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Tight control over cell identity gene expression is necessary for proper adult form and function. The opposing activities of Polycomb and trithorax complexes determine the on/off state of cell identity genes such as the Hox factors. Polycomb group complexes repress target genes, whereas trithorax group complexes are required for their expression. Although trithorax and its orthologs function as methyltransferases specific to histone H3 lysine 4 (H3K4), there is no direct evidence that H3K4 regulates Polycomb group target genes in vivo. Using histone gene replacement in Drosophila , we provide evidence of two key roles for replication-dependent histone H3.2K4 in Polycomb target gene control. First, we found that H3.2K4 mutants mimic H3.2K4me3 in antagonizing methyltransferase activity of the PRC2 Polycomb group complex. Second, we found that H3.2K4 is also required for proper activation of Polycomb targets. We conclude that H3.2K4 directly regulates Polycomb target gene expression., (© 2024 Anyetei-Anum et al.; Published by Cold Spring Harbor Laboratory Press.)

In an unmodified state, positively charged histone N-terminal tails engage nucleosomal DNA in a manner which restricts access to not only the underlying DNA but also key tail residues subject to binding and/or modification. Charge-neutralizing modifications, such as histone acetylation, serve to disrupt this DNA-tail interaction, facilitating access to such residues. We previously showed that a polyacetylation-mediated chromatin "switch" governs the read-write capability of H3K4me3 by the MLL1 methyltransferase complex. Here, we discern the relative contributions of site-specific acetylation states along the H3 tail and extend our interrogation to other chromatin modifiers. We show that the contributions of H3 tail acetylation to H3K4 methylation by MLL1 are highly variable, with H3K18 and H3K23 acetylation exhibiting robust stimulatory effects and that this extends to the related H3K4 methyltransferase complex, MLL4. We show that H3K4me1 and H3K4me3 are found preferentially co-enriched with H3 N-terminal tail proteoforms bearing dual H3K18 and H3K23 acetylation (H3{K18acK23ac}). We further show that this effect is specific to H3K4 methylation, while methyltransferases targeting other H3 tail residues (H3K9, H3K27, & H3K36), a methyltransferase targeting the nucleosome core (H3K79), and a kinase targeting a residue directly adjacent to H3K4 (H3T3) are insensitive to tail acetylation. Together, these findings indicate a unique and robust stimulation of H3K4 methylation by H3K18 and H3K23 acetylation and provide key insight into why H3K4 methylation is often associated with histone acetylation in the context of active gene expression., Competing Interests: Conflict of interest EpiCypher is a commercial developer and supplier of fully defined semi-synthetic nucleosomes as used in this study. N. N. R., B. G., and B. D. S. own shares in EpiCypher with BDS also a board member of the same. All other authors declare that they have no other conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Tight control over cell identity gene expression is necessary for proper adult form and function. The opposing activities of Polycomb and trithorax complexes determine the ON/OFF state of targets like the Hox genes. Trithorax encodes a methyltransferase specific to histone H3 lysine-4 (H3K4). However, there is no direct evidence that H3K4 regulates Polycomb group target genes in vivo . Here, we demonstrate two key roles for replication-dependent histone H3.2K4 in target control. We find that H3.2K4 antagonizes Polycomb group catalytic activity and that it is required for proper target gene activation. We conclude that H3.2K4 directly regulates expression of Polycomb targets.

The NuA3 complex is a major regulator of gene transcription and the cell cycle in yeast. Five core subunits are required for complex assembly and function, but it remains unclear how these subunits interact to form the complex. Here, we report that the Taf14 subunit of the NuA3 complex binds to two other subunits of the complex, Yng1 and Sas3, and describe the molecular mechanism by which the extra-terminal domain of Taf14 recognizes the conserved motif present in Yng1 and Sas3. Structural, biochemical, and mutational analyses show that two motifs are sandwiched between the two extra-terminal domains of Taf14. The head-to-toe dimeric complex enhances the DNA binding activity of Taf14, and the formation of the hetero-dimer involving the motifs of Yng1 and Sas3 is driven by sequence complementarity. In vivo assays in yeast demonstrate that the interactions of Taf14 with both Sas3 and Yng1 are required for proper function of the NuA3 complex in gene transcription and DNA repair. Our findings suggest a potential basis for the assembly of three core subunits of the NuA3 complex, Taf14, Yng1 and Sas3., (© 2024. The Author(s).)

Understanding the mechanisms that drive HIV expression and latency is a key goal for achieving an HIV cure. Here we investigate the role of the SETD2 histone methyltransferase, which deposits H3K36 trimethylation (H3K36me3), in HIV infection. We show that prevention of H3K36me3 by a potent and selective inhibitor of SETD2 (EPZ-719) leads to reduced post-integration viral gene expression and accelerated emergence of latently infected cells. CRISPR/Cas9-mediated knockout of SETD2 in primary CD4 T cells confirmed the role of SETD2 in HIV expression. Transcriptomic profiling of EPZ-719-exposed HIV-infected cells identified numerous pathways impacted by EPZ-719. Notably, depletion of H3K36me3 prior to infection did not prevent HIV integration but resulted in a shift of integration sites from highly transcribed genes to quiescent chromatin regions and to polycomb repressed regions. We also observed that SETD2 inhibition did not apparently affect HIV RNA levels, indicating a post-transcriptional mechanism affecting HIV expression. Viral RNA splicing was modestly reduced in the presence of EPZ-719. Intriguingly, EPZ-719 exposure enhanced responsiveness of latent HIV to the HDAC inhibitor vorinostat, suggesting that H3K36me3 can contribute to a repressive chromatin state at the HIV locus. These results identify SETD2 and H3K36me3 as novel regulators of HIV integration, expression and latency., Competing Interests: BDS is a co-founder and BOD member of EpiCypher and received financial compensation from this company., (Copyright: © 2024 Bussey-Sutton et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In the nucleus, biological processes are driven by proteins that diffuse through and bind to a meshwork of nucleic acid polymers. To better understand this interplay, we present an imaging platform to simultaneously visualize single protein dynamics together with the local chromatin environment in live cells. Together with super-resolution imaging, new fluorescent probes, and biophysical modeling, we demonstrate that nucleosomes display differential diffusion and packing arrangements as chromatin density increases whereas the viscoelastic properties and accessibility of the interchromatin space remain constant. Perturbing nuclear functions impacts nucleosome diffusive properties in a manner that is dependent both on local chromatin density and on relative location within the nucleus. Our results support a model wherein transcription locally stabilizes nucleosomes while simultaneously allowing for the free exchange of nuclear proteins. Additionally, they reveal that nuclear heterogeneity arises from both active and passive processes and highlight the need to account for different organizational principles when modeling different chromatin environments., (© 2024. The Author(s).)

We report a novel translation-regulatory function of G9a, a histone methyltransferase and well-understood transcriptional repressor, in promoting hyperinflammation and lymphopenia; two hallmarks of endotoxin tolerance (ET)-associated chronic inflammatory complications. Using multiple approaches, we demonstrate that G9a interacts with multiple translation regulators during ET, particularly the N6-methyladenosine (m 6 A) RNA methyltransferase METTL3, to co-upregulate expression of certain m 6 A-modified mRNAs that encode immune-checkpoint and anti-inflammatory proteins. Mechanistically, G9a promotes m 6 A methyltransferase activity of METTL3 at translational/post-translational level by regulating its expression, its methylation, and its cytosolic localization during ET. Additionally, from a broader view extended from the G9a-METTL3-m 6 A translation regulatory axis, our translatome proteomics approach identified numerous "G9a-translated" proteins that unite the networks associated with inflammation dysregulation, T cell dysfunction, and systemic cytokine response. In sum, we identified a previously unrecognized function of G9a in protein-specific translation that can be leveraged to treat ET-related chronic inflammatory diseases., Competing Interests: Declaration of interests This invention of the mechanism of inhibitor action is protected by United States Provisional Patent Application filed by the University of North Carolina-Chapel Hill (UNC 21–0060)., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Trimethylation of histone H3 lysine 9 (H3K9me3) is crucial for the regulation of gene repression and heterochromatin formation, cell-fate determination and organismal development 1 . H3K9me3 also provides an essential mechanism for silencing transposable elements 1-4 . However, previous studies have shown that canonical H3K9me3 readers (for example, HP1 (refs. 5-9 ) and MPP8 (refs. 10-12 )) have limited roles in silencing endogenous retroviruses (ERVs), one of the main transposable element classes in the mammalian genome 13 . Here we report that trinucleotide-repeat-containing 18 (TNRC18), a poorly understood chromatin regulator, recognizes H3K9me3 to mediate the silencing of ERV class I (ERV1) elements such as LTR12 (ref. 14 ). Biochemical, biophysical and structural studies identified the carboxy-terminal bromo-adjacent homology (BAH) domain of TNRC18 (TNRC18(BAH)) as an H3K9me3-specific reader. Moreover, the amino-terminal segment of TNRC18 is a platform for the direct recruitment of co-repressors such as HDAC-Sin3-NCoR complexes, thus enforcing optimal repression of the H3K9me3-demarcated ERVs. Point mutagenesis that disrupts the TNRC18(BAH)-mediated H3K9me3 engagement caused neonatal death in mice and, in multiple mammalian cell models, led to derepressed expression of ERVs, which affected the landscape of cis-regulatory elements and, therefore, gene-expression programmes. Collectively, we describe a new H3K9me3-sensing and regulatory pathway that operates to epigenetically silence evolutionarily young ERVs and exert substantial effects on host genome integrity, transcriptomic regulation, immunity and development., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Isochromosomes are mirror-imaged chromosomes with simultaneous duplication and deletion of genetic material which may contain two centromeres to create isodicentric chromosomes. Although isochromosomes commonly occur in cancer and developmental disorders and promote genome instability, mechanisms that prevent isochromosomes are not well understood. We show here that the tumor suppressor and methyltransferase SETD2 is essential to prevent these errors. Using cellular and cytogenetic approaches, we demonstrate that loss of SETD2 or its epigenetic mark, histone H3 lysine 36 trimethylation (H3K36me3), results in the formation of isochromosomes as well as isodicentric and acentric chromosomes. These defects arise during DNA replication and are likely due to faulty homologous recombination by RAD52. These data provide a mechanism for isochromosome generation and demonstrate that SETD2 and H3K36me3 are essential to prevent the formation of this common mutable chromatin structure known to initiate a cascade of genomic instability in cancer.

A critical component of gene regulation is recognition of histones and their post-translational modifications by transcription-associated proteins or complexes. Although many histone-binding reader modules have been characterized, the bromo-adjacent homology (BAH) domain family of readers is still poorly characterized. A pre-eminent member of this family is PBRM1 (BAF180), a component of the PBAF chromatin-remodeling complex. PBRM1 contains two adjacent BAH domains of unknown histone-binding potential. We evaluated the tandem BAH domains for their capacity to associate with histones and to contribute to PBAF-mediated gene regulation. The BAH1 and BAH2 domains of human PBRM1 broadly interacted with histone tails, but they showed a preference for unmodified N-termini of histones H3 and H4. Molecular modeling and comparison of the BAH1 and BAH2 domains with other BAH readers pointed to a conserved binding mode via an extended open pocket and, in general, an aromatic cage for histone lysine binding. Point mutants that were predicted to disrupt the interaction between the BAH domains and histones reduced histone binding in vitro and resulted in dysregulation of genes targeted by PBAF in cellulo. Although the BAH domains in PBRM1 were important for PBAF-mediated gene regulation, we found that overall chromatin targeting of PBRM1 was not dependent on BAH-histone interaction. Our findings identify a function of the PBRM1 BAH domains in PBAF activity that is likely mediated by histone tail interaction., Competing Interests: Conflict of interest B. D. S. is a co-founder and BOD member of EpiCypher, Inc., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

In nucleosomes, histone N-terminal tails exist in dynamic equilibrium between free/accessible and collapsed/DNA-bound states. The latter state is expected to impact histone N-termini availability to the epigenetic machinery. Notably, H3 tail acetylation (e.g. K9ac, K14ac, K18ac) is linked to increased H3K4me3 engagement by the BPTF PHD finger, but it is unknown if this mechanism has a broader extension. Here, we show that H3 tail acetylation promotes nucleosomal accessibility to other H3K4 methyl readers, and importantly, extends to H3K4 writers, notably methyltransferase MLL1. This regulation is not observed on peptide substrates yet occurs on the cis H3 tail, as determined with fully-defined heterotypic nucleosomes. In vivo, H3 tail acetylation is directly and dynamically coupled with cis H3K4 methylation levels. Together, these observations reveal an acetylation 'chromatin switch' on the H3 tail that modulates read-write accessibility in nucleosomes and resolves the long-standing question of why H3K4me3 levels are coupled with H3 acetylation., Competing Interests: KJ, FJ, KP, SC, BT, GF, KN, NY No competing interests declared, MM, JB, SG, IP, NH, AV, SH, HT, LM, UO, EP, EW, MC, MM, MC, ZS is affiliated with EpiCypher, Inc The author has no financial interests to declare, ZG, KR is affiliated with EpiCypher, Inc. The author has no financial interests to declare, KK owns options in EpiCypher, Inc, MC owns stock/serves on the Consultant Advisory Board for Kathera Bioscience Inc and holds266 US patents (8,133,690; 8,715,678; and 10,392,423) for compounds/methods for inhibiting267 SET1/MLL family complexes, MK is a BOD member of EpiCypher Inc, BS is a co-founder and BOD member of EpiCypher, Inc, (© 2023, Jain et al.)

Epigenetic modifications have been gaining in prominence as fundamental components of the chromatin regulatory machinery. In this review, we summarize the molecular and structural mechanisms of reading, writing, and erasing of lysine benzoylation, a recently discovered posttranslational modification (PTM) in histones. We highlight a unique nature of the conjugated π system of benzoyllysine that may aid in the development of benzoyllysine-specific effectors indifferent to the saturated acyllysine modifications. We also discuss transcriptional and metabolic functions associated with benzoylation of histones and implications of ingesting of sodium benzoate for human health., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Histone post-translational modifications (PTMs) are important for regulating various DNA-templated processes. Here, we report the existence of a histone PTM in mammalian cells, namely histone H3 with hydroxylation of proline at residue 16 (H3P16oh), which is catalyzed by the proline hydroxylase EGLN2. We show that H3P16oh enhances direct binding of KDM5A to its substrate, histone H3 with trimethylation at the fourth lysine residue (H3K4me3), resulting in enhanced chromatin recruitment of KDM5A and a corresponding decrease of H3K4me3 at target genes. Genome- and transcriptome-wide analyses show that the EGLN2-KDM5A axis regulates target gene expression in mammalian cells. Specifically, our data demonstrate repression of the WNT pathway negative regulator DKK1 through the EGLN2-H3P16oh-KDM5A pathway to promote WNT/β-catenin signaling in triple-negative breast cancer (TNBC). This study characterizes a regulatory mark in the histone code and reveals a role for H3P16oh in regulating mammalian gene expression., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The Chromosome Passenger Complex (CPC) generates chromosome autonomous signals that regulate mitotic events critical for genome stability. Tip60 is a lysine acetyltransferase that is a tumor suppressor and is targeted for proteasomal degradation by oncogenic papilloma viruses. Mitotic regulation requires the localization of the CPC to inner centromeres, which is driven by the Haspin kinase phosphorylating histone H3 on threonine 3 (H3T3ph). Here we describe how Tip60 acetylates histone H3 at lysine 4 (H3K4ac) to block both the H3T3ph writer and the reader to ensure that this mitotic signaling cannot begin before prophase. Specifically, H3K4ac inhibits Haspin phosphorylation of H3T3 and prevents binding of the Survivin subunit to H3T3ph. Tip60 acetylates H3K4 during S/G2 at centromeres. Inhibition of Tip60 allows the CPC to bind centromeres in G2 cells, and targeting of Tip60 to centromeres prevents CPC localization in mitosis. The H3K4ac mark is removed in prophase by HDAC3 to initiate the CPC localization cascade. Together, our results suggest that Tip60 and HDAC3 temporally control H3K4 acetylation to precisely time the targeting of the CPC to inner centromeres.

The assembly and function of the yeast general transcription factor TFIID complex requires specific contacts between its Taf14 and Taf2 subunits, however, the mechanism underlying these contacts remains unclear. Here, we determined the molecular and structural basis by which the YEATS and ET domains of Taf14 bind to the C-terminal tail of Taf2 and identified a unique DNA-binding activity of the linker region connecting the two domains. We show that in the absence of ligands the linker region of Taf14 is occluded by the surrounding domains, and therefore the DNA binding function of Taf14 is autoinhibited. Binding of Taf2 promotes a conformational rearrangement in Taf14, resulting in a release of the linker for the engagement with DNA and the nucleosome. Genetic in vivo data indicate that the association of Taf14 with both Taf2 and DNA is essential for transcriptional regulation. Our findings provide a basis for deciphering the role of individual TFIID subunits in mediating gene transcription., (© 2022. The Author(s).)

Mono-methylation of histone H4 lysine 20 (H4K20me1) is catalyzed by Set8/KMT5A and regulates numerous aspects of genome organization and function. Loss-of-function mutations in Drosophila melanogaster Set8 or mammalian KMT5A prevent H4K20me1 and disrupt development. Set8/KMT5A also has non-histone substrates, making it difficult to determine which developmental functions of Set8/KMT5A are attributable to H4K20me1 and which to other substrates or to non-catalytic roles. Here, we show that human KMT5A can functionally substitute for Set8 during Drosophila development and that the catalytic SET domains of the two enzymes are fully interchangeable. We also uncovered a role in eye development for the N-terminal domain of Set8 that cannot be complemented by human KMT5A. Whereas Set820/20 null mutants are inviable, we found that an R634G mutation in Set8 predicted from in vitro experiments to ablate catalytic activity resulted in viable adults. Additionally, Set8(R634G) mutants retain significant, albeit reduced, H4K20me1, indicating that the R634G mutation does not eliminate catalytic activity in vivo and is functionally hypomorphic rather than null. Flies engineered to express only unmodifiable H4 histones (H4K20A) can also complete development, but are phenotypically distinct from H4K20R, Set820/20 null, and Set8R634G mutants. Taken together, our results demonstrate functional conservation of KMT5A and Set8 enzymes, as well as distinct roles for Set8 and H4K20me1 in Drosophila development., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Latency reversal strategies for HIV cure using inhibitor of apoptosis protein (IAP) antagonists (IAPi) induce unprecedented levels of latent reservoir expression without immunotoxicity during suppressive antiretroviral therapy (ART). However, full targeting of the reservoir may require combinatorial approaches. A Jurkat latency model screen for IAPi combination partners demonstrated synergistic latency reversal with bromodomain (BD) and extraterminal domain protein inhibitors (BETi). Mechanistic investigations using CRISPR-CAS9 and single-cell RNA-Seq informed comprehensive ex vivo evaluations of IAPi plus pan-BET, bD-selective BET, or selective BET isoform targeting in CD4+ T cells from ART-suppressed donors. IAPi+BETi treatment resulted in striking induction of cell-associated HIV gag RNA, but lesser induction of fully elongated and tat-rev RNA compared with T cell activation-positive controls. IAPi+BETi resulted in HIV protein induction in bulk cultures of CD4+ T cells using an ultrasensitive p24 assay, but did not result in enhanced viral outgrowth frequency using a standard quantitative viral outgrowth assay. This study defines HIV transcriptional elongation and splicing as important barriers to latent HIV protein expression following latency reversal, delineates the roles of BET proteins and their BDs in HIV latency, and provides a rationale for exploration of IAPi+BETi in animal models of HIV latency.

The E3 ubiquitin ligase HERC2 has been linked to neurological diseases and cancer, however it remains a poorly characterized human protein. Here, we show that the ZZ domain of HERC2 (HERC2 ZZ ) recognizes a mimetic of the Nt-R cargo degradation signal. NMR titration experiments and mutagenesis results reveal that the Nt-R mimetic peptide occupies a well-defined binding site of HERC2 ZZ comprising of the negatively charged aspartic acids. We report the crystal structure of the DOC domain of HERC2 (HERC2 DOC ) that is adjacent to HERC2 ZZ and show that a conformational rearrangement in the protein may occur when the two domains are linked. Immunofluorescence microscopy data suggest that the stimulation of autophagy promotes targeting of HERC2 to the proteasome. Our findings suggest a role of cytosolic HERC2 in the ubiquitin-dependent degradation pathways., (© 2022. The Author(s).)

How transcription programs rapidly adjust to changing metabolic and cellular cues remains poorly defined. Here, we reveal a function for the Yaf9 component of the SWR1-C and NuA4 chromatin regulatory complexes in maintaining timely transcription of metabolic genes across the yeast metabolic cycle (YMC). By reading histone acetylation during the oxidative and respiratory phase of the YMC, Yaf9 recruits SWR1-C and NuA4 complexes to deposit H2A.Z and acetylate H4, respectively. Increased H2A.Z and H4 acetylation during the oxidative phase promotes transcriptional initiation and chromatin machinery occupancy and is associated with reduced RNA polymerase II levels at genes-a pattern reversed during transition from oxidative to reductive metabolism. Prevention of Yaf9-H3 acetyl reading disrupted this pattern of transcriptional and chromatin regulator recruitment and impaired the timely transcription of metabolic genes. Together, these findings reveal that Yaf9 contributes to a dynamic chromatin and transcription initiation factor signature that is necessary for the proper regulation of metabolic gene transcription during the YMC. They also suggest that unique regulatory mechanisms of transcription exist at distinct metabolic states., (© 2021 Zhang et al.; Published by Cold Spring Harbor Laboratory Press.)

Bromodomains (BD) are conserved reader modules that bind acetylated lysine residues on histones. Although much has been learned regarding the in vitro properties of these domains, less is known about their function within chromatin complexes. SWI/SNF chromatin-remodeling complexes modulate transcription and contribute to DNA damage repair. Mutations in SWI/SNF subunits have been implicated in many cancers. Here we demonstrate that the BD of Caenorhabditis elegans SMARCA4/BRG1, a core SWI/SNF subunit, recognizes acetylated lysine 14 of histone H3 (H3K14ac), similar to its Homo sapiens ortholog. We identify the interactions of SMARCA4 with the acetylated histone peptide from a 1.29 Å-resolution crystal structure of the CeSMARCA4 BD-H3K14ac complex. Significantly, most of the SMARCA4 BD residues in contact with the histone peptide are conserved with other proteins containing family VIII bromodomains. Based on the premise that binding specificity is conserved among bromodomain orthologs, we propose that loop residues outside of the binding pocket position contact residues to recognize the H3K14ac sequence. CRISPR-Cas9-mediated mutations in the SMARCA4 BD that abolish H3K14ac binding in vitro had little or no effect on C. elegans viability or physiological function in vivo. However, combining SMARCA4 BD mutations with knockdown of the SWI/SNF accessory subunit PBRM-1 resulted in severe developmental defects in animals. In conclusion, we demonstrated an essential function for the SWI/SNF bromodomain in vivo and detected potential redundancy in epigenetic readers in regulating chromatin remodeling. These findings have implications for the development of small-molecule BD inhibitors to treat cancers and other diseases., Competing Interests: Conflict of interest B. D. S. is a cofounder of EpiCypher, KK has an equity interest in EpiCypher., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Measuring protein-protein interaction (PPI) affinities is fundamental to biochemistry. Yet, conventional methods rely upon the law of mass action and cannot measure many PPIs due to a scarcity of reagents and limitations in the measurable affinity ranges. Here, we present a novel technique that leverages the fundamental concept of friction to produce a mechanical signal that correlates to binding potential. The mechanically transduced immunosorbent (METRIS) assay utilizes rolling magnetic probes to measure PPI interaction affinities. METRIS measures the translational displacement of protein-coated particles on a protein-functionalized substrate. The translational displacement scales with the effective friction induced by a PPI, thus producing a mechanical signal when a binding event occurs. The METRIS assay uses as little as 20 pmols of reagents to measure a wide range of affinities while exhibiting a high resolution and sensitivity. We use METRIS to measure several PPIs that were previously inaccessible using traditional methods, providing new insights into epigenetic recognition., Competing Interests: CP, KR, MP, DS, BS, JH No competing interests declared, JS is a cofounder of Tribosense Technologies, (© 2021, Petell et al.)

DNA methylation and trimethylated histone H4 Lysine 20 (H4K20me3) constitute two important heterochromatin-enriched marks that frequently cooperate in silencing repetitive elements of the mammalian genome. However, it remains elusive how these two chromatin modifications crosstalk. Here, we report that DNA methyltransferase 1 (DNMT1) specifically 'recognizes' H4K20me3 via its first bromo-adjacent-homology domain (DNMT1 BAH1 ). Engagement of DNMT1 BAH1 -H4K20me3 ensures heterochromatin targeting of DNMT1 and DNA methylation at LINE-1 retrotransposons, and cooperates with the previously reported readout of histone H3 tail modifications (i.e., H3K9me3 and H3 ubiquitylation) by the RFTS domain to allosterically regulate DNMT1's activity. Interplay between RFTS and BAH1 domains of DNMT1 profoundly impacts DNA methylation at both global and focal levels and genomic resistance to radiation-induced damage. Together, our study establishes a direct link between H4K20me3 and DNA methylation, providing a mechanism in which multivalent recognition of repressive histone modifications by DNMT1 ensures appropriate DNA methylation patterning and genomic stability.

Fueled by key technological innovations during the last several decades, chromatin-based research has greatly advanced our mechanistic understanding of how genes are regulated by epigenetic factors and their associated histone-modifying activities. Most notably, the landmark finding that linked histone acetylation by Gcn5 of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex to gene activation ushered in a new area of chromatin research and a realization that histone-modifying activities have integral genome functions. This review will discuss past and recent studies that have shaped our understanding of how the histone-modifying activities of SAGA are regulated by, and modulate the outcomes of, other histone modifications during gene transcription. Because much of our understanding of SAGA was established with budding yeast, we will focus on yeast as a model. We discuss the actions of cis- and trans-histone crosstalk pathways that involve the histone acetyltransferase, deubiquitylase, and reader domains of SAGA. We conclude by considering unanswered questions about SAGA and related complexes., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Histone acetylation levels are regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) that antagonistically control the overall balance of this post-translational modification. HDAC inhibitors (HDACi) are potent agents that disrupt this balance and are used clinically to treat diseases including cancer. Despite their use, little is known about their effects on chromatin regulators, particularly those that signal through lysine acetylation. We apply quantitative genomic and proteomic approaches to demonstrate that HDACi robustly increases a low-abundance histone 4 polyacetylation state, which serves as a preferred binding substrate for several bromodomain-containing proteins, including BRD4. Increased H4 polyacetylation occurs in transcribed genes and correlates with the targeting of BRD4. Collectively, these results suggest that HDAC inhibition functions, at least in part, through expansion of a rare histone acetylation state, which then retargets lysine-acetyl readers associated with changes in gene expression, partially mimicking the effect of bromodomain inhibition., Competing Interests: Declaration of interests B.D.S. is a cofounder and scientific advisory board member of EpiCypher, Inc., and a scientific advisory board member of Triangle Biotechnology, Inc. I.J.D. is a scientific advisory board member of Triangle Biotechnology, Inc., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

DNA double-strand breaks (DSBs) are among the deleterious lesions that are both endogenous and exogenous in origin and are repaired by nonhomologous end joining or homologous recombination. However, the molecular mechanisms responsible for maintaining genome stability remain incompletely understood. Here, we investigate the role of two E3 ligases, Dma1 and Dma2 (homologs of human RNF8), in the maintenance of genome stability in budding yeast. Using yeast spotting assays, chromatin immunoprecipitation and plasmid and chromosomal repair assays, we establish that Dma1 and Dma2 act in a redundant and a catalysis-dependent manner in the maintenance of genome stability, as well as localize to transcribed regions of the genome and increase in abundance upon phleomycin treatment. In addition, Dma1 and Dma2 are required for the normal kinetics of histone H4 acetylation under DNA damage conditions, genetically interact with RAD9 and SAE2, and are in a complex with Rad53 and histones. Taken together, our results demonstrate the requirement of Dma1 and Dma2 in regulating DNA repair pathway choice, preferentially affecting homologous recombination over nonhomologous end joining, and open up the possibility of using these candidates in manipulating the repair pathways toward precision genome editing., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Determining structures of protein complexes is crucial for understanding cellular functions. Here, we describe an integrative structure determination approach that relies on in vivo measurements of genetic interactions. We construct phenotypic profiles for point mutations crossed against gene deletions or exposed to environmental perturbations, followed by converting similarities between two profiles into an upper bound on the distance between the mutated residues. We determine the structure of the yeast histone H3-H4 complex based on ~500,000 genetic interactions of 350 mutants. We then apply the method to subunits Rpb1-Rpb2 of yeast RNA polymerase II and subunits RpoB-RpoC of bacterial RNA polymerase. The accuracy is comparable to that based on chemical cross-links; using restraints from both genetic interactions and cross-links further improves model accuracy and precision. The approach provides an efficient means to augment integrative structure determination with in vivo observations., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Trimethylated histone H3 lysine 27 (H3K27me3) regulates gene repression, cell-fate determination and differentiation. We report that a conserved bromo-adjacent homology (BAH) module of BAHCC1 (BAHCC1 BAH ) 'recognizes' H3K27me3 specifically and enforces silencing of H3K27me3-demarcated genes in mammalian cells. Biochemical, structural and integrated chromatin immunoprecipitation-sequencing-based analyses demonstrate that direct readout of H3K27me3 by BAHCC1 is achieved through a hydrophobic trimethyl-L-lysine-binding 'cage' formed by BAHCC1 BAH , mediating colocalization of BAHCC1 and H3K27me3-marked genes. BAHCC1 is highly expressed in human acute leukemia and interacts with transcriptional corepressors. In leukemia, depletion of BAHCC1, or disruption of the BAHCC1 BAH -H3K27me3 interaction, causes derepression of H3K27me3-targeted genes that are involved in tumor suppression and cell differentiation, leading to suppression of oncogenesis. In mice, introduction of a germline mutation at Bahcc1 to disrupt its H3K27me3 engagement causes partial postnatal lethality, supporting a role in development. This study identifies an H3K27me3-directed transduction pathway in mammals that relies on a conserved BAH 'reader'.

Histone H3 lysine 36 methylation (H3K36me) is a conserved histone modification associated with transcription and DNA repair. Although the effects of H3K36 methylation have been studied, the genome-wide dynamics of H3K36me deposition and removal are not known. We established rapid and reversible optogenetic control for Set2, the sole H3K36 methyltransferase in yeast, by fusing the enzyme with the light-activated nuclear shuttle (LANS) domain. Light activation resulted in efficient Set2-LANS nuclear localization followed by H3K36me3 deposition in vivo, with total H3K36me3 levels correlating with RNA abundance. Although genes showed disparate levels of H3K36 methylation, relative rates of H3K36me3 accumulation were largely linear and consistent across genes, suggesting that H3K36me3 deposition occurs in a directed fashion on all transcribed genes regardless of their overall transcription frequency. Removal of H3K36me3 was highly dependent on the demethylase Rph1. However, the per-gene rate of H3K36me3 loss weakly correlated with RNA abundance and followed exponential decay, suggesting H3K36 demethylases act in a global, stochastic manner. Altogether, these data provide a detailed temporal view of H3K36 methylation and demethylation that suggests transcription-dependent and -independent mechanisms for H3K36me deposition and removal, respectively., (© 2020 Lerner et al.; Published by Cold Spring Harbor Laboratory Press.)

Human Microrchidia 4 (MORC4) is associated with acute and chronic pancreatitis, inflammatory disorders and cancer but it remains largely uncharacterized. Here, we describe the structure-function relationship of MORC4 and define the molecular mechanism for MORC4 activation. Enzymatic and binding assays reveal that MORC4 has ATPase activity, which is dependent on DNA-binding functions of both the ATPase domain and CW domain of MORC4. The crystal structure of the ATPaseCW cassette of MORC4 and mutagenesis studies show that the DNA-binding site and the histone/ATPase binding site of CW are located on the opposite sides of the domain. The ATPase and CW domains cooperate in binding of MORC4 to the nucleosome core particle (NCP), enhancing the DNA wrapping around the histone core and impeding binding of DNA-associated proteins, such as transcription factors, to the NCP. In cells, MORC4 mediates formation of nuclear bodies in the nucleus and has a role in the progression of S-phase of the cell cycle, and both these functions require CW and catalytic activity of MORC4. Our findings highlight the mechanism for MORC4 activation, which is distinctly different from the mechanisms of action observed in other MORC family members.

The chromatin-binding E3 ubiquitin ligase ubiquitin-like with PHD and RING finger domains 1 (UHRF1) contributes to the maintenance of aberrant DNA methylation patterning in cancer cells through multivalent histone and DNA recognition. The tandem Tudor domain (TTD) of UHRF1 is well-characterized as a reader of lysine 9 di- and tri-methylation on histone H3 (H3K9me2/me3) and, more recently, lysine 126 di- and tri-methylation on DNA ligase 1 (LIG1K126me2/me3). However, the functional significance and selectivity of these interactions remain unclear. In this study, we used protein domain microarrays to search for additional readers of LIG1K126me2, the preferred methyl state bound by the UHRF1 TTD. We show that the UHRF1 TTD binds LIG1K126me2 with high affinity and selectivity compared to other known methyllysine readers. Notably, and unlike H3K9me2/me3, the UHRF1 plant homeodomain (PHD) and its N-terminal linker (L2) do not contribute to multivalent LIG1K126me2 recognition along with the TTD. To test the functional significance of this interaction, we designed a LIG1K126me2 cell-penetrating peptide (CPP). Consistent with LIG1 knockdown, uptake of the CPP had no significant effect on the propagation of DNA methylation patterning across the genomes of bulk populations from high-resolution analysis of several cancer cell lines. Further, we did not detect significant changes in DNA methylation patterning from bulk cell populations after chemical or genetic disruption of lysine methyltransferase activity associated with LIG1K126me2 and H3K9me2. Collectively, these studies identify UHRF1 as a selective reader of LIG1K126me2 in vitro and further implicate the histone and non-histone methyllysine reader activity of the UHRF1 TTD as a dispensable domain function for cancer cell DNA methylation maintenance.

In mammals, repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3), frequently coexist with DNA methylation, producing a more stable and silenced chromatin state. However, it remains elusive how these epigenetic modifications crosstalk. Here, through structural and biochemical characterizations, we identified the replication foci targeting sequence (RFTS) domain of maintenance DNA methyltransferase DNMT1, a module known to bind the ubiquitylated H3 (H3Ub), as a specific reader for H3K9me3/H3Ub, with the recognition mode distinct from the typical trimethyl-lysine reader. Disruption of the interaction between RFTS and the H3K9me3Ub affects the localization of DNMT1 in stem cells and profoundly impairs the global DNA methylation and genomic stability. Together, this study reveals a previously unappreciated pathway through which H3K9me3 directly reinforces DNMT1-mediated maintenance DNA methylation., Competing Interests: Competing interest statement: O.G. and B.D.S are cofounders of EpiCypher, Inc.

A hallmark of human immunodeficiency type-1 (HIV) infection is the integration of the viral genome into host chromatin, resulting in a latent reservoir that persists despite antiviral therapy or immune response. Thus, key priorities toward eradication of HIV infection are to understand the mechanisms that allow HIV latency and to develop latency reversal agents (LRAs) that can facilitate the clearance of latently infected cells. The repressive H3K27me3 histone mark, catalyzed by the PRC2 complex, plays a pivotal role in transcriptional repression at the viral promoter in both cell line and primary CD4+ T cell models of latency. EZH2 inhibitors which block H3K27 methylation have been shown to act as LRAs, suggesting other PRC2 components could also be potential targets for latency reversal. EED, a core component of PRC2, ensures the propagation of H3K27me3 by allosterically activating EZH2 methyltransferase activity. Therefore, we sought to investigate if inhibition of EED would also reverse latency. Inhibitors of EED, EED226 and A-395, demonstrated latency reversal activity as single agents, and this activity was further enhanced when used in combination with other known LRAs. Loss of H3K27me3 following EED inhibition significantly increased the levels of H3K27 acetylation globally and at the HIV LTR. These results further confirm that PRC2 mediated repression plays a significant role in the maintenance of HIV latency and suggest that EED may serve as a promising new target for LRA development.

Set2 co-transcriptionally methylates lysine 36 of histone H3 (H3K36), producing mono-, di-, and trimethylation (H3K36me1/2/3). These modifications recruit or repel chromatin effector proteins important for transcriptional fidelity, mRNA splicing, and DNA repair. However, it was not known whether the different methylation states of H3K36 have distinct biological functions. Here, we use engineered forms of Set2 that produce different lysine methylation states to identify unique and shared functions for H3K36 modifications. Although H3K36me1/2 and H3K36me3 are functionally redundant in many SET2 deletion phenotypes, we found that H3K36me3 has a unique function related to Bur1 kinase activity and FACT (facilitates chromatin transcription) complex function. Further, during nutrient stress, either H3K36me1/2 or H3K36me3 represses high levels of histone acetylation and cryptic transcription that arises from within genes. Our findings uncover the potential for the regulation of diverse chromatin functions by different H3K36 methylation states., Competing Interests: Declaration of Interests B.D.S. acknowledges he is a co-founder and scientific advisory board member of EpiCypher, Inc., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Histone H2B monoubiquitylation (H2Bub1) has central functions in multiple DNA-templated processes, including gene transcription, DNA repair, and replication. H2Bub1 also is required for the trans -histone regulation of H3K4 and H3K79 methylation. Although previous studies have elucidated the basic mechanisms that establish and remove H2Bub1, we have only an incomplete understanding of how H2Bub1 is regulated. We report here that the histone H4 basic patch regulates H2Bub1. Yeast cells with arginine-to-alanine mutations in the H4 basic patch (H4 2RA ) exhibited a significant loss of global H2Bub1. H4 2RA mutant yeast strains also displayed chemotoxin sensitivities similar to, but less severe than, strains containing a complete loss of H2Bub1. We found that the H4 basic patch regulates H2Bub1 levels independently of interactions with chromatin remodelers and separately from its regulation of H3K79 methylation. To measure H2B ubiquitylation and deubiquitination kinetics in vivo , we used a rapid and reversible optogenetic tool, the light-inducible nuclear exporter, to control the subcellular location of the H2Bub1 E3 ligase, Bre1. The ability of Bre1 to ubiquitylate H2B was unaffected in the H4 2RA mutant. In contrast, H2Bub1 deubiquitination by SAGA-associated Ubp8, but not by Ubp10, increased in the H4 2RA mutant. Consistent with a function for the H4 basic patch in regulating SAGA deubiquitinase activity, we also detected increased SAGA-mediated histone acetylation in H4 basic patch mutants. Our findings uncover that the H4 basic patch has a regulatory function in SAGA-mediated histone modifications., (© 2020 Meriesh et al.)

Background: Plant homeodomain (PHD) fingers are central "readers" of histone post-translational modifications (PTMs) with > 100 PHD finger-containing proteins encoded by the human genome. Many of the PHDs studied to date bind to unmodified or methylated states of histone H3 lysine 4 (H3K4). Additionally, many of these domains, and the proteins they are contained in, have crucial roles in the regulation of gene expression and cancer development. Despite this, the majority of PHD fingers have gone uncharacterized; thus, our understanding of how these domains contribute to chromatin biology remains incomplete., Results: We expressed and screened 123 of the annotated human PHD fingers for their histone binding preferences using reader domain microarrays. A subset (31) of these domains showed strong preference for the H3 N-terminal tail either unmodified or methylated at H3K4. These H3 readers were further characterized by histone peptide microarrays and/or AlphaScreen to comprehensively define their H3 preferences and PTM cross-talk., Conclusions: The high-throughput approaches utilized in this study establish a compendium of binding information for the PHD reader family with regard to how they engage histone PTMs and uncover several novel reader domain-histone PTM interactions (i.e., PHRF1 and TRIM66). This study highlights the usefulness of high-throughput analyses of histone reader proteins as a means of understanding how chromatin engagement occurs biochemically.

Histone post-translational modifications (PTMs) are crucial for many cellular processes including mitosis, transcription, and DNA repair. The cellular readout of histone PTMs is dependent on both the chemical modification and histone site, and the array of histone PTMs on chromatin is dynamic throughout the eukaryotic life cycle. Accordingly, methods that report on the presence of PTMs are essential tools for resolving open questions about epigenetic processes and for developing therapeutic diagnostics. Reader domains that recognize histone PTMs have shown potential as advantageous substitutes for anti-PTM antibodies, and engineering efforts aimed at enhancing reader domain affinities would advance their efficacy as antibody alternatives. Here we describe engineered chromodomains from Drosophila melanogaster and humans that bind more tightly to H3K9 methylation (H3K9me) marks and result in the tightest reported reader-H3K9me interaction to date. Point mutations near the binding interface of the HP1 chromodomain were screened in a combinatorial fashion, and a triple mutant was found that binds 20-fold tighter than the native scaffold without any loss in PTM-site selectivity. The beneficial mutations were then translated to a human homologue, CBX1, resulting in an even tighter interaction with H3K9me3. Furthermore, we show that these engineered readers ( e Readers) increase detection of H3K9me marks in several biochemical assays and outperform a commercial anti-H3K9me antibody in detecting H3K9me-containing nucleosomes in vitro , demonstrating the utility of e Readers to complement antibodies in epigenetics research.

Metabolic signaling to chromatin often underlies how adaptive transcriptional responses are controlled. While intermediary metabolites serve as co-factors for histone-modifying enzymes during metabolic flux, how these modifications contribute to transcriptional responses is poorly understood. Here, we utilize the highly synchronized yeast metabolic cycle (YMC) and find that fatty acid β-oxidation genes are periodically expressed coincident with the β-oxidation byproduct histone crotonylation. Specifically, we found that H3K9 crotonylation peaks when H3K9 acetylation declines and energy resources become limited. During this metabolic state, pro-growth gene expression is dampened; however, mutation of the Taf14 YEATS domain, a H3K9 crotonylation reader, results in de-repression of these genes. Conversely, exogenous addition of crotonic acid results in increased histone crotonylation, constitutive repression of pro-growth genes, and disrupted YMC oscillations. Together, our findings expose an unexpected link between metabolic flux and transcription and demonstrate that histone crotonylation and Taf14 participate in the repression of energy-demanding gene expression., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Acetylation of histone H3K23 has emerged as an essential posttranslational modification associated with cancer and learning and memory impairment, yet our understanding of this epigenetic mark remains insufficient. Here, we identify the native MORF complex as a histone H3K23-specific acetyltransferase and elucidate its mechanism of action. The acetyltransferase function of the catalytic MORF subunit is positively regulated by the DPF domain of MORF (MORF DPF ). The crystal structure of MORF DPF in complex with crotonylated H3K14 peptide provides mechanistic insight into selectivity of this epigenetic reader and its ability to recognize both histone and DNA. ChIP data reveal the role of MORF DPF in MORF-dependent H3K23 acetylation of target genes. Mass spectrometry, biochemical and genomic analyses show co-existence of the H3K23ac and H3K14ac modifications in vitro and co-occupancy of the MORF complex, H3K23ac, and H3K14ac at specific loci in vivo. Our findings suggest a model in which interaction of MORF DPF with acylated H3K14 promotes acetylation of H3K23 by the native MORF complex to activate transcription.

Covalent post-translational modification (PTM) of proteins with acyl groups of various carbon chain-lengths regulates diverse biological processes ranging from chromatin dynamics to subcellular localization. While the YEATS domain has been found to be a prominent reader of acetylation and other short acyl modifications, whether additional acyl-lysine reader domains exist, particularly for longer carbon chains, is unclear. Here, we employed a quantitative proteomic approach using various modified peptide baits to identify reader proteins of various acyl modifications. We discovered that proteins harboring HEAT and ARM repeats bind to lysine myristoylated peptides. Recombinant HEAT and ARM repeats bind to myristoylated peptides independent of the peptide sequence or the position of the myristoyl group. Indeed, HEAT and ARM repeats bind directly to medium- and long-chain free fatty acids (MCFA and LCFA). Lipidomic experiments suggest that MCFAs and LCFAs interact with HEAT and ARM repeat proteins in mammalian cells. Finally, treatment of cells with exogenous MCFAs and inhibitors of MCFA-CoA synthases increase the transactivation activity of the ARM repeat protein β-catenin. Taken together, our results suggest an unappreciated role for fatty acids in the regulation of proteins harboring HEAT or ARM repeats.

Histone methyltransferase MLL4 is centrally involved in transcriptional regulation and is often mutated in human diseases, including cancer and developmental disorders. MLL4 contains a catalytic SET domain that mono-methylates histone H3K4 and seven PHD fingers of unclear function. Here, we identify the PHD6 finger of MLL4 (MLL4-PHD6) as a selective reader of the epigenetic modification H4K16ac. The solution NMR structure of MLL4-PHD6 in complex with a H4K16ac peptide along with binding and mutational analyses reveal unique mechanistic features underlying recognition of H4K16ac. Genomic studies show that one third of MLL4 chromatin binding sites overlap with H4K16ac-enriched regions in vivo and that MLL4 occupancy in a set of genomic targets depends on the acetyltransferase activity of MOF, a H4K16ac-specific acetyltransferase. The recognition of H4K16ac is conserved in the PHD7 finger of paralogous MLL3. Together, our findings reveal a previously uncharacterized acetyllysine reader and suggest that selective targeting of H4K16ac by MLL4 provides a direct functional link between MLL4, MOF and H4K16 acetylation.

Reactive and clonal neutrophil expansion has been associated with thrombosis, suggesting that neutrophils play a role in this process. However, although there is no doubt that activated monocytes trigger coagulation in a tissue factor-dependent manner, it remains uncertain whether stimulated neutrophils can also directly activate coagulation. After more than a decade of debate, it is now largely accepted that normal human neutrophils do not synthetize tissue factor, the initiator of the extrinsic pathway of coagulation. However, neutrophils may passively acquire tissue factor from monocytes. Recently, the contact system, which initiates coagulation via the intrinsic pathway, has been implicated in the pathogenesis of thrombosis. After the recent description of neutrophil extracellular trap (NET) release by activated neutrophils, some animal models of thrombosis have demonstrated that coagulation may be enhanced by direct NET-dependent activation of the contact system. However, there is currently no consensus on how to assess or quantify NETosis in vivo, and other experimental animal models have failed to demonstrate a role for neutrophils in thrombogenesis. Nevertheless, it is likely that NETs can serve to localize other circulating coagulation components and can also promote vessel occlusion independent of fibrin formation. This article provides a critical appraisal of the possible roles of neutrophils in thrombosis and highlights some existing knowledge gaps regarding the procoagulant activities of neutrophil-derived extracellular chromatin and its molecular components. A better understanding of these mechanisms could guide future approaches to prevent and/or treat thrombosis., (© 2019 by The American Society of Hematology.)

Histone post-translational modifications contribute to chromatin function largely through the recruitment of effector proteins that contain specialized "reader" domains. While a significant number of reader domains have been characterized for their histone binding specificities, many of these domains remain poorly characterized. Peptide microarrays have been widely employed for the characterization of histone readers, as well as modifying enzymes and histone antibodies. While powerful, this platform has limitations in terms of its sensitivity and they frequently miss low affinity reader domain interactions. Here, we provide several technical changes that improve reader domain detection of low-affinity interactions. We show that 1% non-fat milk in 1X PBST as the blocking reagent during incubation improved reader-domain interaction results. Further, coupling this with post-binding high-salt washes and a brief, low-percentage formaldehyde cross-linking step prior to the high-salt washes provided the optimal balance between resolving specific low-affinity interactions and minimizing background or spurious signals. We expect this improved methodology will lead to the elucidation of previously unreported reader-histone interactions that will be important for chromatin function.

Proper determination of cell fates depends on epigenetic information that is used to preserve memory of decisions made earlier in development. Post-translational modification of histone residues is thought to be a central means by which epigenetic information is propagated. In particular, modifications of histone H3 lysine 27 (H3K27) are strongly correlated with both gene activation and gene repression. H3K27 acetylation is found at sites of active transcription, whereas H3K27 methylation is found at loci silenced by Polycomb group proteins. The histones bearing these modifications are encoded by the replication-dependent H3 genes as well as the replication-independent H3.3 genes. Owing to differential rates of nucleosome turnover, H3K27 acetylation is enriched on replication-independent H3.3 histones at active gene loci, and H3K27 methylation is enriched on replication-dependent H3 histones across silenced gene loci. Previously, we found that modification of replication-dependent H3K27 is required for Polycomb target gene silencing, but it is not required for gene activation. However, the contribution of replication-independent H3.3K27 to these functions is unknown. Here, we used CRISPR/Cas9 to mutate the endogenous replication-independent H3.3K27 to a non-modifiable residue. Surprisingly, we find that H3.3K27 is also required for Polycomb target gene silencing despite the association of H3.3 with active transcription. However, the requirement for H3.3K27 comes at a later stage of development than that found for replication-dependent H3K27, suggesting a greater reliance on replication-independent H3.3K27 in post-mitotic cells. Notably, we find no evidence of global transcriptional defects in H3.3K27 mutants, despite the strong correlation between H3.3K27 acetylation and active transcription., Competing Interests: The authors have declared that no competing interests exist.