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Differential Expression in Isogenic Lines

Glycine 34-to-tryptophan (G34W) substitutions in H3.3 arise in approximately 90% of giant cell tumor of bone (GCT). Here, we show H3.3 G34W is necessary for tumor formation. By profiling the epigenome, transcriptome, and secreted proteome of patient samples and tumor-derived cells CRISPR–Cas9-edited for H3.3 G34W, we show that H3.3K36me3 loss on mutant H3.3 alters the deposition of the repressive H3K27me3 mark from intergenic to genic regions, beyond areas of H3.3 deposition. This promotes redistribution of other chromatin marks and aberrant transcription, altering cell fate in mesenchymal progenitors and hindering differentiation. Single-cell transcriptomics reveals that H3.3 G34W stromal cells recapitulate a neoplastic trajectory from a SPP1+ osteoblast-like progenitor population toward an ACTA2+ myofibroblast-like population, which secretes extracellular matrix ligands predicted to recruit and activate osteoclasts. Our findings suggest that H3.3 G34W leads to GCT by sustaining a transformed state in osteoblast-like progenitors, which promotes neoplastic growth, pathologic recruitment of giant osteoclasts, and bone destruction.Significance:This study shows that H3.3 G34W drives GCT tumorigenesis through aberrant epigenetic remodeling, altering differentiation trajectories in mesenchymal progenitors. H3.3 G34W promotes in neoplastic stromal cells an osteoblast-like progenitor state that enables undue interactions with the tumor microenvironment, driving GCT pathogenesis. These epigenetic changes may be amenable to therapeutic targeting in GCT.See related commentary by Licht, p. 1794.This article is highlighted in the In This Issue feature, p. 1775

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Supplementary Information

Human pre-mRNA splicing is primarily catalyzed by the major spliceosome, comprising five small nuclear ribonucleoprotein complexes, U1, U2, U4, U5, and U6 snRNPs, each of which contains the corresponding U-rich snRNA. These snRNAs are encoded by large gene families exhibiting significant sequence variation, but it remains unknown if most human snRNA genes are untranscribed pseudogenes or produce variant snRNAs with the potential to differentially influence splicing. Since gene duplication and variation are powerful mechanisms of evolutionary adaptation, we sought to address this knowledge gap by systematically profiling human U1, U2, U4, and U5 snRNA variant gene transcripts. We identified 55 transcripts that are detectably expressed in human cells, 38 of which incorporate into snRNPs and spliceosomes in 293T cells. All U1 snRNA variants are more than 1000-fold less abundant in spliceosomes than the canonical U1, whereas at least 1% of spliceosomes contain a variant of U2 or U4. In contrast, eight U5 snRNA sequence variants occupy spliceosomes at levels of 1% to 46%. Furthermore, snRNA variants display distinct expression patterns across five human cell lines and adult and fetal tissues. Different RNA degradation rates contribute to the diverse steady state levels of snRNA variants. Our findings suggest that variant spliceosomes containing noncanonical snRNAs may contribute to different tissue- and cell-type–specific alternative splicing patterns.

BACKGROUND: Histone modifications alter transcriptional gene function and participate in cancer progression. Enhancer-of-Zeste-Homologue-2 (EZH2) and Nuclear-Receptor-Binding-SET-domain2 (NSD2) methylate H3K27 and H3K36, respectively, to regulate transcription. Given the therapeutic interest in these enzymes, we investigated expression and coregulation in hormone-sensitive (HS) and castrate-resistant (CR) prostate cancer (PC). METHODS: EZH2 and NSD2 levels were quantified using VECTRA analysis in HS and CRPC tissue microarrays (n = 105 + 66). Expression data from The Cancer Genome Atlas (n = 498), Memorial Sloan Kettering Cancer Center (n = 240), and Stand Up to Cancer/Prostate Cancer Foundation (n = 444) cBioportal datasets were queried, and associations between EZH2 and NSD2 and clinicopathologic variables determined. RESULTS: Tumour expression of NSD2, but not EZH2, increased in CRPC (p = 0.05, 0.09). Epithelial nuclei co-expressing NSD2 and EZH2 increased in CRPC compared to HSPC (69 vs 42%, p = 0.02), and in metastatic tissue relative to benign (55 vs 35%, p = 0.02). cBioportal analysis revealed collinear NSD2/EZH2 expression (Spearman = 0.57, 0.58, 0.58, all p

Posterior fossa type A (PFA) ependymomas exhibit very low H3K27 methylation and express high levels of EZHIP (Enhancer of Zeste Homologs Inhibitory Protein, also termed CXORF67). Here we find that a conserved sequence in EZHIP is necessary and sufficient to inhibit PRC2 catalytic activity in vitro and in vivo. EZHIP directly contacts the active site of the EZH2 subunit in a mechanism similar to the H3 K27M oncohistone. Furthermore, expression of H3 K27M or EZHIP in cells promotes similar chromatin profiles: loss of broad H3K27me3 domains, but retention of H3K27me3 at CpG islands. We find that H3K27me3-mediated allosteric activation of PRC2 substantially increases the inhibition potential of EZHIP and H3 K27M, providing a mechanism to explain the observed loss of H3K27me3 spreading in tumors. Our data indicate that PFA ependymoma and DIPG are driven in part by the action of peptidyl PRC2 inhibitors, the K27M oncohistone and the EZHIP ‘oncohistone-mimic’, that dysregulate gene silencing to promote tumorigenesis., PFA tumours express high levels of EZHIP (also known as CXORF67). Here the authors find that EZHIP directly interacts with the active site of EZH2 and is a competitive inhibitor of PRC2 and that EZHIP gives rise to H3K27me3 genomic profile similar to the K27M oncohistone.

Nucleosomes, composed of DNA and histone proteins, represent the fundamental repeating unit of the eukaryotic genome1; posttranslational modifications of these histone proteins influence the activity of the associated genomic regions to regulate cell identity2–4. Traditionally, trimethylation of histone 3-lysine 4 (H3K4me3) is associated with transcriptional initiation5–10, whereas trimethylation of H3K27 (H3K27me3) is considered transcriptionally repressive11–15. The apparent juxtaposition of these opposing marks, termed “bivalent domains”16–18, was proposed to specifically demarcate of small set transcriptionally-poised lineage-commitment genes that resolve to one constituent modification through differentiation, thereby determining transcriptional status19–22. Since then, many thousands of studies have canonized the bivalency model as a chromatin hallmark of development in many cell types. However, these conclusions are largely based on chromatin immunoprecipitations (ChIP) with significant methodological problems hampering their interpretation. Absent direct quantitative measurements, it has been difficult to evaluate the strength of the bivalency model. Here, we present reICeChIP, a calibrated sequential ChIP method to quantitatively measure H3K4me3/H3K27me3 bivalency genome-wide, addressing the limitations of prior measurements. With reICeChIP, we profile bivalency through the differentiation paradigm that first established this model16,18: from naïve mouse embryonic stem cells (mESCs) into neuronal progenitor cells (NPCs). Our results cast doubt on every aspect of the bivalency model; in this context, we find that bivalency is widespread, does not resolve with differentiation, and is neither sensitive nor specific for identifying poised developmental genes or gene expression status more broadly. Our findings caution against interpreting bivalent domains as specific markers of developmentally poised genes.

A high percentage of pediatric gliomas and bone tumors reportedly harbor missense mutations at glycine 34 in genes encoding histone variant H3.3. We find that these H3.3 G34 mutations directly alter the enhancer chromatin landscape of mesenchymal stem cells by impeding methylation at lysine 36 on histone H3 (H3K36) by SETD2, but not by the NSD1/2 enzymes. The reduction of H3K36 methylation by G34 mutations promotes an aberrant gain of PRC2-mediated H3K27me2/3 and loss of H3K27ac at active enhancers containing SETD2 activity. This altered histone modification profile promotes a unique gene expression profile that supports enhanced tumor development in vivo. Our findings are mirrored in G34W-containing giant cell tumors of bone where patient-derived stromal cells exhibit gene expression profiles associated with early osteoblastic differentiation. Overall, we demonstrate that H3.3 G34 oncohistones selectively promote PRC2 activity by interfering with SETD2-mediated H3K36 methylation. We propose that PRC2-mediated silencing of enhancers involved in cell differentiation represents a potential mechanism by which H3.3 G34 mutations drive these tumors.

Glycine 34-to-tryptophan (G34W) substitutions in H3.3 arise in approximately 90% of giant cell tumor of bone (GCT). Here, we show H3.3 G34W is necessary for tumor formation. By profiling the epigenome, transcriptome, and secreted proteome of patient samples and tumor-derived cells CRISPR–Cas9-edited for H3.3 G34W, we show that H3.3K36me3 loss on mutant H3.3 alters the deposition of the repressive H3K27me3 mark from intergenic to genic regions, beyond areas of H3.3 deposition. This promotes redistribution of other chromatin marks and aberrant transcription, altering cell fate in mesenchymal progenitors and hindering differentiation. Single-cell transcriptomics reveals that H3.3 G34W stromal cells recapitulate a neoplastic trajectory from a SPP1+ osteoblast-like progenitor population toward an ACTA2+ myofibroblast-like population, which secretes extracellular matrix ligands predicted to recruit and activate osteoclasts. Our findings suggest that H3.3 G34W leads to GCT by sustaining a transformed state in osteoblast-like progenitors, which promotes neoplastic growth, pathologic recruitment of giant osteoclasts, and bone destruction. Significance: This study shows that H3.3 G34W drives GCT tumorigenesis through aberrant epigenetic remodeling, altering differentiation trajectories in mesenchymal progenitors. H3.3 G34W promotes in neoplastic stromal cells an osteoblast-like progenitor state that enables undue interactions with the tumor microenvironment, driving GCT pathogenesis. These epigenetic changes may be amenable to therapeutic targeting in GCT. See related commentary by Licht, p. 1794. This article is highlighted in the In This Issue feature, p. 1775

Diffuse midline gliomas and posterior fossa type-A ependymomas contain the highly recurrent histone H3 K27M mutation and the H3 K27M-mimic EZHIP, respectively. In vitro, H3 K27M and EZHIP are competitive inhibitors of Polycomb Repressive Complex 2 (PRC2) lysine methyltransferase activity. In vivo, these proteins reduce overall H3K27me3 levels, however residual peaks of H3K27me3 remain at CpG islands through an unknown mechanism. Here, we report that EZHIP and H3 K27M preferentially interact with an allosterically activated form of PRC2 in vivo. The formation of H3 K27M- and EZHIP-PRC2 complexes occurs at CpG islands containing H3K27me3 and impedes PRC2 and H3K27me3 spreading. While EZHIP is not found outside of placental mammals, we find that expression of human EZHIP reduces H3K27me3 in Drosophila melanogaster through a conserved molecular mechanism. Our results highlight the mechanistic similarities between EZHIP and H3 K27M in vivo and provide mechanistic insight for the retention of residual H3K27me3 in tumors driven by these oncogenes.

Histone posttranslational modifications control the organization and function of chromatin. In particular, methylation of lysine 36 in histone H3 (H3K36me) has been shown to mediate gene transcription, DNA repair, cell cycle regulation, and pre-mRNA splicing. Notably, mutations at or near this residue have been causally linked to the development of several human cancers. These observations have helped to illuminate the role of histones themselves in disease and to clarify the mechanisms by which they acquire oncogenic properties. This perspective focuses on recent advances in discovery and characterization of histone H3 mutations that impact H3K36 methylation. We also highlight findings that the common cancer-related substitution of H3K36 to methionine (H3K36M) disturbs functions of not only H3K36me-writing enzymes but also H3K36me-specific readers. The latter case suggests that the oncogenic effects could also be linked to the inability of readers to engage H3K36M.

Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of Polycomb Repressor Complex 2 (PRC2), the enzyme that catalyzes monomethylation, dimethylation, and trimethylation of lysine 27 on histone H3 (H3K27). Trimethylation at H3K27 (H3K27me3) is associated with transcriptional silencing of developmentally important genes. Intriguingly, H3K27me3 is mutually exclusive with H3K36 trimethylation on the same histone tail. Disruptions in this cross-talk result in aberrant H3K27/H3K36 methylation patterns and altered transcriptional profiles that have been implicated in tumorigenesis and other disease states. Despite their importance, the molecular details of how PRC2 “senses” H3K36 methylation are unclear. We demonstrate that PRC2 is activated in cis by the unmodified side chain of H3K36, and that this activation results in a fivefold increase in the k(cat) of its enzymatic activity catalyzing H3K27 methylation compared with activity on a substrate methylated at H3K36. Using a photo-cross-linking MS strategy and histone methyltransferase activity assays on PRC2 mutants, we find that EZH2 contains a specific sensing pocket for the H3K36 methylation state that allows the complex to distinguish between modified and unmodified H3K36 residues, altering enzymatic activity accordingly to preferentially methylate the unmodified nucleosome substrate. We also present evidence that this process may be disrupted in some cases of Weaver syndrome.

PURPOSE: Many transformed cells and embryonic stem cells are dependent on the biosynthesis of the universal methyl-donor S-adenosylmethionine (SAM) from methionine by the enzyme MAT2A to maintain their epigenome. We hypothesized that cancer stem cells (CSCs) rely on SAM biosynthesis and that the combination of methionine depletion and MAT2A inhibition would eradicate CSCs. METHODS: Human triple (ER/PR/HER2)-negative breast carcinoma (TNBC) cell lines were cultured as CSC-enriched mammospheres in control or methionine-free media. MAT2A was inhibited with siRNAs or cycloleucine. The effects of methionine restriction and/or MAT2A inhibition on the formation of mammospheres, the expression of CSC markers (CD44(hi)/C24(low)), MAT2A and CSC transcriptional regulators, apoptosis induction, and histone modifications were determined. A murine model of metastatic TNBC was utilized to evaluate the effects of dietary methionine restriction, MAT2A inhibition and the combination. RESULTS: Methionine restriction inhibited mammosphere formation and reduced the CD44(hi)/C24(low) CSC population; these effects were partly rescued by SAM. Methionine depletion induced MAT2A expression (mRNA and protein) and sensitized CSCs to inhibition of MAT2A (siRNAs or cycloleucine). Cycloleucine enhanced the effects of methionine depletion on H3K4me3 demethylation and suppression of Sox9 expression. Dietary methionine restriction induced MAT2A expression in mammary tumors, and the combination of methionine restriction and cycloleucine was more effective than either alone at suppressing primary and lung metastatic tumor burden in a murine TNBC model. CONCLUSIONS: Our findings point to SAM biosynthesis as a unique metabolic vulnerability of CSCs that can be targeted by combining methionine depletion with MAT2A inhibition to eradicate drug resistant-CSCs.

Anti-silencing function 1 (ASF1) is a histone H3-H4 chaperone involved in DNA replication and repair, and transcriptional regulation. Here, we identify ASF1B, the mammalian paralog to ASF1, as a proliferation-inducing histone chaperone in human β-cells. Overexpression of ASF1B led to distinct transcriptional signatures consistent with increased cellular proliferation and reduced cellular death. Using multiple methods of monitoring proliferation and mitotic progression, we show that overexpression of ASF1B is sufficient to induce human β-cell proliferation. Co-expression of histone H3.3 further augmented β-cell proliferation, whereas suppression of endogenous H3.3 attenuated the stimulatory effect of ASF1B. Using the histone binding-deficient mutant of ASF1B (V94R), we show that histone binding to ASF1B is required for the induction of β-cell proliferation. In contrast to H3.3, overexpression of histone H3 variants H3.1 and H3.2 did not have an impact on ASF1B-mediated induction of proliferation. Our findings reveal a novel role of ASF1B in human β-cell replication and show that ASF1B and histone H3.3A synergistically stimulate human β-cell proliferation.

Lys-27-Met mutations in histone 3 genes (H3K27M) characterize a subgroup of deadly gliomas and decrease genome-wide H3K27 trimethylation. Here we use primary H3K27M tumor lines and isogenic CRISPR-edited controls to assess H3K27M effects in vitro and in vivo. We find that whereas H3K27me3 and H3K27me2 are normally deposited by PRC2 across broad regions, their deposition is severely reduced in H3.3K27M cells. H3K27me3 is unable to spread from large unmethylated CpG islands, while H3K27me2 can be deposited outside these PRC2 high-affinity sites but to levels corresponding to H3K27me3 deposition in wild-type cells. Our findings indicate that PRC2 recruitment and propagation on chromatin are seemingly unaffected by K27M, which mostly impairs spread of the repressive marks it catalyzes, especially H3K27me3. Genome-wide loss of H3K27me3 and me2 deposition has limited transcriptomic consequences, preferentially affecting lowly-expressed genes regulating neurogenesis. Removal of H3K27M restores H3K27me2/me3 spread, impairs cell proliferation, and completely abolishes their capacity to form tumors in mice., Lysine27-to-methionine mutations in histone H3 genes (H3K27M) occur in a subgroup of gliomas and decrease genome-wide H3K27 trimethylation. Here the authors utilise primary H3K27M tumour lines and isogenic CRISPR-edited controls and show that H3K27M induces defective chromatin spread of PRC2-mediated repressive H3K27me2/me3.

Reprogramming cell fate during the first stages of embryogenesis requires that transcriptional activators gain access to the genome and remodel the zygotic transcriptome. Nonetheless, it is not clear whether the continued activity of these pioneering factors is required throughout zygotic genome activation or whether they are only required early to establish cis-regulatory regions. To address this question, we developed an optogenetic strategy to rapidly and reversibly inactivate the master regulator of genome activation in Drosophila, Zelda. Using this strategy, we demonstrate that continued Zelda activity is required throughout genome activation. We show that Zelda binds DNA in the context of nucleosomes and suggest that this allows Zelda to occupy the genome despite the rapid division cycles in the early embryo. These data identify a powerful strategy to inactivate transcription factor function during development and suggest that reprogramming in the embryo may require specific, continuous pioneering functions to activate the genome.

The commonly mutated genes in pancreatic neuroendocrine tumors (PanNETs) are ATRX, DAXX, and MEN1. We genotyped 64 PanNETs and found 58% carry ATRX, DAXX, and MEN1 mutations (A-D-M mutant PanNETs) and this correlates with a worse clinical outcome than tumors carrying the wild-type alleles of all three genes (A-D-M WT PanNETs). We performed RNA sequencing and DNA-methylation analysis to reveal two distinct subgroups with one consisting entirely of A-D-M mutant PanNETs. Two genes differentiating A-D-M mutant from A-D-M WT PanNETs were high ARX and low PDX1 gene expression with PDX1 promoter hyper-methylation in the A-D-M mutant PanNETs. Moreover, A-D-M mutant PanNETs had a gene expression signature related to that of alpha-cells (FDR q-value, In pancreatic neuroendocrine tumors (PanNETs) ATRX, DAXX, and MEN1 are commonly mutated (A-D-M mutant PanNETs). Here, the authors find in a cohort of PanNETS 58% are A-D-M mutant PanNETs, with a worse clinical outcome and differences in gene expression and methylation compared to A-D-M wild type cases- these gene expression differences suggest that A-D-M mutant PanNETs potentially originate from a cell type similar to alpha cells.

High-grade gliomas (HGG) defined by histone 3 K27M driver mutations exhibit global loss of H3K27 trimethylation and reciprocal gain of H3K27 acetylation, respectively shaping repressive and active chromatin landscapes. We generated tumor-derived isogenic models bearing this mutation and show that it leads to pervasive H3K27ac deposition across the genome. In turn, active enhancers and promoters are not created de novo and instead reflect the epigenomic landscape of the cell of origin. H3K27ac is enriched at repeat elements, resulting in their increased expression, which in turn can be further amplified by DNA demethylation and histone deacetylase inhibitors providing an exquisite therapeutic vulnerability. These agents may therefore modulate anti-tumor immune responses as a therapeutic modality for this untreatable disease.

Eukaryotic genomes are dynamically regulated through a host of epigenetic stimuli. The substrate for these epigenetic transactions, chromatin, is a polymer of nucleosome building blocks. In native (i.e. cellular) chromatin, each nucleosome can differ from its neighbors through the localized installation of covalent modifications to both the genomic DNA and the histone packaging proteins. The heterotypic nature of chromatin presents a formidable obstacle to biochemical studies seeking to understand the role of context on epigenetic regulation and that, as a consequence, must employ compositionally defined chromatin substrates. Here, we introduce a chemical approach to the production of heterotypic ‘designer’ chromatin that can be used in such studies. Our method involves attachment of a user-defined modified histone peptide to a designated nucleosome within the polymer by using a Peptide Nucleic Acid (PNA) targeting compound. We apply this strategy to dissect the role of chromatin context on both the activation and inhibition of the histone methyltransferase, PRC2, which methylates Lys 27 of histone H3 (H3K27). Our studies show that PRC2 can be stimulated to produce de novo H3K27 methylation from a defined nucleation site. More generally, this technology promises to facilitate biochemical studies that require the use of heterotypic chromatin substrates.

ATRX (the alpha thalassemia/mental retardation syndrome X-linked protein) is a member of the switch2/sucrose nonfermentable2 (SWI2/SNF2) family of chromatin-remodeling proteins and primarily functions at heterochromatic loci via its recognition of “repressive” histone modifications [e.g., histone H3 lysine 9 tri-methylation (H3K9me3)]. Despite significant roles for ATRX during normal neural development, as well as its relationship to human disease, ATRX function in the central nervous system is not well understood. Here, we describe ATRX’s ability to recognize an activity-dependent combinatorial histone modification, histone H3 lysine 9 tri-methylation/serine 10 phosphorylation (H3K9me3S10ph), in postmitotic neurons. In neurons, this “methyl/phos” switch occurs exclusively after periods of stimulation and is highly enriched at heterochromatic repeats associated with centromeres. Using a multifaceted approach, we reveal that H3K9me3S10ph-bound Atrx represses noncoding transcription of centromeric minor satellite sequences during instances of heightened activity. Our results indicate an essential interaction between ATRX and a previously uncharacterized histone modification in the central nervous system and suggest a potential role for abnormal repetitive element transcription in pathological states manifested by ATRX dysfunction.

Studies in pediatric high-grade astrocytomas (HGA) by our group and others have uncovered recurrent somatic mutations affecting highly conserved residues in histone 3 (H3) variants. One of these mutations leads to analogous p.Lys27Met (K27M) mutations in both H3.3 and H3.1 variants, is associated with rapid fatal outcome, and occurs specifically in HGA of the midline in children and young adults. This includes diffuse intrinsic pontine gliomas (80 %) and thalamic or spinal HGA (>90 %), which are surgically challenging locations with often limited tumor material available and critical need for specific histopathological markers. Here, we analyzed formalin-fixed paraffin-embedded tissues from 143 pediatric HGA and 297 other primary brain tumors or normal brain. Immunohistochemical staining for H3K27M was compared to tumor genotype, and also compared to H3 tri-methylated lysine 27 (H3K27me3) staining, previously shown to be drastically decreased in samples carrying this mutation. There was a 100 % concordance between genotype and immunohistochemical analysis of H3K27M in tumor samples. Mutant H3K27M was expressed in the majority of tumor cells, indicating limited intra-tumor heterogeneity for this specific mutation within the limits of our dataset. Both H3.1 and H3.3K27M mutants were recognized by this antibody while non-neoplastic elements, such as endothelial and vascular smooth muscle cells or lymphocytes, did not stain. H3K27me3 immunoreactivity was largely mutually exclusive with H3K27M positivity. These results demonstrate that mutant H3K27M can be specifically identified with high specificity and sensitivity using an H3K27M antibody and immunohistochemistry. Use of this antibody in the clinical setting will prove very useful for diagnosis, especially in the context of small biopsies in challenging midline tumors and will help orient care in the context of the extremely poor prognosis associated with this mutation. Electronic supplementary material The online version of this article (doi:10.1007/s00401-014-1337-4) contains supplementary material, which is available to authorized users.

Diffuse intrinsic pontine glioma (DIPG) is a fatal brain cancer that arises in the brainstem of children, with no effective treatment and near 100% fatality. The failure of most therapies can be attributed to the delicate location of these tumors and to the selection of therapies on the basis of assumptions that DIPGs are molecularly similar to adult disease. Recent studies have unraveled the unique genetic makeup of this brain cancer, with nearly 80% found to harbor a p.Lys27Met histone H3.3 or p.Lys27Met histone H3.1 alteration. However, DIPGs are still thought of as one disease, with limited understanding of the genetic drivers of these tumors. To understand what drives DIPGs, we integrated whole-genome sequencing with methylation, expression and copy number profiling, discovering that DIPGs comprise three molecularly distinct subgroups (H3-K27M, silent and MYCN) and uncovering a new recurrent activating mutation affecting the activin receptor gene ACVR1 in 20% of DIPGs. Mutations in ACVR1 were constitutively activating, leading to SMAD phosphorylation and increased expression of the downstream activin signaling targets ID1 and ID2. Our results highlight distinct molecular subgroups and novel therapeutic targets for this incurable pediatric cancer.

© The Authors, some rights reserved;. Childhood posterior fossa (PF) ependymomas cause substantial morbidity and mortality. These tumors lack recurrent genetic mutations, but a subset of these ependymomas exhibits CpG island (CpGi) hypermethylation [PF group A (PFA)], implicating epigenetic alterations in their pathogenesis. Further, histological grade does not reliably predict prognosis, highlighting the importance of developing more robust prognostic markers. We discovered global H3K27me3 reduction in a subset of these tumors (PF-ve ependymomas) analogous to H3K27M mutant gliomas. PF-ve tumors exhibited many clinical and biological similarities with PFA ependymomas. Genomic H3K27me3 distribution showed an inverse relationship with CpGi methylation, suggesting that CpGi hypermethylation drives low H3K27me3 in PF-ve ependymomas. Despite CpGi hypermethylation and global H3K27me3 reduction, these tumors showed DNA hypomethylation in the rest of the genome and exhibited increased H3K27me3 genomic enrichment at limited genomic loci similar to H3K27M mutant gliomas. Combined integrative analysis of PF-ve ependymomas with H3K27M gliomas uncovered common epigenetic deregulation of select factors that control radial glial biology, and PF radial glia in early human development exhibited reduced H3K27me3. Finally, H3K27me3 immunostaining served as a biomarker of poor prognosis and delineated radiologically invasive tumors, suggesting that reduced H3K27me3 may be a prognostic indicator in PF ependymomas. 2016

Lysine to methionine (K-to-M) mutations in genes encoding histone H3 are thought to drive a subset of pediatric brain and bone cancers. These high-frequency K-to-M mutations occur at sites of methylation on histone H3, and tumors containing the mutant histones exhibit a global loss of specific histone methylation marks. Previous studies showed that K-to-M mutant histones, also known as oncohistones, are potent orthosteric inhibitors of specific Su(var)3-9, Enhancer-of-zeste, Trithorax (SET) domain methyltransferases. However, the biochemical and biophysical details of the interaction between K-to-M mutant histones and the respective SET domain methyltransferases are currently unknown. Here, we use the histone H3K9-directed methyltransferase G9a as a model to explore the mechanism of inhibition by K-to-M oncohistones. X-ray cocrystal structures revealed that the K9M residue of histone H3 occupies the active site cavity of G9a, and kinetic analysis indicates competitive inhibition of G9a by histone H3K9M. Additionally, we find that the cofactor S-adenosyl methionine (SAM) is necessary for stable interaction between G9a and H3K9M histone. Consistent with the formation of a ternary complex, we find that the inhibitory peptide is uncompetitive with regard to SAM. These data and others indicate that K-to-M oncohistones promote global loss of specific lysine methylation through sequestration and inhibition of SAM-bound SET domain methyltransferases.

The Drosophila melanogaster Myb-MuvB/dREAM complex (MMB/dREAM) participates in both the activation and repression of developmentally regulated genes and origins of DNA replication. Mutants in MMB subunits exhibit diverse phenotypes, including lethality, eye defects, reduced fecundity, and sterility. Here, we used P-element excision to generate mutations in lin-52, which encodes the smallest subunit of the MMB/dREAM complex. lin-52 is required for viability, as null mutants die prior to pupariation. The generation of somatic and germ line mutant clones indicates that lin-52 is required for adult eye development and for early embryogenesis via maternal effects. Interestingly, the maternal-effect embryonic lethality, larval lethality, and adult eye defects could be suppressed by mutations in other subunits of the MMB/dREAM complex. These results suggest that a partial MMB/dREAM complex is responsible for the lethality and eye defects of lin-52 mutants. Furthermore, these findings support a model in which the Lin-52 and Myb proteins counteract the repressive activities of the other members of the MMB/dREAM complex at specific genomic loci in a developmentally controlled manner.

Ependymomas in children occur most commonly in the posterior fossa of the brain and cause significant morbidity and mortality. The pathogenesis of these tumors remains obscure as recent whole genome and whole exome sequencing efforts have not yielded recurrent genetic mutations. These tumors exhibit abnormal CpG island methylation, suggesting that epigenetic alterations may be a significant driver. To gain insights into the epigenetics of childhood ependymomas, we performed mass spectroscopy for histone modifications to discover that H3K27 trimethylation was globally reduced in these tumors. Despite global reduction in H3K27me3, ChIP-sequencing for H3K27me3 revealed genomic enrichment at several loci important for neurodevelopment. In a cohort of 195 childhood posterior fossa ependymoma samples, reduction in H3K27me3 was associated with worse prognosis (p

Embryonic development is regulated by both genetic and epigenetic mechanisms, with nearly all DNA-templated processes influenced by chromatin architecture. Sequence variations in histone proteins, core components of chromatin, provide a means to generate diversity in the chromatin structure, resulting in distinct and profound biological outcomes in the developing embryo. Emerging literature suggests that epigenetic contributions from histone variants play key roles in a number of developmental processes such as the initiation and maintenance of pericentric heterochromatin, X-inactivation, and germ cell differentiation. Here, we review the role of histone variants in the embryo with particular emphasis on early mammalian development.

Covalent modifications to both the DNA and the histone proteins allow chromatin to act as a dynamic information hub that integrates diverse biochemical stimuli to regulate genomic DNA access to the transcription machinery and ultimately establish and maintain cellular phenotypes. Moreover, there is increasing appreciation that chromatin alterations per se, including DNA and histone modifications, are involved in the pathogenesis of cancer. Nowhere is this better supported than with the groundbreaking discoveries of high-frequency, somatic mutations in histones that are drivers of oncogenesis. These mutations (collectively called "oncohistones") cause amino acid substitutions that localize to conserved residues in the N-terminal tail of histone H3 and all seem to be linked, either directly or indirectly, to disruption of normal levels and distribution of histone H3 methylation and thus genomic regulation. Specifically, oncohistones directly or indirectly promote aberrant genome-wide distribution of lysine 27 methylation on histone H3. H3K27 methylation, catalyzed by the Polycomb Repressive Complex 2 (PRC2), is mechanistically linked to establishment and maintenance of gene repression. We are currently using a combination of biochemical and genomic approaches to investigate how oncohistone-driven changes in histone H3 K27 methylation lead to altered chromatin states that profoundly influence gene expression patterns. I will discuss some of our recent mechanistic and functional work on various oncohistone mutations. Citation Format: Peter W. Lewis. Polycomb dysregulation by oncohistones [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY05-02.

The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state. Using genome editing with zinc finger nucleases to tag endogenous H3.3, we report genome-wide profiles of H3 variants in mammalian embryonic stem (ES) cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acid sequence, and change with cellular differentiation at developmentally regulated loci. The H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for localization of H3.3 at telomeres and many transcription factor binding sites. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Atrx is required for Hira-independent localization of H3.3 at telomeres, and for the repression of telomeric RNA. Our data demonstrate that multiple and distinct factors are responsible for H3.3 localization at specific genomic locations in mammalian cells.

Several types of bone/cartilage tumors reportedly contain high-frequency missense mutations in histone H3, yet the underlying oncogenic mechanism remains poorly characterized. We find that the H3 lysine 36 to methionine (H3K36M) mutation, identified in >90% of chondroblastomas, impairs the differentiation of mesenchymal progenitor cells and generates undifferentiated sarcoma in vivo. H3K36M mutant nucleosomes inhibit the enzymatic activities of several H3K36 methyltransferases, resulting in globally diminished H3K36 methylation. Depletion of H3K36 methyltransferases, or expressing an H3K36I mutant that similarly inhibits H3K36 methylation, is sufficient to phenocopy H3K36M mutation. Following the loss of H3K36 methylation, a genome-wide gain in H3K27 methylation leads to a redistribution of polycomb repressive complex 1 and de-repression of polycomb target genes known to block mesenchymal differentiation. Our findings are mirrored in human undifferentiated sarcomas where novel K36M/I mutations in H3.1 are identified in rare pediatric cases. This abstract is also being presented as Poster A12. Citation Format: Chao Lu, Siddhant Jain, Dominik Hoelper, Denise Bechet, Leili Ran, Irene Andrulis, Brendan Dickson, Jacek Majewski, Craig Thompson, Ping Chi, Benjamin Garcia, Nada Jabado, Peter Lewis, C. David Allis. Histone H3K36 mutations promote sarcomagenesis through altered histone methylation landscape [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr PR12.

Human papillomavirus (HPV)-negative head and neck squamous cell carcinomas (HNSCCs) are deadly and common cancers. Recent genomic studies implicate multiple genetic pathways, including cell signaling, cell cycle and immune evasion, in their development. Here we analyze public data sets and uncover a previously unappreciated role of epigenome deregulation in the genesis of 13% of HPV-negative HNSCCs. Specifically, we identify novel recurrent mutations encoding p.Lys36Met (K36M) alterations in multiple H3 histone genes. We further validate the presence of these alterations in multiple independent HNSCC data sets and show that, along with previously described NSD1 mutations, they correspond to a specific DNA methylation cluster. The K36M substitution and NSD1 defects converge on altering methylation of histone H3 at K36 (H3K36), subsequently blocking cellular differentiation and promoting oncogenesis. Our data further indicate limited redundancy for NSD family members in HPV-negative HNSCCs and suggest a potential role for impaired H3K36 methylation in their development. Further investigation of drugs targeting chromatin regulators is warranted in HPV-negative HNSCCs driven by aberrant H3K36 methylation. Citation Format: Chao Lu, Simon Papillon-Cavanagh, Tenzin Gayden, Leonie G. Mikael, Denise Bechet, Christina Karamboulas, Laurie Ailles, Jason Karamchandani, Dylan M. Marchione, Benjamin A. Garcia, Ilan Weinreb, David Goldstein, Peter W. Lewis, Octavia-Maria Dancu, Sandeep Dhaliwal, William Stecho, Christopher J. Howlett, Joe S. Mymryk, John W. Barrett, Anthony C. Nichols, C David Allis, Jacek Majewski, Nada Jabado. Impaired H3K36 methylation defines a subset of head and neck squamous cell carcinomas [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 08.

Pediatric posterior fossa ependymomas are poorly understood childhood brain tumors and have no effective treatments. The biology of these tumors is obscure as recent sequencing efforts suggest that they lack recurrent genetic alterations. A subset of these tumors termed PF-A ependymomas exhibits CpG-island hypermethylation implicating epigenetic alterations in their pathogenesis. Through comprehensive analyses of histone modification, we discovered global H3K27me3 reduction in a subset of these tumors. Tumors with lowered H3K27me3 showed many clinical and biologic similarities with PFA-ependymomas. Global reduction in H3K27me3 is likewise observed in pediatric gliomas that bear histone H3K27M mutations termed diffuse intrinsic pontine gliomas (DIPG) that also arise in the posterior fossa of young children. Analyses of ependymomas with reduced H3K27me3 and H3K27M mutant DIPGs showed many similarities in DNA methylation and enrichment of H3K27me3 in many genomic loci important for neuroglial specification. Combined integrative analysis of both tumor types uncovered common epigenetic deregulation of select factors that control radial glial biology and radial glia in the developing posterior fossa showed reduced H3K27me3. Finally, PF ependymomas with lowered H3K27me3 were more invasive radiologically and exhibited poor prognosis in three independent cohorts (P300). These data have clinical implications for biomarker development and to inform epigenetic approaches to treat PF ependymomas. Citation Format: Sriram Venneti, Jill Bayliss, Piali Mukherjee, Chao Lu, Siddhant Jain, Chan Chung, Daniel Martinez, Benjamin Sabari, Ashley Margol, Pooja Panwalkar, Abhijit Paroloia, Melike Pekmezci, Richard Mc Eachin, Marcin Cieslik, Benita Tamrazi, Benjamin Garcia, Gaspare La Rocca, Mariarita Santi, Peter Lewis, Cynthia Hawkins, Ari Melnick, C David Allis, Craig B. Thompson, Arul Chinnaiyan, Alexander R. Judkins. A subset of poorly prognostic pediatric posterior fossa ependymomas exhibit lowered H3K27me3 and DNA hypomethylation and show epigenetic similarities with H3K27M mutant diffuse intrinsic pontine gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3863. doi:10.1158/1538-7445.AM2017-3863

The histone methyltransferase PRC2 plays a central role in genomic stability and cellular development. Consequently, its misregulation has been implicated in several cancers. Recent work has shown that a histone H3 mutant, where the PRC2 substrate residue Lys27 is replaced by methionine, is also associated with cancer phenotypes and functions as an inhibitor of PRC2. Here we investigate the mechanism of this PRC2 inhibition through kinetic studies and photo-cross-linking. Efficient inhibition is dependent on (1) hydrophobic lysine isosteres blocking the active site, (2) proximal residues, and (3) the H3 tail forming extensive contacts with the EZH2 subunit of PRC2. We further show that naturally occurring post-translational modifications of the same H3 tail, both proximal and distal to K27M, can greatly diminish the inhibition of PRC2. These results suggest that this potent gain of function mutation may be “detoxified” by modulating alternate chromatin modification pathways.

Pediatric glioblastomas (GBM) are highly aggressive and lethal tumors. Recent sequencing studies have shown that ~30 % of pediatric GBM and ~80 % of diffuse intrinsic pontine gliomas show K27M mutations in the H3F3A gene, a variant encoding histone H3.3. H3F3A K27M mutations lead to global reduction in H3K27me3. Our goal was to develop biomarkers for the histopathologic detection of these tumors. Therefore, we evaluated the utility of measuring H3K27me3 global reduction as a histopathologic and prognostic biomarker and tested an antibody directed specifically against the H3.3 K27M mutation in 290 samples. The study cohort included 203 pediatric (including 38 pediatric high-grade astrocytomas) and 38 adult brain tumors of various subtypes and grades and 49 non-neoplastic reactive brain tissues. Detection of H3.3 K27M by immunohistochemistry showed 100 % sensitivity and specificity and was superior to global reduction in H3K27me3 as a biomarker in diagnosing H3F3A K27M mutations. Moreover, cases that stained positive for H3.3 K27M showed a significantly poor prognosis compared to corresponding negative tumors. These results suggest that immunohistochemical detection of H3.3 K27M is a sensitive and specific surrogate for the H3F3A K27M mutation and defines a prognostically poor subset of pediatric GBM. Electronic supplementary material The online version of this article (doi:10.1007/s00401-014-1338-3) contains supplementary material, which is available to authorized users.

SummaryAcquired chromosomal instability and copy number alterations are hallmarks of cancer. Enzymes capable of promoting site-specific copy number changes have yet to be identified. Here, we demonstrate that H3K9/36me3 lysine demethylase KDM4A/JMJD2A overexpression leads to localized copy gain of 1q12, 1q21, and Xq13.1 without global chromosome instability. KDM4A-amplified tumors have increased copy gains for these same regions. 1q12h copy gain occurs within a single cell cycle, requires S phase, and is not stable but is regenerated each cell division. Sites with increased copy number are rereplicated and have increased KDM4A, MCM, and DNA polymerase occupancy. Suv39h1/KMT1A or HP1γ overexpression suppresses the copy gain, whereas H3K9/K36 methylation interference promotes gain. Our results demonstrate that overexpression of a chromatin modifier results in site-specific copy gains. This begins to establish how copy number changes could originate during tumorigenesis and demonstrates that transient overexpression of specific chromatin modulators could promote these events.

Histone chaperones represent a structurally and functionally diverse family of histone-binding proteins that prevent promiscuous interactions of histones before their assembly into chromatin. DAXX is a metazoan histone chaperone specific to the evolutionarily conserved histone variant H3.3. Here we report the crystal structures of the DAXX histone-binding domain with a histone H3.3-H4 dimer, including mutants within DAXX and H3.3, together with in vitro and in vivo functional studies that elucidate the principles underlying H3.3 recognition specificity. Occupying 40% of the histone surface-accessible area, DAXX wraps around the H3.3-H4 dimer, with complex formation accompanied by structural transitions in the H3.3-H4 histone fold. DAXX uses an extended α-helical conformation to compete with major inter-histone, DNA and ASF1 interaction sites. Our structural studies identify recognition elements that read out H3.3-specific residues, and functional studies address the contributions of Gly 90 in H3.3 and Glu 225 in DAXX to chaperone-mediated H3.3 variant recognition specificity.

Pancreatic Neuroendocrine Tumors (PanNETs) are a rare but clinically important form of pancreatic neoplasia. To explore the genetic basis of PanNETs, we determined the exomic sequences of ten non-familial PanNETs and then screened the most commonly mutated genes in 58 additional PanNETs. Remarkably, the most frequently mutated genes specify proteins implicated in chromatin remodeling: 44% of the tumors had somatic inactivating mutations in MEN-1, which encodes menin, a component of a histone methyltransferase complex; and 43% had mutations in genes encoding either of the two subunits of a transcription/chromatin remodeling complex consisting of DAXX (death-domain associated protein) and ATRX (alpha thalassemia/mental retardation syndrome X-linked). Clinically, mutations in the MEN1 and DAXX/ATRX genes were associated with better prognosis. We also found mutations in genes in the mTOR (mammalian target of rapamycin) pathway in 14% of the tumors, a finding that could potentially be used to stratify patients for treatment with mTOR inhibitors.

BACKGROUND: Up to 30% of pediatric glioblastomas (GB) demonstrate mutations in H3F3A, a gene variant encoding histone H3.3. Global reduction in H3K27me3 is associated with H3F3A K27M mutation. METHODS: We evaluated the utility of histopathological detection of this reduction in H3K27me3 as a prognostic biomarker. H3K27me3 expression was established by immunohistochemistry in 160 (118 pediatric and 42 adult) brain tumors of various subtypes and grades and 45 non-neoplastic reactive brain tissues. RESULTS: Global reduction in H3K27me3 was specific to GB cases with the H3F3A K27M mutantion, and was not observed in all other cases examined. In addition, H3F3A K27M mutant GB with reduced H3K27me3 exhibited a poor prognosis compared to H3F3A wild type tumors with preserved H3K27me3 (Log-rank (Mantel-Cox) test hazards ratio = 2.9, p = 0.0021). CONCLUSIONS: Global reduction in H3K27me3 is a histopathologically detectable surrogate for H3F3A K27M mutant GB that defines a poor prognosis subset of pediatric GB. SECONDARY CATEGORY: Pediatrics.

ATR-X (alpha-thalassemia/mental retardation, X-linked) syndrome is a human congenital disorder that causes severe intellectual disabilities. Mutations in the ATRX gene, which encodes an ATP-dependent chromatin-remodeler, are responsible for the syndrome. Approximately 50% of the missense mutations in affected persons are clustered in a cysteine-rich domain termed ADD (ATRX-DNMT3-DNMT3L, ADD(ATRX)), whose function has remained elusive. Here we identify ADD(ATRX) as a previously unknown histone H3-binding module, whose binding is promoted by lysine 9 trimethylation (H3K9me3) but inhibited by lysine 4 trimethylation (H3K4me3). The cocrystal structure of ADD(ATRX) bound to H3(1-15)K9me3 peptide reveals an atypical composite H3K9me3-binding pocket, which is distinct from the conventional trimethyllysine-binding aromatic cage. Notably, H3K9me3-pocket mutants and ATR-X syndrome mutants are defective in both H3K9me3 binding and localization at pericentromeric heterochromatin; thus, we have discovered a unique histone-recognition mechanism underlying the ATR-X etiology.