Discovery of NSD2-Degraders from Novel and Selective DEL Hits.

NSD2 is a histone methyltransferase predominantly catalyzing di-methylation of histone H3 on lysine K36. Increased NSD2 activity due to mutations or fusion-events affecting the gene encoding NSD2 is considered an oncogenic event and a driver in various cancers, including multiple myelomas carrying t(4;14) chromosomal translocations and acute lymphoblastic leukemia's expressing the hyperactive NSD2 mutant E1099 K. Using DNA-encoded libraries, we have identified small molecule ligands that selectively and potently bind to the PWWP1 domain of NSD2, inhibit NSD2 binding to H3K36me2-bearing nucleosomes, but do not inhibit the methyltransferase activity. The ligands were subsequently converted to selective VHL1-recruiting NSD2 degraders and by using one of the most efficacious degraders in cell lines, we show that it leads to NSD2 degradation, decrease in K3 K36me2 levels and inhibition of cell proliferation.

Novel CIC variants identified in individuals with neurodevelopmental phenotypes.

Heterozygous pathogenic variants in CIC, which encodes a transcriptional repressor, have been identified in individuals with neurodevelopmental phenotypes. To date, 11 CIC variants have been associated with the CIC-related neurodevelopmental syndrome. Here, we describe three novel and one previously reported CIC variants in four individuals with neurodevelopmental delay. Notably, we report for the first time a de novo frameshift variant specific to the long isoform of CIC (CIC-L, NM_001304815.1:c.1100dup, p.Pro368AlafsTer16) in an individual with speech delay, intellectual disability, and autism spectrum disorder. Our investigation into the function of CIC-L reveals that partial loss of CIC-L leads to transcriptional derepression of CIC target genes. We also describe a missense variant (NM_015125.3:c.683G>A, p.Arg228Gln) in an individual with a history of speech delay and relapsed pre-B acute lymphoblastic leukemia. Functional studies of this variant suggest a partial loss of CIC transcriptional repressor activity. Our study expands the list of CIC pathogenic variants and contributes to the accumulating evidence that CIC haploinsufficiency or partial loss of function is a pathogenic mechanism causing neurodevelopmental phenotypes.

PR-DUB maintains the expression of critical genes through FOXK1/2- and ASXL1/2/3-dependent recruitment to chromatin and H2AK119ub1 deubiquitination.

Polycomb group proteins are important for maintaining gene expression patterns and cell identity in metazoans. The mammalian Polycomb repressive deubiquitinase (PR-DUB) complexes catalyze removal of monoubiquitination on lysine 119 of histone H2A (H2AK119ub1) through a multiprotein core comprised of BAP1, HCFC1, FOXK1/2, and OGT in combination with either of ASXL1, 2, or 3. Mutations in PR-DUB components are frequent in cancer. However, mechanistic understanding of PR-DUB function in gene regulation is limited. Here, we show that BAP1 is dependent on the ASXL proteins and FOXK1/2 in facilitating gene activation across the genome. Although PR-DUB was previously shown to cooperate with PRC2, we observed minimal overlap and functional interaction between BAP1 and PRC2 in embryonic stem cells. Collectively, these results demonstrate that PR-DUB, by counteracting accumulation of H2AK119ub1, maintains chromatin in an optimal configuration ensuring expression of genes important for general functions such as cell metabolism and homeostasis.

Mutant FOXL2C134W Hijacks SMAD4 and SMAD2/3 to Drive Adult Granulosa Cell Tumors.

The mutant protein FOXL2C134W is expressed in at least 95% of adult-type ovarian granulosa cell tumors (AGCT) and is considered to be a driver of oncogenesis in this disease. However, the molecular mechanism by which FOXL2C134W contributes to tumorigenesis is not known. Here, we show that mutant FOXL2C134W acquires the ability to bind SMAD4, forming a FOXL2C134W/SMAD4/SMAD2/3 complex that binds a novel hybrid DNA motif AGHCAHAA, unique to the FOXL2C134W mutant. This binding induced an enhancer-like chromatin state, leading to transcription of nearby genes, many of which are characteristic of epithelial-to-mesenchymal transition. FOXL2C134W also bound hybrid loci in primary AGCT. Ablation of SMAD4 or SMAD2/3 resulted in strong reduction of FOXL2C134W binding at hybrid sites and decreased expression of associated genes. Accordingly, inhibition of TGFß mitigated the transcriptional effect of FOXL2C134W. Our results provide mechanistic insight into AGCT pathogenesis, identifying FOXL2C134W and its interaction with SMAD4 as potential therapeutic targets to this condition. SIGNIFICANCE: FOXL2C134W hijacks SMAD4 and leads to the expression of genes involved in EMT, stemness, and oncogenesis in AGCT, making FOXL2C134W and the TGFß pathway therapeutic targets in this condition. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/17/3466/F1.large.jpg.

The Tumor Suppressor CIC Directly Regulates MAPK Pathway Genes via Histone Deacetylation.

Oligodendrogliomas are brain tumors accounting for approximately 10% of all central nervous system cancers. CIC is a transcription factor that is mutated in most patients with oligodendrogliomas; these mutations are believed to be a key oncogenic event in such cancers. Analysis of the Drosophila melanogaster ortholog of CIC, Capicua, indicates that CIC loss phenocopies activation of the EGFR/RAS/MAPK pathway, and studies in mammalian cells have demonstrated a role for CIC in repressing the transcription of the PEA3 subfamily of ETS transcription factors. Here, we address the mechanism by which CIC represses transcription and assess the functional consequences of CIC inactivation. Genome-wide binding patterns of CIC in several cell types revealed that CIC target genes were enriched for MAPK effector genes involved in cell-cycle regulation and proliferation. CIC binding to target genes was abolished by high MAPK activity, which led to their transcriptional activation. CIC interacted with the SIN3 deacetylation complex and, based on our results, we suggest that CIC functions as a transcriptional repressor through the recruitment of histone deacetylases. Independent single amino acid substitutions found in oligodendrogliomas prevented CIC from binding its target genes. Taken together, our results show that CIC is a transcriptional repressor of genes regulated by MAPK signaling, and that ablation of CIC function leads to increased histone acetylation levels and transcription at these genes, ultimately fueling mitogen-independent tumor growth.Significance: Inactivation of CIC inhibits its direct repression of MAPK pathway genes, leading to their increased expression and mitogen-independent growth.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/15/4114/F1.large.jpg Cancer Res; 78(15); 4114-25. ©2018 AACR.

The demethylase JMJD2C localizes to H3K4me3-positive transcription start sites and is dispensable for embryonic development.

The histone demethylase JMJD2C, also known as KDM4C/GASC1, has activity against methylated H3K9 and H3K36 and is amplified and/or overexpressed in human cancers. By the generation of Jmjd2c knockout mice, we demonstrate that loss of Jmjd2c is compatible with cellular proliferation, embryonic stem cell (ESC) self-renewal, and embryonic development. Moreover, we report that JMJD2C localizes to H3K4me3-positive transcription start sites in both primary cells and in the human carcinoma KYSE150 cell line containing an amplification of the JMJD2C locus. Binding is dependent on the double Tudor domain of JMJD2C, which recognizes H3K4me3 but not H4K20me2/me3 in vitro, showing a binding specificity different from that of the double Tudor domains of JMJD2A and JMJD2B. Depletion of JMJD2C in KYSE150 cells has a modest effect on H3K9me3 and H3K36me3 levels but impairs proliferation and leads to deregulated expression of a subset of target genes involved in cell cycle progression. Taking these findings together, we show that JMJD2C is targeted to H3K4me3-positive transcription start sites, where it can contribute to transcriptional regulation, and report that the putative oncogene JMJD2C generally is not required for cellular proliferation or embryonic development.

Posttranslational modifications of the histone 3 tail and their impact on the activity of histone lysine demethylases in vitro.

Posttranslational modifications (PTMs) of the histone H3 tail such as methylation, acetylation and phosphorylation play important roles in epigenetic signaling. Here we study the effect of some of these PTMs on the demethylation rates of methylated lysine 9 in vitro using peptide substrates mimicking histone H3. Various combinations with other PTMs were employed to study possible cross-talk effects by comparing enzyme kinetic characteristics. We compared the kinetics of histone tail substrates for truncated histone lysine demethylases KDM4A and KDM4C containing only the catalytic core (cc) and some combinations were characterized on full length (FL) KDM4A and KDM4C. We found that the substrates combining trimethylated K4 and K9 resulted in a significant increase in the catalytic activity for FL-KDM4A. For the truncated versions of KDM4A and KDM4C a two-fold increase in the catalytic activity toward bis-trimethylated substrates could be observed. Furthermore, a significant difference in the catalytic activity between dimethylated and trimethylated substrates was found for full length demethylases in line with what has been reported previously for truncated demethylases. Histone peptide substrates phosphorylated at T11 could not be demethylated by neither truncated nor full length KDM4A and KDM4C, suggesting that phosphorylation of threonine 11 prevents demethylation of the H3K9me3 mark on the same peptide. Acetylation of K14 was also found to influence demethylation rates significantly. Thus, for truncated KDM4A, acetylation on K14 of the substrate leads to an increase in enzymatic catalytic efficiency (k cat/K m), while for truncated KDM4C it induces a decrease, primarily caused by changes in K m. This study demonstrates that demethylation activities towards trimethylated H3K9 are significantly influenced by other PTMs on the same peptide, and emphasizes the importance of studying these interactions at the peptide level to get a more detailed understanding of the dynamics of epigenetic marks.

Identification of catechols as histone-lysine demethylase inhibitors.

Identification of inhibitors of histone-lysine demethylase (HDM) enzymes is important because of their involvement in the development of cancer. An ELISA-based assay was developed for identification of inhibitors of the HDM KDM4C in a natural products library. Based on one of the hits with affinity in the low µM range (1, a catechol), a subset of structurally related compounds was selected and tested against a panel of HDMs. In this subset, two inhibitors (2 and 10) had comparable affinities towards KDM4C and KDM6A but no effect on PHF8. One inhibitor restored H3K9me3 levels in KDM4C transfected U2-OS cells.

Studies of H3K4me3 demethylation by KDM5B/Jarid1B/PLU1 reveals strong substrate recognition in vitro and identifies 2,4-pyridine-dicarboxylic acid as an in vitro and in cell inhibitor.

Dynamic methylations and demethylations of histone lysine residues are important for gene regulation and are facilitated by histone methyltransferases and histone demethylases (HDMs). KDM5B/Jarid1B/PLU1 is an H3K4me3/me2-specific lysine demethylase belonging to the JmjC domain-containing family of histone demethylases (JHDMs). Several studies have linked KDM5B to breast, prostate and skin cancer, highlighting its potential as a drug target. However, most inhibitor studies have focused on other JHDMs, and inhibitors for KDM5B remain to be explored. Here, we report the expression, purification and characterization of the catalytic core of recombinant KDM5B (ccKDM5B, residues 1-769). We show that ccKDM5B, recombinantly expressed in insect cells, demethylates H3K4me3 and H3K4me2 in vitro. The kinetic characterization showed that ccKDM5B has an apparent Michaelis constant (K(m) (app) ) value of 0.5 µm for its trimethylated substrate H3(1-15)K4me3, a considerably increased apparent substrate affinity than reported for related HDMs. Despite the presence of a PHD domain, the catalytic activity was not affected by additional methylation at the H3K9 position, suggesting that in vitro chromatin cross-talk between H3K4 and H3K9 does not occur for ccKDM5B. Inhibition studies of ccKDM5B showed both in vitro and in cell inhibition of ccKDM5B by 2,4-pyridinedicarboxylic acid (2,4-PDCA) with a potency similar to that reported for the HDM KDM4C. Structure-guided sequence alignment indicated that the binding mode of 2,4-PDCA is conserved between KDM4A/C and KDM5B.

TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity.

Enzymes catalysing the methylation of the 5-position of cytosine (mC) have essential roles in regulating gene expression and maintaining cellular identity. Recently, TET1 was found to hydroxylate the methyl group of mC, converting it to 5-hydroxymethyl cytosine (hmC). Here we show that TET1 binds throughout the genome of embryonic stem cells, with the majority of binding sites located at transcription start sites (TSSs) of CpG-rich promoters and within genes. The hmC modification is found in gene bodies and in contrast to mC is also enriched at CpG-rich TSSs. We provide evidence further that TET1 has a role in transcriptional repression. TET1 binds a significant proportion of Polycomb group target genes. Furthermore, TET1 associates and colocalizes with the SIN3A co-repressor complex. We propose that TET1 fine-tunes transcription, opposes aberrant DNA methylation at CpG-rich sequences and thereby contributes to the regulation of DNA methylation fidelity.

JARID2 regulates binding of the Polycomb repressive complex 2 to target genes in ES cells.

The Polycomb group (PcG) proteins have an important role in controlling the expression of genes essential for development, differentiation and maintenance of cell fates. The Polycomb repressive complex 2 (PRC2) is believed to regulate transcriptional repression by catalysing the di- and tri-methylation of lysine 27 on histone H3 (H3K27me2/3). At present, it is unknown how the PcG proteins are recruited to their target promoters in mammalian cells. Here we show that PRC2 forms a stable complex with the Jumonji- and ARID-domain-containing protein, JARID2 (ref. 4). Using genome-wide location analysis, we show that JARID2 binds to more than 90% of previously mapped PcG target genes. Notably, we show that JARID2 is sufficient to recruit PcG proteins to a heterologous promoter, and that inhibition of JARID2 expression leads to a major loss of PcG binding and to a reduction of H3K27me3 levels on target genes. Consistent with an essential role for PcG proteins in early development, we demonstrate that JARID2 is required for the differentiation of mouse embryonic stem cells. Thus, these results demonstrate that JARID2 is essential for the binding of PcG proteins to target genes and, consistent with this, for the proper differentiation of embryonic stem cells and normal development.

The H3K27me3 demethylase JMJD3 contributes to the activation of the INK4A-ARF locus in response to oncogene- and stress-induced senescence.

The tumor suppressor proteins p16INK4A and p14ARF, encoded by the INK4A-ARF locus, are key regulators of cellular senescence. The locus is epigenetically silenced by the repressive H3K27me3 mark in normally growing cells, but becomes activated in response to oncogenic stress. Here, we show that expression of the histone H3 Lys 27 (H3K27) demethylase JMJD3 is induced upon activation of the RAS-RAF signaling pathway. JMJD3 is recruited to the INK4A-ARF locus and contributes to the transcriptional activation of p16INK4A in human diploid fibroblasts. Additionally, inhibition of Jmjd3 expression in mouse embryonic fibroblasts results in suppression of p16Ink4a and p19Arf expression and in their immortalization.

Coordinated regulation of transcriptional repression by the RBP2 H3K4 demethylase and Polycomb-Repressive Complex 2.

Polycomb group (PcG) proteins regulate important cellular processes such as embryogenesis, cell proliferation, and stem cell self-renewal through the transcriptional repression of genes determining cell fate decisions. The Polycomb-Repressive Complex 2 (PRC2) is highly conserved during evolution, and its intrinsic histone H3 Lys 27 (K27) trimethylation (me3) activity is essential for PcG-mediated transcriptional repression. Here, we show a functional interplay between the PRC2 complex and the H3K4me3 demethylase Rbp2 (Jarid1a) in mouse embryonic stem (ES) cells. By genome-wide location analysis we found that Rbp2 is associated with a large number of PcG target genes in mouse ES cells. We show that the PRC2 complex recruits Rbp2 to its target genes, and that this interaction is required for PRC2-mediated repressive activity during ES cell differentiation. Taken together, these results demonstrate an elegant mechanism for repression of developmental genes by the coordinated regulation of epigenetic marks involved in repression and activation of transcription.

Erasing the methyl mark: histone demethylases at the center of cellular differentiation and disease.

The enzymes catalyzing lysine and arginine methylation of histones are essential for maintaining transcriptional programs and determining cell fate and identity. Until recently, histone methylation was regarded irreversible. However, within the last few years, several families of histone demethylases erasing methyl marks associated with gene repression or activation have been identified, underscoring the plasticity and dynamic nature of histone methylation. Recent discoveries have revealed that histone demethylases take part in large multiprotein complexes synergizing with histone deacetylases, histone methyltransferases, and nuclear receptors to control developmental and transcriptional programs. Here we review the emerging biochemical and biological functions of the histone demethylases and discuss their potential involvement in human diseases, including cancer.

The emerging functions of histone demethylases.

Epigenetic information refers to heritable changes in gene function that are stable between cell divisions but which is not a result of changes in the DNA sequence. Part of the epigenetic mechanism has been ascribed to modifications of histones or DNA that affects the transcription of specific genes. In this context, post-translational modifications of histone tails, in particular methylation of lysines, are regarded as important for the storage of epigenetic information. Regulation of this information plays an important role during cellular differentiation where cells with different characteristic features evolve from the same ancestor, despite identical genomic material. The characterization of several enzymes catalyzing histone lysine methylation have supported this concept by showing the requirement of these enzymes for normal development and their involvement in diseases such as cancer. The recent identification of proteins with histone demethylase activity has shown that the methylated mark is much more dynamic than previously anticipated, thereby potentially challenging the concept of histone-methylation in stable epigenetic programming.

Posttranslational Protein Modifications in Type 1 Diabetes - Genetic Studies with PCMT1, the Repair Enzyme Protein Isoaspartate Methyltransferase (PIMT) Encoding Gene.

BACKGROUND: Posttranslational protein modifications have been implicated in the development of autoimmunity. Protein L-isoaspartate (D-aspartate) O-methyltransferase (PIMT) repairs modified proteins and is encoded by PCMT1, located in a region linked to type 1 diabetes (T1D), namely IDDM5. AIM: To evaluate the association between genetic variations in the PCMT1 gene and T1D. METHODS: Firstly, PCMT1 was sequenced in 26 patients with T1D (linked to IDDM5) and 10 control subjects. The variations found in PCMT1 were then tested (alone and interacting with a functional polymorphism in SUMO4 and with HLA) for association with T1D in 253 families (using transmission disequilibrium test). In a third step, the association of the functional variation in PCMT1 (rs4816) with T1D was analyzed in 778 T1D patients and 749 controls (using chi-square test). In vitro promoter activity was assessed by transfecting INS-1E cells with PCMT1 promoter constructs and a reporter gene, with or without cytokine stimulation. RESULTS: Four polymorphisms in complete linkage disequilibrium were identified in PCMT1 (5' to the gene (rs11155676), exon 5 (rs4816) and exon 8 (rs7818 and rs4552)). In the whole cohort of 253 families, the allele associated with increased PIMT enzyme activity (rs4816, allele A) was less frequently transmitted to the affected than to the non-affected offspring (46% vs. 53%, p = 0.099). This finding was even more evident in the subset of families where the proband had high-risk SUMO4 (p = 0.069) or low-risk HLA (p = 0.086). Surprisingly, in the case-control study with 778 cases and 749 controls, an inverse trend was found (40.36% of patients and 36.98% of controls had the allele, p = 0.055). PCMT1 promoter activity increased with cytokine stimulation, but no differences were detected between the constructs adjacent to rs11155676. CONCLUSION: PCMT1 was virtually associated with T1D in groups defined by other risk genes (SUMO4 and HLA). A general association in a not further defined sample of T1D patients was not evident. Verification in a larger population is needed.

UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development.

The trithorax and the polycomb group proteins are chromatin modifiers, which play a key role in the epigenetic regulation of development, differentiation and maintenance of cell fates. The polycomb repressive complex 2 (PRC2) mediates transcriptional repression by catalysing the di- and tri-methylation of Lys 27 on histone H3 (H3K27me2/me3). Owing to the essential role of the PRC2 complex in repressing a large number of genes involved in somatic processes, the H3K27me3 mark is associated with the unique epigenetic state of stem cells. The rapid decrease of the H3K27me3 mark during specific stages of embryogenesis and stem-cell differentiation indicates that histone demethylases specific for H3K27me3 may exist. Here we show that the human JmjC-domain-containing proteins UTX and JMJD3 demethylate tri-methylated Lys 27 on histone H3. Furthermore, we demonstrate that ectopic expression of JMJD3 leads to a strong decrease of H3K27me3 levels and causes delocalization of polycomb proteins in vivo. Consistent with the strong decrease in H3K27me3 levels associated with HOX genes during differentiation, we show that UTX directly binds to the HOXB1 locus and is required for its activation. Finally mutation of F18E9.5, a Caenorhabditis elegans JMJD3 orthologue, or inhibition of its expression, results in abnormal gonad development. Taken together, these results suggest that H3K27me3 demethylation regulated by UTX/JMJD3 proteins is essential for proper development. Moreover, the recent demonstration that UTX associates with the H3K4me3 histone methyltransferase MLL2 (ref. 8) supports a model in which the coordinated removal of repressive marks, polycomb group displacement, and deposition of activating marks are important for the stringent regulation of transcription during cellular differentiation.

RBP2 belongs to a family of demethylases, specific for tri-and dimethylated lysine 4 on histone 3.

Methylation of histones has been regarded as a stable modification defining the epigenetic program of the cell, which regulates chromatin structure and transcription. However, the recent discovery of histone demethylases has challenged the stable nature of histone methylation. Here we demonstrate that the JARID1 proteins RBP2, PLU1, and SMCX are histone demethylases specific for di- and trimethylated histone 3 lysine 4 (H3K4). Consistent with a role for the JARID1 Drosophila homolog Lid in regulating expression of homeotic genes during development, we show that RBP2 is displaced from Hox genes during embryonic stem (ES) cell differentiation correlating with an increase of their H3K4me3 levels and expression. Furthermore, we show that mutation or RNAi depletion of the C. elegans JARID1 homolog rbr-2 leads to increased levels of H3K4me3 during larval development and defects in vulva formation. Taken together, these results suggest that H3K4me3/me2 demethylation regulated by the JARID1 family plays an important role during development.