Epigenetic Therapy for Epithelioid Sarcoma.

Tazemetostat is the first epigenetic therapy to gain FDA approval in a solid tumor. This lysine methyltransferase inhibitor targets EZH2, the enzymatic subunit of the PRC2 transcriptional silencing complex. Tumors with mutations in subunits of the SWI/SNF chromatin remodeling complex, inclusive of most epithelioid sarcomas, are sensitive to EZH2 inhibition.

Ketolysis drives CD8+ T cell effector function through effects on histone acetylation.

Environmental nutrient availability influences T cell metabolism, impacting T cell function and shaping immune outcomes. Here, we identified ketone bodies (KBs)-including ß-hydroxybutyrate (ßOHB) and acetoacetate (AcAc)-as essential fuels supporting CD8+ T cell metabolism and effector function. ßOHB directly increased CD8+ T effector (Teff) cell cytokine production and cytolytic activity, and KB oxidation (ketolysis) was required for Teff cell responses to bacterial infection and tumor challenge. CD8+ Teff cells preferentially used KBs over glucose to fuel the tricarboxylic acid (TCA) cycle in vitro and in vivo. KBs directly boosted the respiratory capacity and TCA cycle-dependent metabolic pathways that fuel CD8+ T cell function. Mechanistically, ßOHB was a major substrate for acetyl-CoA production in CD8+ T cells and regulated effector responses through effects on histone acetylation. Together, our results identify cell-intrinsic ketolysis as a metabolic and epigenetic driver of optimal CD8+ T cell effector responses.

LKB1 controls inflammatory potential through CRTC2-dependent histone acetylation.

Deregulated inflammation is a critical feature driving the progression of tumors harboring mutations in the liver kinase B1 (LKB1), yet the mechanisms linking LKB1 mutations to deregulated inflammation remain undefined. Here, we identify deregulated signaling by CREB-regulated transcription coactivator 2 (CRTC2) as an epigenetic driver of inflammatory potential downstream of LKB1 loss. We demonstrate that LKB1 mutations sensitize both transformed and non-transformed cells to diverse inflammatory stimuli, promoting heightened cytokine and chemokine production. LKB1 loss triggers elevated CRTC2-CREB signaling downstream of the salt-inducible kinases (SIKs), increasing inflammatory gene expression in LKB1-deficient cells. Mechanistically, CRTC2 cooperates with the histone acetyltransferases CBP/p300 to deposit histone acetylation marks associated with active transcription (i.e., H3K27ac) at inflammatory gene loci, promoting cytokine expression. Together, our data reveal a previously undefined anti-inflammatory program, regulated by LKB1 and reinforced through CRTC2-dependent histone modification signaling, that links metabolic and epigenetic states to cell-intrinsic inflammatory potential.

A trivalent nucleosome interaction by PHIP/BRWD2 is disrupted in neurodevelopmental disorders and cancer.

Mutations in the PHIP/BRWD2 chromatin regulator cause the human neurodevelopmental disorder Chung-Jansen syndrome, while alterations in PHIP expression 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.

Lysine Methylation Regulators Moonlighting outside the Epigenome.

Landmark discoveries made nearly two decades ago identified known transcriptional regulators as histone lysine methyltransferases. Since then, the field of lysine methylation signaling has been dominated by studies of how this small chemical posttranslational modification regulates gene expression and other chromatin-based processes. However, recent advances in mass-spectrometry-based proteomics have revealed that histones are just a subset of the thousands of eukaryotic proteins marked by lysine methylation. As the writers, erasers, and readers of histone lysine methylation are emerging as a promising therapeutic target class for cancer and other diseases, a key challenge for the field is to define the full spectrum of activities for these proteins. Here we summarize recent discoveries implicating non-histone lysine methylation as a major regulator of diverse cellular processes. We further discuss recent technological innovations that are enabling the expanded study of lysine methylation signaling. Collectively, these findings are shaping our understanding of the fundamental mechanisms of non-histone protein regulation through this dynamic and multi-functional posttranslational modification.

Histone H3.3 phosphorylation amplifies stimulation-induced transcription.

Complex organisms can rapidly induce select genes in response to diverse environmental cues. This regulation occurs in the context of large genomes condensed by histone proteins into chromatin. The sensing of pathogens by macrophages engages conserved signalling pathways and transcription factors to coordinate the induction of inflammatory genes1-3. Enriched integration of histone H3.3, the ancestral histone H3 variant, is a general feature of dynamically regulated chromatin and transcription4-7. However, how chromatin is regulated at induced genes, and what features of H3.3 might enable rapid and high-level transcription, are unknown. The amino terminus of H3.3 contains a unique serine residue (Ser31) that is absent in 'canonical' H3.1 and H3.2. Here we show that this residue, H3.3S31, is phosphorylated (H3.3S31ph) in a stimulation-dependent manner along rapidly induced genes in mouse macrophages. This selective mark of stimulation-responsive genes directly engages the histone methyltransferase SETD2, a component of the active transcription machinery, and 'ejects' the elongation corepressor ZMYND118,9. We propose that features of H3.3 at stimulation-induced genes, including H3.3S31ph, provide preferential access to the transcription apparatus. Our results indicate dedicated mechanisms that enable rapid transcription involving the histone variant H3.3, its phosphorylation, and both the recruitment and the ejection of chromatin regulators.

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 Regulation through Ubiquitin and Ubiquitin-like Histone Modifications.

Chromatin functions are influenced by the addition, removal, and recognition of histone post-translational modifications (PTMs). Ubiquitin and ubiquitin-like (UBL) PTMs on histone proteins can function as signaling molecules by mediating protein-protein interactions. Fueled by the identification of novel ubiquitin and UBL sites and the characterization of the writers, erasers, and readers, the breadth of chromatin functions associated with ubiquitin signaling is emerging. Here, we highlight recently appreciated roles for histone ubiquitination in DNA methylation control, PTM crosstalk, nucleosome structure, and phase separation. We also discuss the expanding diversity and functions associated with histone UBL modifications. We conclude with a look toward the future and pose key questions that will drive continued discovery at the interface of epigenetics and ubiquitin signaling.

Structural basis for DNMT3A-mediated de novo DNA methylation.

DNA methylation by de novo DNA methyltransferases 3A (DNMT3A) and 3B (DNMT3B) at cytosines is essential for genome regulation and development. Dysregulation of this process is implicated in various diseases, notably cancer. However, the mechanisms underlying DNMT3 substrate recognition and enzymatic specificity remain elusive. Here we report a 2.65-ångström crystal structure of the DNMT3A-DNMT3L-DNA complex in which two DNMT3A monomers simultaneously attack two cytosine-phosphate-guanine (CpG) dinucleotides, with the target sites separated by 14 base pairs within the same DNA duplex. The DNMT3A-DNA interaction involves a target recognition domain, a catalytic loop, and DNMT3A homodimeric interface. Arg836 of the target recognition domain makes crucial contacts with CpG, ensuring DNMT3A enzymatic preference towards CpG sites in cells. Haematological cancer-associated somatic mutations of the substrate-binding residues decrease DNMT3A activity, induce CpG hypomethylation, and promote transformation of haematopoietic cells. Together, our study reveals the mechanistic basis for DNMT3A-mediated DNA methylation and establishes its aetiological link to human disease.

Lysine methylation signaling in skeletal muscle biology: from myogenesis to clinical insights.

Lysine methylation signaling is well studied for its key roles in the regulation of transcription states through modifications on histone proteins. While histone lysine methylation has been extensively studied, recent discoveries of lysine methylation on thousands of non-histone proteins has broadened our appreciation for this small chemical modification in the regulation of protein function. In this review, we highlight the significance of histone and non-histone lysine methylation signaling in skeletal muscle biology, spanning development, maintenance, regeneration, and disease progression. Furthermore, we discuss potential future implications for its roles in skeletal muscle biology as well as clinical applications for the treatment of skeletal muscle-related diseases.

In silico APC/C substrate discovery reveals cell cycle-dependent degradation of UHRF1 and other chromatin regulators.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to globally map APC/C substrates. By combining orthogonal features of known substrates, we predicted APC/C substrates in silico. This analysis identified many known substrates and suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among putative substrates, and we show experimentally that several chromatin proteins bind APC/C, oscillate during the cell cycle, and are degraded following APC/C activation, consistent with being direct APC/C substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences in normal cell physiology, where the chromatin environment changes depending on proliferative state, as well as in disease.

An Interactive Database for the Assessment of Histone Antibody Specificity.

Access to high-quality antibodies is a necessity for the study of histones and their posttranslational modifications (PTMs). Here we debut the Histone Antibody Specificity Database (http://www.histoneantibodies.com), an online and expanding resource cataloging the behavior of widely used, commercially available histone antibodies by peptide microarray. This interactive web portal provides a critical resource to the biological research community that routinely uses these antibodies as detection reagents for a wide range of applications.

ARID1A-dependent maintenance of H3.3 is required for repressive CHD4-ZMYND8 chromatin interactions at super-enhancers.

BACKGROUND: SWI/SNF (BAF) chromatin remodeling complexes regulate lineage-specific enhancer activity by promoting accessibility for diverse DNA-binding factors and chromatin regulators. Additionally, they are known to modulate the function of the epigenome through regulation of histone post-translational modifications and nucleosome composition, although the way SWI/SNF complexes govern the epigenome remains poorly understood. Here, we investigate the function of ARID1A, a subunit of certain mammalian SWI/SNF chromatin remodeling complexes associated with malignancies and benign diseases originating from the uterine endometrium. RESULTS: Through genome-wide analysis of human endometriotic epithelial cells, we show that more than half of ARID1A binding sites are marked by the variant histone H3.3, including active regulatory elements such as super-enhancers. ARID1A knockdown leads to H3.3 depletion and gain of canonical H3.1/3.2 at ARID1A-bound active regulatory elements, and a concomitant redistribution of H3.3 toward genic elements. ARID1A interactions with the repressive chromatin remodeler CHD4 (NuRD) are associated with H3.3, and ARID1A is required for CHD4 recruitment to H3.3. ZMYND8 interacts with CHD4 to suppress a subset of ARID1A, CHD4, and ZMYND8 co-bound, H3.3+ H4K16ac+ super-enhancers near genes governing extracellular matrix, motility, adhesion, and epithelial-to-mesenchymal transition. Moreover, these gene expression alterations are observed in human endometriomas. CONCLUSIONS: These studies demonstrate that ARID1A-containing BAF complexes are required for maintenance of the histone variant H3.3 at active regulatory elements, such as super-enhancers, and this function is required for the physiologically relevant activities of alternative chromatin remodelers.

Binding specificity and function of the SWI/SNF subunit SMARCA4 bromodomain interaction with acetylated histone H3K14.

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.

A physical basis for quantitative ChIP-sequencing.

ChIP followed by next-generation sequencing (ChIP-Seq) is a key technique for mapping the distribution of histone posttranslational modifications (PTMs) and chromatin-associated factors across genomes. There is a perceived challenge to define a quantitative scale for ChIP-Seq data, and as such, several approaches making use of exogenous additives, or "spike-ins," have recently been developed. Herein, we report on the development of a quantitative, physical model defining ChIP-Seq. The quantitative scale on which ChIP-Seq results should be compared emerges from the model. To test the model and demonstrate the quantitative scale, we examine the impacts of an EZH2 inhibitor through the lens of ChIP-Seq. We report a significant increase in immunoprecipitation of presumed off-target histone PTMs after inhibitor treatment, a trend predicted by the model but contrary to spike-in-based indications. Our work also identifies a sensitivity issue in spike-in normalization that has not been considered in the literature, placing limitations on its utility and trustworthiness. We call our new approach the sans-spike-in method for quantitative ChIP-sequencing (siQ-ChIP). A number of changes in community practice of ChIP-Seq, data reporting, and analysis are motivated by this work.

Study of mitotic chromatin supports a model of bookmarking by histone modifications and reveals nucleosome deposition patterns.

Mitosis encompasses key molecular changes including chromatin condensation, nuclear envelope breakdown, and reduced transcription levels. Immediately after mitosis, the interphase chromatin structure is reestablished and transcription resumes. The reestablishment of the interphase chromatin is probably achieved by "bookmarking," i.e., the retention of at least partial information during mitosis. To gain a deeper understanding of the contribution of histone modifications to the mitotic bookmarking process, we merged proteomics, immunofluorescence, and ChIP-seq approaches. We focused on key histone modifications and employed HeLa-S3 cells as a model system. Generally, in spite of the general hypoacetylation observed during mitosis, we observed a global concordance between the genomic organization of histone modifications in interphase and mitosis, suggesting that the epigenomic landscape may serve as a component of the mitotic bookmarking process. Next, we investigated the nucleosome that enters nucleosome depleted regions (NDRs) during mitosis. We observed that in ∼60% of the NDRs, the entering nucleosome is distinct from the surrounding highly acetylated nucleosomes and appears to have either low levels of acetylation or high levels of phosphorylation in adjacent residues (since adjacent phosphorylation may interfere with the ability to detect acetylation). Inhibition of histone deacetylases (HDACs) by the small molecule TSA reverts this pattern, suggesting that these nucleosomes are specifically deacetylated during mitosis. Altogether, by merging multiple approaches, our study provides evidence to support a model where histone modifications may play a role in mitotic bookmarking and uncovers new insights into the deposition of nucleosomes during mitosis.

An H3K36 methylation-engaging Tudor motif of polycomb-like proteins mediates PRC2 complex targeting.

Polycomb repressive complex 2 (PRC2) regulates pluripotency, differentiation, and tumorigenesis through catalysis of histone H3 lysine 27 trimethylation (H3K27me3) on chromatin. However, the mechanisms that underlie PRC2 recruitment and spreading on chromatin remain unclear. Here we report that histone H3 lysine 36 trimethylation (H3K36me3) binding activity is harbored in the Tudor motifs of PRC2-associated polycomb-like (PCL) proteins PHF1/PCL1 and PHF19/PCL3. Ectopically expressed PHF1 induced Tudor-dependent stabilization of PRC2 complexes on bulk chromatin and mediated spreading of PRC2 and H3K27me3 into H3K36me3-containing chromatin regions. In murine pluripotent stem cells, we identified coexistence of H3K36me3, H3K27me3, and PHF19/PCL3 at a subset of poised developmental genes and demonstrated that PHF19/PCL3 Tudor function is required for optimal H3K27me3 and repression of these loci. Collectively, our data suggest that PCL recognition of H3K36me3 promotes intrusion of PRC2 complexes into active chromatin regions to promote gene silencing and modulate the chromatin landscape during development.

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.

Multivalent histone engagement by the linked tandem Tudor and PHD domains of UHRF1 is required for the epigenetic inheritance of DNA methylation.

Histone post-translational modifications regulate chromatin structure and function largely through interactions with effector proteins that often contain multiple histone-binding domains. While significant progress has been made characterizing individual effector domains, the role of paired domains and how they function in a combinatorial fashion within chromatin are poorly defined. Here we show that the linked tandem Tudor and plant homeodomain (PHD) of UHRF1 (ubiquitin-like PHD and RING finger domain-containing protein 1) operates as a functional unit in cells, providing a defined combinatorial readout of a heterochromatin signature within a single histone H3 tail that is essential for UHRF1-directed epigenetic inheritance of DNA methylation. These findings provide critical support for the "histone code" hypothesis, demonstrating that multivalent histone engagement plays a key role in driving a fundamental downstream biological event in chromatin.

The finger loop of the SRA domain in the E3 ligase UHRF1 is a regulator of ubiquitin targeting and is required for the maintenance of DNA methylation.

The Su(var)3-9, enhancer of zeste, and trithorax (SET) and really interesting new gene (RING) finger-associated (SRA) protein domain is conserved across bacteria and eukaryota and coordinates extrahelical or "flipped" DNA bases. A functional SRA domain is required for ubiquitin-like with PHD and RING finger domains 1 (UHRF1) E3 ubiquitin ligase activity toward histone H3, a mechanism for recruiting the DNA methylation maintenance enzyme DNA methyltransferase 1 (DNMT1). The SRA domain supports UHRF1 oncogenic activity in colon cancer cells, highlighting that UHRF1 SRA antagonism could be a cancer therapeutic strategy. Here we used molecular dynamics simulations, DNA binding assays, in vitro ubiquitination reactions, and DNA methylation analysis to identify the SRA finger loop as a regulator of UHRF1 ubiquitin targeting and DNA methylation maintenance. A chimeric UHRF1 (finger swap) with diminished E3 ligase activity toward nucleosomal histones, despite tighter binding to unmodified or asymmetric or symmetrically methylated DNA, uncouples DNA affinity from regulation of E3 ligase activity. Our model suggests that SRA domains sample DNA bases through flipping in the presence or absence of a cytosine modification and that specific interactions of the SRA finger loop with DNA are required for downstream host protein function. Our findings provide insight into allosteric regulation of UHRF1 E3 ligase activity, suggesting that UHRF1's SRA finger loop regulates its conformation and function.