Nathan R. Rose

The oncometabolite 2-hydroxyglutarate inhibits histone lysine demethylases.

Mutations in isocitrate dehydrogenases (IDHs) have a gain‐of‐function effect leading to R(−)‐2‐hydroxyglutarate (R‐2HG) accumulation. By using biochemical, structural and cellular assays, we show that either or both R‐ and S‐2HG inhibit 2‐oxoglutarate (2OG)‐dependent oxygenases with varying potencies. Half‐maximal inhibitory concentration (IC50) values for the R‐form of 2HG varied from approximately 25 μM for the histone Nε‐lysine demethylase JMJD2A to more than 5 mM for the hypoxia‐inducible factor (HIF) prolyl hydroxylase. The results indicate that candidate oncogenic pathways in IDH‐associated malignancy should include those that are regulated by other 2OG oxygenases than HIF hydroxylases, in particular those involving the regulation of histone methylation.

Crystal structures of histone demethylase JMJD2A reveal basis for substrate specificity.

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.

KDM2B links the Polycomb Repressive Complex 1 (PRC1) to recognition of CpG islands

CpG islands (CGIs) are associated with most mammalian gene promoters. A subset of CGIs act as polycomb response elements (PREs) and are recognized by the polycomb silencing systems to regulate expression of genes involved in early development. How CGIs function mechanistically as nucleation sites for polycomb repressive complexes remains unknown. Here we discover that KDM2B (FBXL10) specifically recognizes non-methylated DNA in CGIs and recruits the polycomb repressive complex 1 (PRC1). This contributes to histone H2A lysine 119 ubiquitylation (H2AK119ub1) and gene repression. Unexpectedly, we also find that CGIs are occupied by low levels of PRC1 throughout the genome, suggesting that the KDM2B-PRC1 complex may sample CGI-associated genes for susceptibility to polycomb-mediated silencing. These observations demonstrate an unexpected and direct link between recognition of CGIs by KDM2B and targeting of the polycomb repressive system. This provides the basis for a new model describing the functionality of CGIs as mammalian PREs. DOI: http://dx.doi.org/10.7554/eLife.00205.001

Inhibitor Scaffolds for 2-Oxoglutarate-Dependent Histone Lysine Demethylases.

The dynamic methylation of histone lysyl residues plays an important role in biology by regulating transcription, maintaining genomic integrity, and by contributing to epigenetic effects. Here we describe a variety of inhibitor scaffolds that inhibit the human 2-oxoglutarate-dependent JMJD2 subfamily of histone demethylases. Combined with structural data, these chemical starting points will be useful to generate small-molecule probes to analyze the physiological roles of these enzymes in epigenetic signaling.

Understanding the relationship between DNA methylation and histone lysine methylation

DNA methylation acts as an epigenetic modification in vertebrate DNA. Recently it has become clear that the DNA and histone lysine methylation systems are highly interrelated and rely mechanistically on each other for normal chromatin function in vivo. Here we examine some of the functional links between these systems, with a particular focus on several recent discoveries suggesting how lysine methylation may help to target DNA methylation during development, and vice versa. In addition, the emerging role of non-methylated DNA found in CpG islands in defining histone lysine methylation profiles at gene regulatory elements will be discussed in the context of gene regulation. This article is part of a Special Issue entitled: Methylation: A Multifaceted Modification — looking at transcription and beyond.

Quantitative high-throughput screening identifies 8-hydroxyquinolines as cell-active histone demethylase inhibitors.

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.

Selective Inhibitors of the JMJD2 Histone Demethylases: Combined Nondenaturing Mass Spectrometric Screening and Crystallographic Approaches

Ferrous ion and 2-oxoglutarate (2OG) oxygenases catalyze the demethylation of Nε-methylated lysine residues in histones. Here we report studies on the inhibition of the JMJD2 subfamily of histone demethylases, employing binding analyses by nondenaturing mass spectrometry (MS), dynamic combinatorial chemistry coupled to MS, turnover assays, and crystallography. The results of initial binding and inhibition assays directed the production and analysis of a set of N-oxalyl-d-tyrosine derivatives to explore the extent of a subpocket at the JMJD2 active site. Some of the inhibitors were shown to be selective for JMJD2 over the hypoxia-inducible factor prolyl hydroxylase PHD2. A crystal structure of JMJD2A in complex with one of the potent inhibitors was obtained; modeling other inhibitors based on this structure predicts interactions that enable improved inhibition for some compounds.

PHF8, a gene associated with cleft lip/palate and mental retardation, encodes for an Nε-dimethyl lysine demethylase

Mutations of human PHF8 cluster within its JmjC encoding exons and are linked to mental retardation (MR) and a cleft lip/palate phenotype. Sequence comparisons, employing structural insights, suggest that PHF8 contains the double stranded beta-helix fold and ferrous iron binding residues that are present in 2-oxoglutarate-dependent oxygenases. We report that recombinant PHF8 is an Fe(II) and 2-oxoglutarate-dependent N(epsilon)-methyl lysine demethylase, which acts on histone substrates. PHF8 is selective in vitro for N(epsilon)-di- and mono-methylated lysine residues and does not accept trimethyl substrates. Clinically observed mutations to the PHF8 gene cluster in exons encoding for the double stranded beta-helix fold and will therefore disrupt catalytic activity. The PHF8 missense mutation c.836C>T is associated with mild MR, mild dysmorphic features, and either unilateral or bilateral cleft lip and cleft palate in two male siblings. This mutant encodes a F279S variant of PHF8 that modifies a conserved hydrophobic region; assays with both peptides and intact histones reveal this variant to be catalytically inactive. The dependence of PHF8 activity on oxygen availability is interesting because the occurrence of fetal cleft lip has been demonstrated to increase with maternal hypoxia in mouse studies. Cleft lip and other congenital anomalies are also linked indirectly to maternal hypoxia in humans, including from maternal smoking and maternal anti-hypertensive treatment. Our results will enable further studies aimed at defining the molecular links between developmental changes in histone methylation status, congenital disorders and MR.

Targeting Polycomb systems to regulate gene expression: modifications to a complex story.

Polycomb group proteins are transcriptional repressors that are essential for normal gene regulation during development. Recent studies suggest that Polycomb repressive complexes (PRCs) recognize and are recruited to their genomic target sites through a range of different mechanisms, which involve transcription factors, CpG island elements and non-coding RNAs. Together with the realization that the interplay between PRC1 and PRC2 is more intricate than was previously appreciated, this has increased our understanding of the vertebrate Polycomb system at the molecular level.

Conversion of Cysteine into Dehydroalanine Enables Access to Synthetic Histones Bearing Diverse Post-Translational Modifications**

Histones undergo many post-translational modifications (PTMs) at multiple sites and residue types. 4] Lysine methylation, acetylation, ubiquitination, and several other acylations (including succinylation and crotonylation), serine and threonine phosphorylation and glycosylation, as well as arginine methylation and deimination are PTMs common to histones. Many are dynamic and regulate chromatin structure, transcription, and DNA replication and repair. A biochemical understanding of how histone PTMs regulate such function is a current frontier in biology. To enable these studies, access to histones bearing PTMs is required. 11, 12] The precision of chemical synthesis is presently unrivaled in accessing purified histones with multiple PTMs, which may be especially valuable when the associated modifying enzyme or co-substrates are unknown or hard to acquire. Native chemical ligation (NCL) and expressed protein ligation (EPL) 15] are two powerful methods. 16] However, when PTMs lie outside of the first 50 residues of either terminus, or are present at both termini, multiple ligations may be required—a significant challenge. 18] Amber codon suppression technology can also provide modified histones. Acetyllysine and latent forms of monoand dimethyllysine can be incorporated into histones using evolved pyrrolysyl-tRNA synthetase/tRNA pairs. This route to modified histones is promising, but several chemical steps are sometimes required after expression to access the final protein, and it is difficult to apply to multiple modifications. Moreover, many histone modifications— lysine trimethylation, serine and threonine phosphorylation and glycosylation, for example—are not generally accessible using this technology. Site-selective modification of a preformed, full-length histone scaffold is an alternative strategy. This approach would allow, in principle, rapid, diverse elaboration of fulllength proteins as common synthetic intermediates. Divergence of late-stage intermediates is common practice (because of advantages in strategy and efficiency) in smallmolecule synthetic chemistry, but explored less frequently in synthetic biology. Notably, other than Cys110 of histone H3, the core human histones do not contain cysteines. A unique Cys can therefore be positioned at desired sites of modification. Cys alkylation can then provide methylated lysine mimics or a nonhydrolyzable thiocarbamate analogue of acetylated lysine (Scheme 1 a). These aminoethylation protocols, which target protein nucleophiles, benefit from operational simplicity, thus enabling the study of lysine modifications in several contexts. However, whilst