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The maintenance of gene expression patterns during metazoan development is achieved by the actions of Polycomb group (PcG) complexes. An essential modification marking silenced genes is monoubiquitination of histone H2A lysine 119 (H2AK119Ub) deposited by the E3 ubiquitin ligase activity of the non-canonical Polycomb Repressive Complex 1. The Polycomb Repressive Deubiquitinase (PR-DUB) complex cleaves monoubiquitin from histone H2A lysine 119 (H2AK119Ub) to restrict focal H2AK119Ub at Polycomb target sites and to protect active genes from aberrant silencing. BAP1 and ASXL1, subunits that form active PR-DUB, are among the most frequently mutated epigenetic factors in human cancers, underscoring their biological importance. How PR-DUB achieves specificity for H2AK119Ub to regulate Polycomb silencing is unknown, and the mechanisms of most of the mutations in BAP1 and ASXL1 found in cancer have not been established. Here we determine a cryo-EM structure of human BAP1 bound to the ASXL1 DEUBAD domain in complex with a H2AK119Ub nucleosome. Our structural, biochemical, and cellular data reveal the molecular interactions of BAP1 and ASXL1 with histones and DNA that are critical for remodeling the nucleosome and thus establishing specificity for H2AK119Ub. These results further provide a molecular explanation for how >50 mutations in BAP1 and ASXL1 found in cancer can dysregulate H2AK119Ub deubiquitination, providing new insight into understanding cancer etiology.One Sentence SummaryWe reveal the molecular mechanism of nucleosomal H2AK119Ub deubiquitination by human BAP1/ASXL1.

Chromatin remodeling is mediated by ATP-dependent enzymes that play key roles regulating gene expression and genome replication/repair. Aberrant nucleosome organization from dysregulated chromatin remodeling can severely alter chromatin accessibility and disrupt these important processes, thereby driving various cancers. Remarkably, nearly 20% of all human cancers contain mutations in subunits from the SWI/SNF family of chromatin remodeling complexes, making them of great interest to basic research and therapeutic intervention. In vitro studies on the remodeling enzymes (and their multi-subunit complexes) are challenging, partially due to the strong preference for nucleosome-based substrates (the physiological target of these enzymes). We have created the EpiDyne® nucleosome portfolio to examine chromatin remodeler activity in biochemical assays, and here present the development of novel readouts (-PicoGreenTM and -TR-FRET). These nonradioactive plate-based assays are automation adaptable, ready for high-throughput inhibitor screening, and can be customized for various remodeling enzymes that exhibit preferences in nucleosome composition (e.g. histone type or DNA linker length).For parallel in vivo studies we note that genome-wide remodeler localization and open chromatin mapping are fundamental for understanding the function/activity of these enzymes in cancer development and inhibitor response. However, traditional genomic approaches have significant issues: e.g. ChIP-seq is unable to effectively map ATPases without heavily modified high-noise protocols; while ATAC-seq to map open regions cannot deal with cross-linking that could stabilize transient states on interest. To this end, we have optimized the CUTANA™ CUT&RUN approach to efficiently capture the localization of all major classes of chromatin remodelers with high signal to background. We have also adopted NicE-seq for chromatin accessibility profiling in cross-linked material. As complementary tools to the EpiDyne platform, CUT&RUN and NicE-seq facilitate epigenomic research on chromatin remodelers in cancer therapeutic intervention. Keywords: Chromatin Remodeling, EpiDyne, Nucleosome repositioning, high-throughput screening (HTS), CUTANA™ CUT&RUN, NicE-seq Citation Format: Lu Sun, Hannah Willis, Matthew R. Marunde, Vishnu U. Kumary, Matthew J. Meiners, Saarang Gopinath, Jonathan M. Burg, Bryan J. Venters, Allison Hickman, Zu-Wen Sun, Martis W. Cowles, Pierre Esteve, Hang Gyeong Chin, Chaithanya Ponnaluri, Sriharsa Pradhan, Michael-Christopher Keogh. Biochemical and genomic approaches for high throughput drug discovery in chromatin remodeling research. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4728.

Background Recent data propose a diagnostic and prognostic capacity for citrullinated histone H3 (H3Cit), a marker of neutrophil extracellular traps (NETs), in pathologic conditions such as cancer and thrombosis. However, current research is hampered by lack of standardized assays. Objectives We aimed to develop an assay to reliably quantify nucleosomal H3Cit in human plasma. Methods We assessed the common practice of in vitro enzymatically modified histone H3 as calibration standards and the specificity of available intrapeptidyl citrulline antibodies. Based on our findings, we developed and validated a novel assay to quantify nucleosomal H3Cit in human plasma. Results We show that enzymatically citrullinated H3 proteins are compromised by high enzyme-dependent lot variability as well as instability in plasma. We furthermore demonstrate that the majority of commercially available antibodies against intrapeptidyl citrulline display poor specificity for their reported target when tested against a panel of semi-synthetic nucleosomes containing distinct histone H3 citrullinations. Finally, we present a novel assay utilizing highly specific monoclonal antibodies and semi-synthetic nucleosomes containing citrulline in place of arginine at histone H3, arginine residues 2, 8, and 17 (H3R2,8,17Cit) as calibration standards. Rigorous validation of this assay shows its capacity to accurately and reliably quantify nucleosomal H3Cit levels in human plasma with clear elevations in cancer patients compared to healthy individuals. Conclusions Our novel approach using defined nucleosome controls enables reliable quantification of H3Cit in human plasma. This assay will be broadly applicable to study the role of histone citrullination in disease and its utility as a biomarker.

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 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 thecisH3 tail, as determined with fully-defined heterotypic nucleosomes.In vivo, H3 tail acetylation is directly and dynamically coupled withcisH3K4 methylation levels. Together, these observations reveal an acetylation ‘chromatin switch’ on the H3 tail that modulates read-write accessibility in nucleosomes and resolve the long-standing question of why H3K4me3 levels are coupled with H3 acetylation.

Histone post-translational modifications (PTMs) play a critical role in chromatin regulation. It has been proposed that these PTMs form localized ‘codes’ that are read by specialized regions (reader domains) in chromatin associated proteins (CAPs) to regulate downstream function. Substantial effort has been made to define [CAP-histone PTM] specificity, and thus decipher the histone code / guide epigenetic therapies. However, this has largely been done using a reductive approach of isolated reader domains and histone peptides, with the assumption that PTM readout is unaffected by any higher order factors. Here we show that CAP-histone PTM interaction is in fact dependent on nucleosome context. Our results indicate this is due to histone tail accessibility and the associated impact on binding potential of reader domains. We further demonstrate that the in vitro specificity of a tandem reader for PTM-defined nucleosomes is recapitulated in a cellular context. This necessitates we refine the ‘histone code’ concept and interrogate it at the nucleosome level.

Chromatin remodeling, driven by ATP-dependent remodeling enzymes, plays key roles in regulating gene expression, genome replication and repair. Aberrant nucleosome organization can severely disrupt these important processes, driving various cancers and developmental disorders. Remarkably, nearly 20% of all human cancers contain mutations in subunits from the SWI/SNF family of chromatin remodeling complexes, making them attractive therapeutic targets and interests of basic research. However, studies on the remodeling enzymes (and their multi-subunit complexes) in vitro are challenging partially due to the requirement for nucleosome-based substrates. We have created the EpiDyne® nucleosome remodeling substrate portfolio for use in biochemical assays to examine nucleosome immobilizing activities of chromatin remodelers. Here we present the development of multiple remodeling assays on the EpiDyne® platform (EpiDyne®-FRET, EpiDyne®-PicoGreenTM and EpiDyne®-TR-FRET). These nonradioactive plate-based assays are easy to use, ready for high-throughput inhibitor screening, and can be customized for various remodeling enzymes that exhibit distinct preferences in nucleosome compositions. Furthermore, genome-wide mapping of chromatin remodelers is fundamental for understanding their functions in diseases and responses to inhibitors in cells, but is often obscured by unreliable ChIP-seq approaches. To this end, we have optimized the CUTANA™ CUT&RUN approach to efficiently capture localizations of all major classes of chromatin remodelers with high signal to background. As a complementary research tool to the EpiDyne® platform, CUT&RUN facilitates the epigenomic research on chromatin remodelers for drug discoveries. Citation Format: Natalia L. Husby, Lu Sun, Hannah Willis, Matthew R. Marunde, Matt J. Meiners, Zachary Gillespie, Rachel Watson, Saarang Gopinath, Jonathan M. Burg, Marcus A. Cheek, Katherine Novitzky, Bryan J. Venters, Zu-Wen Sun, Michael-Christopher Keogh. Versatile biochemical and genomic platforms for drug discovery in chromatin remodeling research. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B015.

Mutations in thePHIP/BRWD2chromatin regulator cause the human neurodevelopmental disorder Chung-Jansen syndrome, while alterations inPHIPexpression are linked to cancer. Precisely how PHIP functions in these contexts is not fully understood. Here we demonstrate that PHIP is a chromatin-associated CRL4 ubiquitin ligase substrate receptor and is required for CRL4 recruitment to chromatin. PHIP binds to chromatin through a trivalent reader domain consisting of a H3K4-methyl binding Tudor domain and two bromodomains (BD1 and BD2). Using semisynthetic nucleosomes with defined histone post-translational modifications, we characterize PHIPs BD1 and BD2 as respective readers of H3K14ac and H4K12ac, and identify human disease-associated mutations in each domain and the intervening linker region that likely disrupt chromatin binding. These findings provide new insight into the biological function of this enigmatic chromatin protein and set the stage for the identification of both upstream chromatin modifiers and downstream targets of PHIP in human disease.

CPAF (chlamydial protease-like activity factor) is a Chlamydia trachomatis protease that is translocated into the host cytosol during infection. CPAF activity results in dampened host inflammation signaling, cytoskeletal remodeling, and suppressed neutrophil activation. Although CPAF is an emerging antivirulence target, its catalytic mechanism has been unexplored to date. Steady state kinetic parameters were obtained for recombinant CPAF with vimentin-derived peptide substrates using a high-performance liquid chromatography-based discontinuous assay (kcat = 45 ± 0.6 s-1; kcat/Km = 0.37 ± 0.02 μM-1 s-1) or a new fluorescence-based continuous assay (kcat = 23 ± 0.7 s-1; kcat/Km = 0.29 ± 0.03 μM-1 s-1). Residues H105, S499, E558, and newly identified D103 were found to be indispensable for autoproteolytic processing by mutagenesis, while participation of C500 was ruled out despite its proximity to the S499 nucleophile. Pre-steady state kinetics indicated a burst kinetic profile, with fast acylation (kacyl = 110 ± 2 s-1) followed by slower, partially rate-limiting deacylation (kdeacyl = 57 ± 1 s-1). Both kcat- and kcat/Km-pH profiles showed single acidic limb ionizations with pKa values of 6.2 ± 0.1 and 6.5 ± 0.1, respectively. A forward solvent deuterium kinetic isotope effect of 2.6 ± 0.1 was observed for D2Okcatapp, but a unity effect was found for D2Okcat/Kmapp. The kcat proton inventory was linear, indicating transfer of a single proton in the rate-determining transition state, most likely from H105. Collectively, these data provide support for the classification of CPAF as a serine protease and provide a mechanistic foundation for the future design of inhibitors.

Current proteomic approaches disassemble and digest nucleosome particles, blurring readouts of the 'histone code'. To preserve nucleosome-level information, we developed Nuc-MS, which displays the landscape of histone variants and their post-translational modifications (PTMs) in a single mass spectrum. Combined with immunoprecipitation, Nuc-MS quantified nucleosome co-occupancy of histone H3.3 with variant H2A.Z (sixfold over bulk) and the co-occurrence of oncogenic H3.3K27M with euchromatic marks (for example, a >15-fold enrichment of dimethylated H3K79me2). Nuc-MS is highly concordant with chromatin immunoprecipitation-sequencing (ChIP-seq) and offers a new readout of nucleosome-level biology.

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.

Flavin-dependent, lysine-specific protein demethylases (KDM1s) are a subfamily of amine oxidases that catalyze the selective posttranslational oxidative demethylation of methyllysine side chains within protein and peptide substrates. KDM1s participate in the widespread epigenetic regulation of both normal and disease state transcriptional programs. Their activities are central to various cellular functions, such as hematopoietic and neuronal differentiation, cancer proliferation and metastasis, and viral lytic replication and establishment of latency. Interestingly, KDM1s function as catalytic subunits within complexes with coregulatory molecules that modulate enzymatic activity of the demethylases and coordinate their access to specific substrates at distinct sites within the cell and chromatin. Although several classes of KDM1-selective small molecule inhibitors have been recently developed, these pan-active site inhibition strategies lack the ability to selectively discriminate between KDM1 activity in specific, and occasionally opposing, functional contexts within these complexes. Here we review the discovery of this class of demethylases, their structures, chemical mechanisms, and specificity. Additionally, we review inhibition of this class of enzymes as well as emerging interactions with coregulatory molecules that regulate demethylase activity in highly specific functional contexts of biological and potential therapeutic importance.