Previously, we described a novel alternative to chromatin immunoprecipitation, CUT&RUN, in which unfixed permeabilized cells are incubated with antibody, followed by binding of a protein A-Micrococcal Nuclease (pA/MNase) fusion protein (Skene and Henikoff, 2017). Here we introduce three enhancements to CUT&RUN: A hybrid protein A-Protein G-MNase construct that expands antibody compatibility and simplifies purification, a modified digestion protocol that inhibits premature release of the nuclease-bound complex, and a calibration strategy based on carry-over of E. coli DNA introduced with the fusion protein. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling.

Improved CUT&RUN chromatin profiling tools.

Cooperative binding between distant transcription factors is a hallmark of active enhancers.

Polycomb group proteins are evolutionary conserved chromatin-modifying complexes, essential for the regulation of developmental and cell-identity genes. Polycomb-mediated transcriptional regulation is provided by two multi-protein complexes known as Polycomb repressive complex 1 (PRC1) and 2 (PRC2). Recent studies positioned PRC1 as a foremost executer of Polycomb-mediated transcriptional control. Mammalian PRC1 complexes can form multiple sub-complexes that vary in their core and accessory subunit composition, leading to fascinating and diverse transcriptional regulatory mechanisms employed by PRC1 complexes. These mechanisms include PRC1-catalytic activity toward monoubiquitination of histone H2AK119, a well-established hallmark of PRC1 complexes, whose importance has been long debated. In this review, the central roles that PRC1-catalytic activity plays in transcriptional repression are emphasized and the recent evidence supporting a role for PRC1 complexes in gene activation is discussed.

Activity of PRC1 and Histone H2AK119 Monoubiquitination: Revising Popular Misconceptions

Polycomb group (PcG) proteins silence master regulatory genes required to properly confer cell identity during the development of both Drosophila and mammals. They may act through chromatin compaction and higher-order folding of chromatin inside the cell nucleus. During the last decade, analysis on interphase chromosome architecture discovered self-interacting regions named topologically associated domains (TADs). TADs result from the 3D chromatin folding of a succession of transcribed and repressed epigenomic domains and from loop extrusion mediated by cohesin/CTCF in mammals. Polycomb silenced chromatin constitutes one type of repressed epigenomic domains which form compacted nano-compartments inside cell nuclei. Recruitment of canonical PcG proteins on chromatin relies on initial binding to discrete elements and further spreading into large chromatin domains covered with H3K27me3. Some of these discrete elements have a bivalent nature both in mammals and Drosophila and are dynamically regulated during development. Loops can occur between them, suggesting that their interaction plays both functional and structural roles. Formation of large chromatin domains covered by H3K27me3 seems crucial for PcG silencing and PcG proteins might exert their function through compaction of these domains in both mammals and flies, rather than by directly controlling the nucleosomal accessibility of discrete regulatory elements. In addition, PcG chromatin domains interact over long genomic distances, shaping a higher-order chromatin network. Therefore, PcG silencing might rely on multiscale chromatin folding to maintain cell identity during differentiation.

The multiscale effects of polycomb mechanisms on 3D chromatin folding.

Development proceeds by the activation of genes by transcription factors and the inactivation of others by chromatin-mediated gene silencing. In certain cases development can be reversed or redirected by mis-expression of master regulator transcription factors. This must involve the activation of previously silenced genes, and such developmental aberrations are thought to underlie a variety of cancers. Here, we express the wing-specific Vestigial master regulator to reprogram the developing eye, and test the role of silencing in reprogramming using an H3.3K27M oncohistone mutation that dominantly inhibits histone H3K27 trimethylation. We find that production of the oncohistone blocks eye-to-wing reprogramming. CUT&Tag chromatin profiling of mutant tissues shows that H3K27me3 of domains is generally reduced upon oncohistone production, suggesting that a previous developmental program must be silenced for effective transformation. Strikingly, Vg and H3.3K27M synergize to stimulate overgrowth of eye tissue, a phenotype that resembles that of mutations in Polycomb silencing components. Transcriptome profiling of elongating RNA Polymerase II implicates the mis-regulation of signaling factors in overgrowth. Our results demonstrate that growth dysregulation can result from the simple combination of crippled silencing and transcription factor mis-expression, an effect that may explain the origins of oncohistone-bearing cancers.

/pdf/the-h3-3k27m-oncohistone-antagonizes-reprogramming-in-1mel5i4k1n.pdf

The H3.3K27M oncohistone antagonizes reprogramming in Drosophila.

Patterned expression of many developmental genes is specified by transcription factor gene expression, but is thought to be refined by chromatin-mediated repression. Regulatory DNA sequences called Polycomb Response Elements (PREs) are required to repress some developmental target genes, and are widespread in genomes, suggesting that they broadly affect developmental programs. While PREs in transgenes can nucleate trimethylation on lysine 27 of the histone H3 tail (H3K27me3), none have been demonstrated to be necessary at endogenous chromatin domains. This failure is thought to be due to the fact that most endogenous H3K27me3 domains contain many PREs, and individual PREs may be redundant. In contrast to these ideas, we show here that PREs near the wing selector gene vestigial have distinctive roles at their endogenous locus, even though both PREs are repressors in transgenes. First, a PRE near the promoter is required for vestigial activation and not for repression. Second, only the distal PRE contributes to H3K27me3, but even removal of both PREs does not eliminate H3K27me3 across the vestigial domain. Thus, endogenous chromatin domains appear to be intrinsically marked by H3K27me3, and PREs appear required to enhance this chromatin modification to high levels at inactive genes. Author summary Eukaryotic genes are packaged in chromatin, and their transcription relies on activators that recruit RNA polymerases and on repressive factors. Some of these factors factors that modulate chromatin structure to promote or inhibit gene transcription. In multicellular organisms, cell types have distinct patterns of gene expression, and these patterns are controlled by by the expression of cell-type-specific transcription factors and by modulating chromatin structure. The Polycomb system is one major system for the chromatin-mediated silencing of developmental gene expression, where a histone methyltransferase marks extended chromatin domains with trimethylation of lysine-27 of the histone H3 tail (H3K27me3) and forms repressed chromatin. In Drosophila, repressive regulatory elements called Polycomb Response Elements (PREs) are thought to nucleate histone methyltransferase binding which then spreads across these domains. In this study, we demonstrate that two PREs near the developmental vestigial gene have distinct and separable effects on gene activation and chromatin structure. Both PREs are functional repressors in transgenes, but the PRE located near the vestigial promoter is required for gene transcription. This PRE has no effect on histone methylation of the domain. The second PRE, located in the middle of the chromatin domain is required for high-level H3K27me3 of the domain, and this methylation is not required to refine vestigial gene expression. Strikingly, a significant chromatin methylation remains when both PREs are deleted. Our findings imply that PREs near promoters may often play activating roles in gene expression in the Drosophila genome. We suggest that some domains of H3K27me3 occur in regions that lack histone acetylation, and has little consequence for correctly patterning gene expression.

/pdf/separate-polycomb-response-elements-control-chromatin-state-2ygdbfm09f.pdf

Separate Polycomb Response Elements control chromatin state and activation of the vestigial gene

Patterned expression of many developmental genes is specified by transcription factor gene expression, but is thought to be refined by chromatin-mediated repression. Regulatory DNA sequences called Polycomb Response Elements (PREs) are required to repress some developmental target genes, and are widespread in genomes, suggesting that they broadly affect developmental programs. While PREs in transgenes can nucleate trimethylation on lysine 27 of the histone H3 tail (H3K27me3), none have been demonstrated to be necessary at endogenous chromatin domains. This failure is thought to be due to the fact that most endogenous H3K27me3 domains contain many PREs, and individual PREs may be redundant. In contrast to these ideas, we show here that PREs near the wing selector gene vestigial have distinctive roles at their endogenous locus, even though both PREs are repressors in transgenes. First, a PRE near the promoter is required for vestigial activation and not for repression. Second, only the distal PRE contributes to H3K27me3, but even removal of both PREs does not eliminate H3K27me3 across the vestigial domain. Thus, endogenous chromatin domains appear to be intrinsically marked by H3K27me3, and PREs appear required to enhance this chromatin modification to high levels at inactive genes.

/pdf/separate-polycomb-response-elements-control-chromatin-state-4bn8woyvi4.pdf

Separate Polycomb Response Elements control chromatin state and activation of the vestigial gene.

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Separate Polycomb Response Elements control chromatin state and activation of the vestigial gene

Separate Polycomb Response Elements control chromatin state and activation of the vestigial gene.