Robert J. Klose

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.

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.

Targeting Polycomb to Pericentric Heterochromatin in Embryonic Stem Cells Reveals a Role for H2AK119u1 in PRC2 Recruitment

Summary The mechanisms by which the major Polycomb group (PcG) complexes PRC1 and PRC2 are recruited to target sites in vertebrate cells are not well understood. Building on recent studies that determined a reciprocal relationship between DNA methylation and Polycomb activity, we demonstrate that, in methylation-deficient embryonic stem cells (ESCs), CpG density combined with antagonistic effects of H3K9me3 and H3K36me3 redirects PcG complexes to pericentric heterochromatin and gene-rich domains. Surprisingly, we find that PRC1-linked H2A monoubiquitylation is sufficient to recruit PRC2 to chromatin in vivo, suggesting a mechanism through which recognition of unmethylated CpG determines the localization of both PRC1 and PRC2 at canonical and atypical target sites. We discuss our data in light of emerging evidence suggesting that PcG recruitment is a default state at licensed chromatin sites, mediated by interplay between CpG hypomethylation and counteracting H3 tail modifications.

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.

ZF-CxxC domain-containing proteins, CpG islands and the chromatin connection

Vertebrate DNA can be chemically modified by methylation of the 5 position of the cytosine base in the context of CpG dinucleotides. This modification creates a binding site for MBD (methyl-CpG-binding domain) proteins which target chromatin-modifying activities that are thought to contribute to transcriptional repression and maintain heterochromatic regions of the genome. In contrast with DNA methylation, which is found broadly across vertebrate genomes, non-methylated DNA is concentrated in regions known as CGIs (CpG islands). Recently, a family of proteins which encode a ZF-CxxC (zinc finger-CxxC) domain have been shown to specifically recognize non-methylated DNA and recruit chromatin-modifying activities to CGI elements. For example, CFP1 (CxxC finger protein 1), MLL (mixed lineage leukaemia protein), KDM (lysine demethylase) 2A and KDM2B regulate lysine methylation on histone tails, whereas TET (ten-eleven translocation) 1 and TET3 hydroxylate methylated cytosine bases. In the present review, we discuss the most recent advances in our understanding of how ZF-CxxC domain-containing proteins recognize non-methylated DNA and describe their role in chromatin modification at CGIs.

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.

CpG island chromatin: a platform for gene regulation.

The majority of mammalian gene promoters are encompassed within regions of the genome called CpG islands that have an elevated level of non-methylated CpG dinucleotides. Despite over 20 years of study, the precise mechanisms by which CpG islands contribute to regulatory element function remain poorly understood. Recently it has been demonstrated that specific histone modifying enzymes are recruited directly to CpG islands through recognition of non-methylated CpG dinucleotide sequence. These enzymes then impose unique chromatin architecture on CpG islands that distinguish them from the surrounding genome. In the context of this work we discuss how CpG island elements may contribute to the function of gene regulatory elements through the utilization of chromatin and epigenetic processes.

Chromatin Sampling—An Emerging Perspective on Targeting Polycomb Repressor Proteins

Polycomb group (PcG) repressor proteins play a central role in gene regulation through differentiation and development, conferring repressive chromatin configurations at target gene promoters through their inherent histone modification activities. Recruitment of Polycomb repressor proteins to defined targets has been attributed to instructive mechanisms in which sequence-specific binding proteins and/or noncoding RNAs interact biochemically with the major Polycomb repressive complexes and thus define their sites of action. Here we highlight that this viewpoint is increasingly incompatible with experimental observations. We propose an alternative perspective based on the concept that Polycomb recruitment is responsive rather than instructive. Specifically, we suggest that Polycomb complexes sample permissive chromatin sites, and through positive feedback mechanisms, accumulate at those sites lacking antagonistic chromatin modifying activities linked to ongoing transcription.

Plant Growth Regulator Daminozide Is a Selective Inhibitor of Human KDM2/7 Histone Demethylases

The JmjC oxygenases catalyze the N-demethylation of N(ε)-methyl lysine residues in histones and are current therapeutic targets. A set of human 2-oxoglutarate analogues were screened using a unified assay platform for JmjC demethylases and related oxygenases. Results led to the finding that daminozide (N-(dimethylamino)succinamic acid, 160 Da), a plant growth regulator, selectively inhibits the KDM2/7 JmjC subfamily. Kinetic and crystallographic studies reveal that daminozide chelates the active site metal via its hydrazide carbonyl and dimethylamino groups.