Nucleosome conformation dictates the histone code.

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] specificities, and thus decipher the histone code and guide epigenetic therapies. However, this has largely been done using the reductive approach of isolated reader domains and histone peptides, which cannot account for any higher-order factors. Here, we show that the [BPTF PHD finger and bromodomain: histone PTM] interaction is dependent on nucleosome context. The tandem reader selectively associates with nucleosomal H3K4me3 and H3K14ac or H3K18ac, a combinatorial engagement that despite being in cis is not predicted by peptides. This in vitro specificity of the BPTF tandem reader for PTM-defined nucleosomes is recapitulated in a cellular context. We propose that regulatable histone tail accessibility and its impact on the binding potential of reader domains necessitates we refine the 'histone code' concept and interrogate it at the nucleosome level.

Beyond the tail: the consequence of context in histone post-translational modification and chromatin research.

The role of histone post-translational modifications (PTMs) in chromatin structure and genome function has been the subject of intense debate for more than 60 years. Though complex, the discourse can be summarized in two distinct - and deceptively simple - questions: What is the function of histone PTMs? And how should they be studied? Decades of research show these queries are intricately linked and far from straightforward. Here we provide a historical perspective, highlighting how the arrival of new technologies shaped discovery and insight. Despite their limitations, the tools available at each period had a profound impact on chromatin research, and provided essential clues that advanced our understanding of histone PTM function. Finally, we discuss recent advances in the application of defined nucleosome substrates, the study of multivalent chromatin interactions, and new technologies driving the next era of histone PTM research.

Structural basis of histone H2A lysine 119 deubiquitination by Polycomb repressive deubiquitinase BAP1/ASXL1.

Histone H2A lysine 119 (H2AK119Ub) is monoubiquitinated by Polycomb repressive complex 1 and deubiquitinated by Polycomb repressive deubiquitinase complex (PR-DUB). PR-DUB cleaves H2AK119Ub to restrict focal H2AK119Ub at Polycomb target sites and to protect active genes from aberrant silencing. The PR-DUB subunits (BAP1 and ASXL1) are among the most frequently mutated epigenetic factors in human cancers. How PR-DUB establishes specificity for H2AK119Ub over other nucleosomal ubiquitination sites and how disease-associated mutations of the enzyme affect activity are unclear. Here, we determine a cryo-EM structure of human BAP1 and the ASXL1 DEUBAD 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 restructuring 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 insight into understanding cancer etiology.

An acetylation-mediated chromatin switch governs H3K4 methylation read-write capability.

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.

Independent transcriptomic and proteomic regulation by type I and II protein arginine methyltransferases.

Protein arginine methyltransferases (PRMTs) catalyze the post-translational monomethylation (Rme1), asymmetric (Rme2a), or symmetric (Rme2s) dimethylation of arginine. To determine the cellular consequences of type I (Rme2a) and II (Rme2s) PRMTs, we developed and integrated multiple approaches. First, we determined total cellular dimethylarginine levels, revealing that Rme2s was ∼3% of total Rme2 and that this percentage was dependent upon cell type and PRMT inhibition status. Second, we quantitatively characterized in vitro substrates of the major enzymes and expanded upon PRMT substrate recognition motifs. We also compiled our data with publicly available methylarginine-modified residues into a comprehensive database. Third, we inhibited type I and II PRMTs and performed proteomic and transcriptomic analyses to reveal their phenotypic consequences. These experiments revealed both overlapping and independent PRMT substrates and cellular functions. Overall, this study expands upon PRMT substrate diversity, the arginine methylome, and the complex interplay of type I and II PRMTs.

Quantification of citrullinated histones: Development of an improved assay to reliably quantify nucleosomal H3Cit in human plasma.

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.

A functional proteomics platform to reveal the sequence determinants of lysine methyltransferase substrate selectivity.

Lysine methylation is a key regulator of histone protein function. Beyond histones, few connections have been made to the enzymes responsible for the deposition of these posttranslational modifications. Here, we debut a high-throughput functional proteomics platform that maps the sequence determinants of lysine methyltransferase (KMT) substrate selectivity without a priori knowledge of a substrate or target proteome. We demonstrate the predictive power of this approach for identifying KMT substrates, generating scaffolds for inhibitor design, and predicting the impact of missense mutations on lysine methylation signaling. By comparing KMT selectivity profiles to available lysine methylome datasets, we reveal a disconnect between preferred KMT substrates and the ability to detect these motifs using standard mass spectrometry pipelines. Collectively, our studies validate the use of this platform for guiding the study of lysine methylation signaling and suggest that substantial gaps exist in proteome-wide curation of lysine methylomes.

Examining the Roles of H3K4 Methylation States with Systematically Characterized Antibodies.

Histone post-translational modifications (PTMs) are important genomic regulators often studied by chromatin immunoprecipitation (ChIP), whereby their locations and relative abundance are inferred by antibody capture of nucleosomes and associated DNA. However, the specificity of antibodies within these experiments has not been systematically studied. Here, we use histone peptide arrays and internally calibrated ChIP (ICeChIP) to characterize 52 commercial antibodies purported to distinguish the H3K4 methylforms (me1, me2, and me3, with each ascribed distinct biological functions). We find that many widely used antibodies poorly distinguish the methylforms and that high- and low-specificity reagents can yield dramatically different biological interpretations, resulting in substantial divergence from the literature for numerous H3K4 methylform paradigms. Using ICeChIP, we also discern quantitative relationships between enhancer H3K4 methylation and promoter transcriptional output and can measure global PTM abundance changes. Our results illustrate how poor antibody specificity contributes to the "reproducibility crisis," demonstrating the need for rigorous, platform-appropriate validation.

Chromatin structure and its chemical modifications regulate the ubiquitin ligase substrate selectivity of UHRF1.

Mitotic inheritance of DNA methylation patterns is facilitated by UHRF1, a DNA- and histone-binding E3 ubiquitin ligase that helps recruit the maintenance DNA methyltransferase DNMT1 to replicating chromatin. The DNA methylation maintenance function of UHRF1 is dependent on its ability to bind chromatin, where it facilitates monoubiquitination of histone H3 at lysines 18 and 23, a docking site for DNMT1. Because of technical limitations, this model of UHRF1-dependent DNA methylation inheritance has been constructed largely based on genetics and biochemical observations querying methylated DNA oligonucleotides, synthetic histone peptides, and heterogeneous chromatin extracted from cells. Here, we construct semisynthetic mononucleosomes harboring defined histone and DNA modifications and perform rigorous analysis of UHRF1 binding and enzymatic activity with these reagents. We show that multivalent engagement of nucleosomal linker DNA and dimethylated lysine 9 on histone H3 directs UHRF1 ubiquitin ligase activity toward histone substrates. Notably, we reveal a molecular switch, stimulated by recognition of hemimethylated DNA, which redirects UHRF1 ubiquitin ligase activity away from histones in favor of robust autoubiquitination. Our studies support a noncompetitive model for UHRF1 and DNMT1 chromatin recruitment to replicating chromatin and define a role for hemimethylated linker DNA as a regulator of UHRF1 ubiquitin ligase substrate selectivity.

Using oriented peptide array libraries to evaluate methylarginine-specific antibodies and arginine methyltransferase substrate motifs.

Signal transduction in response to stimuli relies on the generation of cascades of posttranslational modifications that promote protein-protein interactions and facilitate the assembly of distinct signaling complexes. Arginine methylation is one such modification, which is catalyzed by a family of nine protein arginine methyltransferases, or PRMTs. Elucidating the substrate specificity of each PRMT will promote a better understanding of which signaling networks these enzymes contribute to. Although many PRMT substrates have been identified, and their methylation sites mapped, the optimal target motif for each of the nine PRMTs has not been systematically addressed. Here we describe the use of Oriented Peptide Array Libraries (OPALs) to methodically dissect the preferred methylation motifs for three of these enzymes - PRMT1, CARM1 and PRMT9. In parallel, we show that an OPAL platform with a fixed methylarginine residue can be used to validate the methyl-specific and sequence-specific properties of antibodies that have been generated against different PRMT substrates, and can also be used to confirm the pan nature of some methylarginine-specific antibodies.

Decoding the trans-histone crosstalk: methods to analyze H2B ubiquitination, H3 methylation and their regulatory factors.

Regulation of histone H3 lysine 4 and 79 methylation by histone H2B lysine 123 monoubiquitination is an evolutionarily conserved trans-histone crosstalk mechanism, which demonstrates a functional role for histone ubiquitination within the cell. The regulatory enzymes, factors and processes involved in the establishment and dynamic modulation of these modifications and their genome-wide distribution patterns have been determined in many model systems. Rapid progress in understanding this trans-histone crosstalk has been made using the standard experimental tools of chromatin biology in budding yeast (Saccharomyces cerevisiae), a highly tractable model organism. Here, we provide a set of modified and refined experimental procedures that can be used to gain further insights into the underlying mechanisms that govern this crosstalk in budding yeast. Importantly, the improved procedures and their underlying principles can also be applied to other model organisms. Methods presented here provide a rapid and efficient means to prepare enriched protein extracts to better preserve and assess the steady state levels of histones, non-histone proteins and their modifications. Improved chromatin immunoprecipitation and double immunoprecipitation protocols are provided to measure the occupancy and distribution of proteins and their modified forms at specific chromatin regions or loci. A quick and easy method to measure overall protein abundance and changes in protein-protein and protein-DNA interactions on native chromatin is also described.

Regulation of histone H2A and H2B deubiquitination and Xenopus development by USP12 and USP46.

Post-translational histone modifications play important roles in regulating gene expression programs, which in turn determine cell fate and lineage commitment during development. One such modification is histone ubiquitination, which primarily targets histone H2A and H2B. Although ubiquitination of H2A and H2B has been generally linked to gene silencing and gene activation, respectively, the functions of histone ubiquitination during eukaryote development are not well understood. Here, we identified USP12 and USP46 as histone H2A and H2B deubiquitinases that regulate Xenopus development. USP12 and USP46 prefer nucleosomal substrates and deubiquitinate both histone H2A and H2B in vitro and in vivo. WDR48, a WD40 repeat-containing protein, interacts with USP12 and USP46 and is required for the histone deubiquitination activity. Overexpression of either gene leads to gastrulation defects without affecting mesodermal cell fate, whereas knockdown of USP12 in Xenopus embryos results in reduction of a subset of mesodermal genes at gastrula stages. Immunohistochemical staining and chromatin immunoprecipitation assays revealed that USP12 regulates histone deubiquitination in the mesoderm and at specific gene promoters during Xenopus development. Taken together, this study identifies USP12 and USP46 as histone deubiquitinases for H2A and H2B and reveals that USP12 regulates Xenopus development during gastrula stages.

Hdac3 is essential for the maintenance of chromatin structure and genome stability.

Hdac3 is essential for efficient DNA replication and DNA damage control. Deletion of Hdac3 impaired DNA repair and greatly reduced chromatin compaction and heterochromatin content. These defects corresponded to increases in histone H3K9,K14ac; H4K5ac; and H4K12ac in late S phase of the cell cycle, and histone deposition marks were retained in quiescent Hdac3-null cells. Liver-specific deletion of Hdac3 culminated in hepatocellular carcinoma. Whereas HDAC3 expression was downregulated in only a small number of human liver cancers, the mRNA levels of the HDAC3 cofactor NCOR1 were reduced in one-third of these cases. siRNA targeting of NCOR1 and SMRT (NCOR2) increased H4K5ac and caused DNA damage, indicating that the HDAC3/NCOR/SMRT axis is critical for maintaining chromatin structure and genomic stability.

The JmjN domain of Jhd2 is important for its protein stability, and the plant homeodomain (PHD) finger mediates its chromatin association independent of H3K4 methylation.

Histone lysine methylation is a dynamic process that plays an important role in regulating chromatin structure and gene expression. Recent studies have identified Jhd2, a JmjC domain-containing protein, as an H3K4-specific demethylase in budding yeast. However, important questions regarding the regulation and functions of Jhd2 remain unanswered. In this study, we show that Jhd2 has intrinsic activity to remove all three states of H3K4 methylation in vivo and can dynamically associate with chromatin to modulate H3K4 methylation levels on both active and repressed genes and at the telomeric regions. We found that the plant homeodomain (PHD) finger of Jhd2 is important for its chromatin association in vivo. However, this association is not dependent on H3K4 methylation and the H3 N-terminal tail, suggesting the presence of an alternative mechanism by which Jhd2 binds nucleosomes. We also provide evidence that the JmjN domain and its interaction with the JmjC catalytic domain are important for Jhd2 function and that Not4 (an E3 ligase) monitors the structural integrity of this interdomain interaction to maintain the overall protein levels of Jhd2. We show that the S451R mutation in human SMCX (a homolog of Jhd2), which has been linked to mental retardation, and the homologous T359R mutation in Jhd2 affect the protein stability of both of these proteins. Therefore, our findings provide a mechanistic explanation for the observed defects in patients harboring this SMCX mutant and suggest the presence of a conserved pathway involving Not4 that modulates the protein stability of both yeast Jhd2 and human SMCX.

The ubiquitin-conjugating enzyme HR6B is required for maintenance of X chromosome silencing in mouse spermatocytes and spermatids.

BACKGROUND: The ubiquitin-conjugating enzyme HR6B is required for spermatogenesis in mouse. Loss of HR6B results in aberrant histone modification patterns on the trancriptionally silenced X and Y chromosomes (XY body) and on centromeric chromatin in meiotic prophase. We studied the relationship between these chromatin modifications and their effects on global gene expression patterns, in spermatocytes and spermatids. RESULTS: HR6B is enriched on the XY body and on centromeric regions in pachytene spermatocytes. Global gene expression analyses revealed that spermatid-specific single- and multicopy X-linked genes are prematurely expressed in Hr6b knockout spermatocytes. Very few other differences in gene expression were observed in these cells, except for upregulation of major satellite repeat transcription. In contrast, in Hr6b knockout spermatids, 7298 genes were differentially expressed; 65% of these genes was downregulated, but we observed a global upregulation of gene transcription from the X chromosome. In wild type spermatids, approximately 20% of the single-copy X-linked genes reach an average expression level that is similar to the average expression from autosomes. CONCLUSIONS: Spermatids maintain an enrichment of repressive chromatin marks on the X chromosome, originating from meiotic prophase, but this does not interfere with transcription of the single-copy X-linked genes that are reactivated or specifically activated in spermatids. HR6B represses major satellite repeat transcription in spermatocytes, and functions in the maintenance of X chromosome silencing in spermatocytes and spermatids. It is discussed that these functions involve modification of chromatin structure, possibly including H2B ubiquitylation.

Histone H2B ubiquitination and beyond: Regulation of nucleosome stability, chromatin dynamics and the trans-histone H3 methylation.

Regulation of Set1-COMPASS-mediated H3K4 methylation and Dot1-mediated H3K79 methylation by H2BK123 ubiquitination (H2Bub1) is an evolutionarily conserved trans-histone crosstalk mechanism. How H2Bub1 impacts chromatin structure and affects Set1-COMPASS/Dot1 functions has not been fully defined. Ubiquitin was proposed to bind proteins to physically bridge H2Bub1 with Set1-COMPASS/Dot1. Alternatively, the bulky ubiquitin was thought to be a "wedge" that loosens the nucleosome for factor access. Contrary to the latter possibility, recent discoveries provide evidence for nucleosome stabilization by H2Bub1 via preventing the constant H2A-H2B eviction. Recent data has also uncovered a "docking-site" on H2B for Set1-COMPASS. Collectively, these findings invoke a model, where ubiquitin acts as a "glue" to bind the nucleosome together for supporting Set1-COMPASS/Dot1 functions. This review provides an overview of these novel findings. Additionally, how H2Bub1 and its deubiquitination might alter the chromatin dynamics during transcription is discussed. Possible models for nucleosome stabilization by ubiquitin are also provided.

Histone H2B C-terminal helix mediates trans-histone H3K4 methylation independent of H2B ubiquitination.

The trans-histone regulatory cross talk between H2BK123 ubiquitination (H2Bub1) and H3K4 and H3K79 methylation is not fully understood. In this study, we report that the residues arginine 119 and threonine 122 in the H2B C-terminal helix are important for transcription and cell growth and play a direct role in controlling H2Bub1 and H3K4 methylation. These residues modulate H2Bub1 levels by controlling the chromatin binding and activities of the deubiquitinases. Furthermore, we find an uncoupling of the H2Bub1-mediated coregulation of both H3K4 and -K79 methylation, as these H2B C-terminal helix residues are part of a distinct surface that affects only Set1-COMPASS (complex proteins associated with Set1)-mediated H3K4 methylation without affecting the functions of Dot1. Importantly, we also find that these residues interact with Spp1 and control the chromatin association, integrity, and overall stability of Set1-COMPASS independent of H2Bub1. Therefore, we have uncovered a novel role for the H2B C-terminal helix in the trans-histone cross talk as a binding surface for Set1-COMPASS. We provide further insight into the trans-histone cross talk and propose that H2Bub1 stabilizes the nucleosome by preventing H2A-H2B eviction and, thereby, retains the "docking site" for Set1-COMPASS on chromatin to maintain its stable chromatin association, complex stability, and processive methylation.

Ubiquitination of histone H2B regulates chromatin dynamics by enhancing nucleosome stability.

The mechanism by which ubiquitination of histone H2B (H2Bub1) regulates H3-K4 and -K79 methylation and the histone H2A-H2B chaperone Spt16-mediated nucleosome dynamics during transcription is not fully understood. Upon investigating the effect of H2Bub1 on chromatin structure, we find that contrary to the supposed role for H2Bub1 in opening up chromatin, it is important for nucleosome stability. First, we show that H2Bub1 does not function as a "wedge" to non-specifically unfold chromatin, as replacement of ubiquitin with a bulkier SUMO molecule conjugated to the C-terminal helix of H2B cannot functionally support H3-K4 and -K79 methylation. Second, using a series of biochemical analyses, we demonstrate that nucleosome stability is reduced or enhanced, when the levels of H2Bub1 are abolished or increased, respectively. Besides transcription elongation, we show that H2Bub1 regulates initiation by stabilizing nucleosomes positioned over the promoters of repressed genes. Collectively, our study reveals an intrinsic difference in the property of chromatin assembled in the presence or absence of H2Bub1 and implicates the regulation of nucleosome stability as the mechanism by which H2Bub1 modulates nucleosome dynamics and histone methylation during transcription.

Histone H2BK123 monoubiquitination is the critical determinant for H3K4 and H3K79 trimethylation by COMPASS and Dot1.

Histone H2B monoubiquitination by Rad6/Bre1 is required for the trimethylation of both histone H3K4 and H3K79 by COMPASS and Dot1 methyltransferases, respectively. The dependency of methylation at H3K4 and H3K79 on the monoubiquitination of H2BK123 was recently challenged, and extragenic mutations in the strain background used for previous studies or epitope-tagged proteins were suggested to be the sources of this discrepancy. In this study, we show that H3K4 and H3K79 methylation is solely dependent on H2B monoubiquitination regardless of any additional alteration to the H2B sequence or genome. Furthermore, we report that Y131, one of the yeast histone H2A/H2B shuffle strains widely used for the last decade in the field of chromatin and transcription biology, carries a wild-type copy of each of the HTA2 and HTB2 genes under the GAL1/10 promoter on chromosome II. Therefore, we generated the entire histone H2A and H2B alanine-scanning mutant strains in another background, which does not express wild-type histones.

Deletion of histone deacetylase 3 reveals critical roles in S phase progression and DNA damage control.

Histone deacetylases (HDACs) are enzymes that modify key residues in histones to regulate chromatin architecture, and they play a vital role in cell survival, cell-cycle progression, and tumorigenesis. To understand the function of Hdac3, a critical component of the N-CoR/SMRT repression complex, a conditional allele of Hdac3 was engineered. Cre-recombinase-mediated inactivation of Hdac3 led to a delay in cell-cycle progression, cell-cycle-dependent DNA damage, and apoptosis in mouse embryonic fibroblasts (MEFs). While no overt defects in mitosis were observed in Hdac3-/- MEFs, including normal H3Ser10 phosphorylation, DNA damage was observed in Hdac3-/- interphase cells, which appears to be associated with defective DNA double-strand break repair. Moreover, we noted that Hdac3-/- MEFs were protected from DNA damage when quiescent, which may provide a mechanistic basis for the action of HDAC inhibitors on cycling tumor cells.