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The neoplastic stromal cells of giant cell tumor of bone (GCTB) carry a mutation in H3F3A, leading to a mutant histone variant, H3.3-G34W, as a sole recurrent genetic alteration. We show that in patient-derived stromal cells H3.3-G34W is incorporated into the chromatin and associates with massive epigenetic alterations on the DNA methylation, chromatin accessibility and histone modification level, that can be partially recapitulated in an orthogonal cell line system by the introduction of H3.3-G34W. These epigenetic alterations affect mainly heterochromatic and bivalent regions and provide possible explanations for the genomic instability, as well as the osteolytic phenotype of GCTB. The mutation occurs in differentiating mesenchymal stem cells and associates with an impaired osteogenic differentiation. We propose that the observed epigenetic alterations reflect distinct differentiation stages of H3.3 WT and H3.3 MUT stromal cells and add to H3.3-G34W-associated changes.

T helper (Th) cells are CD4 + effector T cells that play a critical role in immunity by shaping the inflammatory cytokine environment in a variety of physiological and pathological situations. Using a combined chemico-genetic approach, we identify histone H3K27 demethylases KDM6A and KDM6B as central regulators of human Th subsets. The prototypic KDM6 inhibitor GSK-J4 increases genome-wide levels of the repressive H3K27me3 chromatin mark and leads to suppression of the key transcription factor RORγt during Th17 differentiation. In mature Th17 cells, GSK-J4 induces an altered transcriptional program with a profound metabolic reprogramming and concomitant suppression of IL-17 cytokine levels and reduced proliferation. Single-cell analysis reveals a specific shift from highly inflammatory cell subsets toward a resting state upon demethylase inhibition. The root cause of the observed antiinflammatory phenotype in stimulated Th17 cells is reduced expression of key metabolic transcription factors, such as PPRC1. Overall, this leads to reduced mitochondrial biogenesis, resulting in a metabolic switch with concomitant antiinflammatory effects. These data are consistent with an effect of GSK-J4 on Th17 T cell differentiation pathways directly related to proliferation and include regulation of effector cytokine profiles. This suggests that inhibiting KDM6 demethylases may be an effective, even in the short term, therapeutic target for autoimmune diseases, including ankylosing spondylitis., Competing Interests: Competing interest statement: B.S. was an employee of Metabolon. P.K.M. and R.K.P. are employees of GlaxoSmithKline. M.L., S.O., and H.O. were at Roche. U.O. and S.G. are coauthors on a 2016 meeting report., (Copyright © 2020 the Author(s). Published by PNAS.)

Plant homeodomain (PHD) containing proteins are important epigenetic regulators and are of interest as potential drug targets. Inspired by the amiodarone derivatives reported to inhibit the PHD finger 3 of KDM5A (KDM5A(PHD3)), a set of compounds were synthesised. Amiodarone and its derivatives were observed to weakly disrupt the interactions of a histone H3K4me3 peptide with KDM5A(PHD3). Selected amiodarone derivatives inhibited catalysis of KDM5A, but in a PHD-finger independent manner. Amiodarone derivatives also bind to H3K4me3-binding PHD-fingers from the KDM7 subfamily. Further work is required to develop potent and selective PHD finger inhibitors., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Natural killer (NK) cells are innate lymphocytes, important in immune surveillance and elimination of stressed, transformed, or virus-infected cells. They critically shape the inflammatory cytokine environment to orchestrate interactions of cells of the innate and adaptive immune systems. Some studies have reported that NK cell activation and cytokine secretion are controlled epigenetically but have yielded only limited insight into the mechanisms. Using chemical screening with small-molecule inhibitors of chromatin methylation and acetylation, further validated by knockdown approaches, we here identified Jumonji-type histone H3K27 demethylases as key regulators of cytokine production in human NK cell subsets. The prototypic JMJD3/UTX (Jumonji domain-containing protein 3) H3K27 demethylase inhibitor GSK-J4 increased global levels of the repressive H3K27me3 mark around transcription start sites of effector cytokine genes. Moreover, GSK-J4 reduced IFN-γ, TNFα, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-10 levels in cytokine-stimulated NK cells while sparing their cytotoxic killing activity against cancer cells. The anti-inflammatory effect of GSK-J4 in NK cell subsets, isolated from peripheral blood or tissue from individuals with rheumatoid arthritis (RA), coupled with an inhibitory effect on formation of bone-resorbing osteoclasts, suggested that histone demethylase inhibition has broad utility for modulating immune and inflammatory responses. Overall, our results indicate that H3K27me3 is a dynamic and important epigenetic modification during NK cell activation and that JMJD3/UTX-driven H3K27 demethylation is critical for NK cell function., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

The retinoblastoma tumor suppressor protein pRb is a master regulator of cellular proliferation, principally through interaction with E2F and regulation of E2F target genes. Here, we describe the H1.2 linker histone as a major pRb interaction partner. We establish that H1.2 and pRb are found in a chromatin-bound complex on diverse E2F target genes. Interrogating the global influence of H1.2 on the genome-wide distribution of pRb indicated that the E2F target genes affected by H1.2 are functionally linked to cell-cycle control, consistent with the ability of H1.2 to hinder cell proliferation and the elevated levels of chromatin-bound H1-pRb complex, which occur in growth-arrested cells. Our results define a network of E2F target genes as susceptible to the regulatory influence of H1.2, where H1.2 augments global association of pRb with chromatin, enhances transcriptional repression by pRb, and facilitates pRb-dependent cell-cycle arrest., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Methylation of lysine residues on histone tail is a dynamic epigenetic modification that plays a key role in chromatin structure and gene regulation. Members of the KDM5 (also known as JARID1) sub-family are 2-oxoglutarate (2-OG) and Fe 2+ -dependent oxygenases acting as histone 3 lysine 4 trimethyl (H3K4me3) demethylases, regulating proliferation, stem cell self-renewal, and differentiation. Here we present the characterization of KDOAM-25, an inhibitor of KDM5 enzymes. KDOAM-25 shows biochemical half maximal inhibitory concentration values of <100 nM for KDM5A-D in vitro, high selectivity toward other 2-OG oxygenases sub-families, and no off-target activity on a panel of 55 receptors and enzymes. In human cell assay systems, KDOAM-25 has a half maximal effective concentration of ∼50 μM and good selectivity toward other demethylases. KDM5B is overexpressed in multiple myeloma and negatively correlated with the overall survival. Multiple myeloma MM1S cells treated with KDOAM-25 show increased global H3K4 methylation at transcriptional start sites and impaired proliferation., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Epigenetic modifications of histone tails play an essential role in the regulation of eukaryotic transcription. Writer and eraser enzymes establish and maintain the epigenetic code by creating or removing posttranslational marks. Specific binding proteins, called readers, recognize the modifications and mediate epigenetic signalling. Here, we present a versatile assay platform for the investigation of the interaction between methyl lysine readers and their ligands. This can be utilized for the screening of small-molecule inhibitors of such protein-protein interactions and the detailed characterization of the inhibition. Our platform is constructed in a modular way consisting of orthogonal in vitro binding assays for ligand screening and verification of initial hits and biophysical, label-free techniques for further kinetic characterization of confirmed ligands. A stability assay for the investigation of target engagement in a cellular context complements the platform. We applied the complete evaluation chain to the Tudor domain containing protein Spindlin1 and established the in vitro test systems for the double Tudor domain of the histone demethylase JMJD2C. We finally conducted an exploratory screen for inhibitors of the interaction between Spindlin1 and H3K4me3 and identified A366 as the first nanomolar small-molecule ligand of a Tudor domain containing methyl lysine reader., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Small molecule modulators of epigenetic processes are currently sought as basic probes for biochemical mechanisms, and as starting points for development of therapeutic agents. N(ε)-Methylation of lysine residues on histone tails is one of a number of post-translational modifications that together enable transcriptional regulation. Histone lysine demethylases antagonize the action of histone methyltransferases in a site- and methylation state-specific manner. N(ε)-Methyllysine demethylases that use 2-oxoglutarate as co-factor are associated with diverse human diseases, including cancer, inflammation and X-linked mental retardation; they are proposed as targets for the therapeutic modulation of transcription. There are few reports on the identification of templates that are amenable to development as potent inhibitors in vivo and large diverse collections have yet to be exploited for the discovery of demethylase inhibitors., Principal Findings: High-throughput screening of a ∼236,000-member collection of diverse molecules arrayed as dilution series was used to identify inhibitors of the JMJD2 (KDM4) family of 2-oxoglutarate-dependent histone demethylases. Initial screening hits were prioritized by a combination of cheminformatics, counterscreening using a coupled assay enzyme, and orthogonal confirmatory detection of inhibition by mass spectrometric assays. Follow-up studies were carried out on one of the series identified, 8-hydroxyquinolines, which were shown by crystallographic analyses to inhibit by binding to the active site Fe(II) and to modulate demethylation at the H3K9 locus in a cell-based assay., Conclusions: These studies demonstrate that diverse compound screening can yield novel inhibitors of 2OG dependent histone demethylases and provide starting points for the development of potent and selective agents to interrogate epigenetic regulation.

Summary: The structural genomics of histone tail recognition web server is an open access resource that presents within mini articles all publicly available experimental structures of histone tails in complex with human proteins. Each article is composed of interactive 3D slides that dissect the structural mechanism underlying the recognition of specific sequences and histone marks. A concise text html-linked to interactive graphics guides the reader through the main features of the interaction. This resource can be used to analyze and compare binding modes across multiple histone recognition modules, to evaluate the chemical tractability of binding sites involved in epigenetic signaling and design small molecule inhibitors., Availability: http://www.thesgc.org/resources/histone&#95;tails/, Contact: matthieu.schapira@utoronto.ca, Supplementary Information: Supplementary data are available at Bioinformatics online.

Methylation of lysine residues on the tails of histone proteins is a major determinant of the transcription state of associated DNA coding regions. The interplay among methylation states and other histone modifications to direct transcriptional outcome is referred to as the histone code. In addition to histone methyltransferases and demethylases which function to modify the methylation state of lysine sidechains, other proteins recognize specific histone methylation marks essentially serving as code readers. While these interactions are highly specific with respect to site and methylation state of particular lysine residues, they are generally weak and therefore difficult to monitor by traditional assay techniques. Herein, we present the design and implementation of a homogeneous, miniaturizable, and sensitive assay for histone methylation-dependent interactions. We use AlphaScreen, a chemiluminescence-based technique, to monitor the interactions of chromodomains (MPP8, HP1beta and CHD1), tudor domains (JMJD2A) and plant homeodomains (RAG2) with their cognate trimethyllysine histone partners. The utility of the method was demonstrated by profiling the binding specificities of chromo- and tudor domains toward several histone marks. The simplicity of design and the sensitive and robust nature of this assay should make it applicable to a range of epigenetic studies, including the search for novel inhibitors of methylation-dependent interactions.

Post-translational modification of chromatin is emerging as an increasingly important regulator of chromosomal processes. In particular, histone lysine and arginine methylation play important roles in regulating transcription, maintaining genomic integrity, and contributing to epigenetic memory. Recently, the use of new approaches to analyse histone methylation, the generation of genetic model systems, and the ability to interrogate genome wide histone modification profiles has aided in defining how histone methylation contributes to these processes. Here we focus on the recent advances in our understanding of the histone methylation system and examine how dynamic histone methylation contributes to normal cellular function in mammals.

The reversible thermal unfolding of the archaeal histone-like protein Ssh10b from the extremophile Sulfolobus shibatae was studied using differential scanning calorimetry and circular dichroism spectroscopy. Analytical ultracentrifugation and gel filtration showed that Ssh10b is a stable dimer in the pH range 2.5-7.0. Thermal denaturation data fit into a two-state unfolding model, suggesting that the Ssh10 dimer unfolds as a single cooperative unit with a maximal melting temperature of 99.9 degrees C and an enthalpy change of 134 kcal/mol at pH 7.0. The heat capacity change upon unfolding determined from linear fits of the temperature dependence of DeltaH(cal) is 2.55 kcal/(mol K). The low specific heat capacity change of 13 cal/(mol K residue) leads to a considerable flattening of the protein stability curve (DeltaG (T)) and results in a maximal DeltaG of only 9.5 kcal/mol at 320 K and a DeltaG of only 6.0 kcal/mol at the optimal growth temperature of Sulfolobus.

Post-translational histone modification has a fundamental role in chromatin biology and is proposed to constitute a 'histone code' in epigenetic regulation. Differential methylation of histone H3 and H4 lysyl residues regulates processes including heterochromatin formation, X-chromosome inactivation, genome imprinting, DNA repair and transcriptional regulation. The discovery of lysyl demethylases using flavin (amine oxidases) or Fe(II) and 2-oxoglutarate as cofactors (2OG oxygenases) has changed the view of methylation as a stable epigenetic marker. However, little is known about how the demethylases are selective for particular lysyl-containing sequences in specific methylation states, a key to understanding their functions. Here we reveal how human JMJD2A (jumonji domain containing 2A), which is selective towards tri- and dimethylated histone H3 lysyl residues 9 and 36 (H3K9me3/me2 and H3K36me3/me2), discriminates between methylation states and achieves sequence selectivity for H3K9. We report structures of JMJD2A-Ni(II)-Zn(II) inhibitor complexes bound to tri-, di- and monomethyl forms of H3K9 and the trimethyl form of H3K36. The structures reveal a lysyl-binding pocket in which substrates are bound in distinct bent conformations involving the Zn-binding site. We propose a mechanism for achieving methylation state selectivity involving the orientation of the substrate methyl groups towards a ferryl intermediate. The results suggest distinct recognition mechanisms in different demethylase subfamilies and provide a starting point to develop chemical tools for drug discovery and to study and dissect the complexity of reversible histone methylation and its role in chromatin biology.