cells using flow cytometry and perform LC-MS/MS to identify chromosome-bound proteins. A quantitative proteomic comparison between metaphase-arrested cell lysates and chromosome-sorted samples reveals a cohort of proteins that were significantly enriched on mitotic ESC chromosomes. These include pluripotency-associated transcription factors, repressive chromatin-modifiers such as PRC2 and DNA methyl-transferases, and proteins governing chromosome architecture. Deletion of PRC2, Dnmt1/3a/3b or Mecp2 in ESCs leads to an increase in the size of individual mitotic chromosomes, consistent with de-condensation. Similar results were obtained by the experimental cleavage of cohesin. Thus, we identify chromosome-bound factors in pluripotent stem cells during mitosis and reveal that PRC2, DNA methylation and Mecp2 are required to maintain chromosome compaction.

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.