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Histone H3 lysine-to-methionine mutants as a paradigm to study chromatin signaling

ABSTRACT: Histone H3 lysine27-to-methionine (H3K27M) gain-of-function mutations occur in highly aggressive pediatric gliomas. Here, we establish a Drosophila animal model for the pathogenic histone H3K27M mutation and show that its overexpression resembles Polycomb repressive complex 2 (PRC2) loss-of-function phenotypes, causing de-repression of PRC2 target genes and developmental perturbations. Similarly, a H3K9M mutant depletes H3K9 methylation levels and suppresses position-effect variegation in various Drosophila tissues. The histone H3K9 demethylase KDM3B/JHDM2 associates with H3K9M nucleosomes and its overexpression in Drosophila results in loss of H3K9 methylation levels and heterochromatic silencing defects. Here we establish histone lysine-to-methionine mutants as robust in vivo tools for inhibiting methylation pathways that also function as biochemical reagents for capturing site-specific histone-modifying enzymes, thus providing molecular insight into chromatin-signaling pathways.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE59891 | GEO | 2014/09/02

SECONDARY ACCESSION(S): PRJNA256995

REPOSITORIES: GEO

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Similar Datasets

Histone H3 lysine-to-methionine mutants as a paradigm to study chromatin signaling

Project description:Histone H3 lysine27-to-methionine (H3K27M) gain-of-function mutations occur in highly aggressive pediatric gliomas. Here, we establish a Drosophila animal model for the pathogenic histone H3K27M mutation and show that its overexpression resembles Polycomb repressive complex 2 (PRC2) loss-of-function phenotypes, causing de-repression of PRC2 target genes and developmental perturbations. Similarly, a H3K9M mutant depletes H3K9 methylation levels and suppresses position-effect variegation in various Drosophila tissues. The histone H3K9 demethylase KDM3B/JHDM2 associates with H3K9M nucleosomes and its overexpression in Drosophila results in loss of H3K9 methylation levels and heterochromatic silencing defects. Here we establish histone lysine-to-methionine mutants as robust in vivo tools for inhibiting methylation pathways that also function as biochemical reagents for capturing site-specific histone-modifying enzymes, thus providing molecular insight into chromatin-signaling pathways. RNA-seq of wing imaginal discs expressing either H3.3WT-FLAG-HA or H3.3K27M-FLAG-HA.

2014-09-02 | E-GEOD-59891 | biostudies-arrayexpress

Cxorf67-FLAG co-immunoprecipitation analysis from 293T cells

Project description:The regulation of gene expression is controlled in part by post-translational modifications to histone proteins. Methylation at histone H3, lysine 27 (H3K27), which is catalyzed by Polycomb repressive complex 2 (PRC2), is associated with silenced chromatin. Previous studies have identified dysregulation of H3K27 methylation in pediatric diffuse intrinsic pontine gliomas (DIPGs), the majority of which feature mutation of lysine 27 to methionine. This “oncohistone” potently inhibits PRC2 activity and leads to a global reduction in H3K27 methylation. Similar to DIPG, posterior fossa type A (PFA) ependymomas also show low levels of H3K27 methylation. Although PFAs do not possess the H3K27M oncohistone mutation, they do show increased expression of Cxorf67. Interestingly, Cxorf67 contains a C-terminal sequence that resembles the sequence surrounding H3K27, and we find that this portion of Cxorf67 inhibits PRC2 activity to an even greater extent than the H3K27M oncohistone. Thus, we suggest re-naming Cxorf67 as EZHIP (Enhancer of Zeste Homologs Inhibitory Protein). Furthermore, when expressed in 293T cells, Cxorf67 interacts with several members of PRC2 and induces changes in H3K27 methylation patterns that mirror the changes in H3K27 methylation induced by expression of H3K27M. We propose that PFAs have dysregulated H3K27 methylation by a mechanism that involves inhibition of PRC2 by Cxorf67, which could drive tumorigenesis.

2019-04-09 | PXD013390 | Pride

Heterotypic nucleosomes and PRC2 drive DIPG oncogenesis

Project description:Diffuse intrinsic pontine gliomas (DIPG) are characterized by a heterozygous lysine-to-methionine mutation of histone H3 (H3K27M) that potently reduces Polycomb Repressive Complex 2 (PRC2) methylation of wild-type histone H3K27 (H3K27wt). The role of H3K27M and reduced H3K27wt methylation in DIPG pathogenesis has yet to be determined. Here, we have performed epigenomic profiling of patient-derived H3K27M mutant DIPG cells and demonstrate that H3K27M resides in nucleosomes with H3K27wt acetylation (H3K27ac), and H3K27M-H3K27ac containing nucleosomes co-localize with bromodomain proteins at actively transcribed genes and that PRC2 is excluded from H3K27M occupied regions. With respect to therapeutic implications of these observations, we demonstrate that pharmacologic bromodomain protein inhibition suppresses tumor growth in vivo. In total, our results indicate that H3K27M promotes H3K27ac at the expense of H3K27 methylation, and points to bromodomain protein inhibition as a clinical strategy for treating DIPG.

2017-02-27 | GSE78801 | GEO

Mechanism for the selective sequestration of a histone H3K9 methyltransferase at heterochromatin by the H3K9M mutation

Project description:Oncogenic histone lysine-to-methionine mutations block the methylation of their corresponding lysine residues on wild type histones, but the mechanisms by which these mutations function are highly controversial. We have previously shown that in fission yeast, H3K9M containing nucleosomes sequesters the H3K9 methyltransferase Clr4 to block H3K9 methylation{Shan, 2016 #119}. Using ChIP-sequencing, here we show that even though H3K9M containing nucleosomes are broadly distributed across the genome, Clr4 is mainly sequestered at pericentric repeats. The selective sequestration of Clr4 depends not only on H3K9M, but also on H3K14 ubiquitylation (H3K14ub), a modification deposited by a Clr4 associated E3 ubiquitin ligase complex. In vitro, H3K14ub enhances the interaction between H3K9M and Clr4 and potentiates the inhibitory effects of H3K9M on Clr4 enzymatic activity. Using bio-layer interferometry, we show that the H3K9M mutation reduced association rates (kon) of Clr4 with histone tails, suggesting that Clr4 intrinsically disfavors H3K9M. More importantly, H3K9M reduced the dissociation rate (koff) of Clr4 with histone tails, demonstrating that H3K9M slows down the turnover of Clr4 once it is bound. Finally, we generated mutations on Clr4 that specifically disrupt its interaction with H3K14ub and show that they reduce the inhibitory effects of H3K9MK14ub on Clr4 enzymatic activity in vitro and relieve the sequestration of Clr4 at pericentric repeats in vivo. Therefore, the selective sequestration of Clr4 at pericentric repeats by H3K9M is due to the presence of H3K14ub at these regions, which overcomes Clr4’s aversion of H3K9M and reduces the dissociation of Clr4.

2020-10-26 | GSE159192 | GEO

Multiple modes of PRC2 inhibition elicit global chromatin alterations in H3K27M pediatric glioma

Project description:A methionine substitution at lysine 27 on histone H3 variants (H3K27M) characterizes ~80% of diffuse intrinsic pontine gliomas (DIPG) and inhibits PRC2 in a dominant negative fashion. Yet, the mechanisms for this inhibition and abnormal epigenomic landscape have not been resolved. Using quantitative proteomics, we discovered that robust PRC2 inhibition requires levels of H3K27M greatly exceeding those of PRC2, seen in DIPG. While PRC2 inhibition requires interaction with H3K27M, we found this interaction on chromatin is transient with PRC2 largely being released from H3K27M. Unexpectedly, inhibition persisted even after PRC2 dissociated from H3K27M-chromatin suggesting a lasting impact on PRC2. Furthermore, allosterically activated PRC2 is particularly sensitive to K27M leading to a failure to spread H3K27me3 at distinct foci. In turn, levels of Polycomb antagonists such as H3K36me2 are elevated suggesting a more global, downstream effect on the epigenome. Together, these findings reveal the conditions required for H3K27M-mediated PRC2 inhibition and reconcile seemingly paradoxical effects of H3K27M on PRC2 recruitment and activity.

2018-08-31 | GSE118954 | GEO

Distribution of H3K27me2, dUTX, and histone H3 in Sg4 cells

Project description:PcG protein complex PRC2 is a methyltransferase specific for histone H3 lysine27, and H3K27me3 is essential for stable transcription silencing. Less well known but quantitatively much more important is the genome-wide role of PRC2 that dimethylates more than half of total H3K27. Here we have determined genomic distributions of H3K27me2, dUTX, and histone H3 in cultured Drosophila Sg4 cells by hybridization of ChIP products with tiling microarrays. Genomic distributions of H3K27me2, dUTX, and histone H3 in cultured Drosophila Sg4 cells by hybridization of ChIP products with tiling microarrays

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Distribution of H3K27me2, dUTX, and histone H3 in Sg4 cells

2015-05-14 | GSE61261 | GEO

The H3K36me2 writer-reader dependency in H3K27M-DIPG

Project description:The lysine-to-methionine mutation at residue 27 of histone H3 (H3K27M) is a driving mutation in Diffuse Intrinsic Pontine Glioma (DIPG), a highly aggressive form of pediatric brain tumor with no effective treatment and little chance of survival. H3K27M reshapes the epigenome through a global inhibition of PRC2 catalytic activity, displacement of methylation at lysine 27 of histone H3 (H3K27me2/3), and thus promoting oncogenesis of DIPG. As a consequence, a histone modification H3K36me2, antagonistic to H3K27me2/3, is aberrantly elevated. Here, we investigate the role of H3K36me2 in H3K27M-DIPG by tackling its upstream catalyzing enzymes (writers) and downstream binding factors (readers). We determine that NSD1 and NSD2 are the key writers for H3K36me2. Loss of NSD1/2 in H3K27M-DIPG impedes cellular proliferation in vitro and tumorigenesis in vivo, and disrupts tumor-promoting gene expression programs. Further, we demonstrate that LEDGF and HDGF2 are the main readers that mediate the pro-tumorigenic effects downstream of NSD1/2-H3K36me2. Treatment with a chemically modified peptide mimicking endogenous H3K36me2 dislodges LEDGF/HDGF2 from chromatin and specifically inhibits the proliferation of H3K27M-DIPG. Together, our results indicate a functional pathway of NSD1/2-H3K36me2-LEDGF/HDGF2 as an acquired dependency in H3K27M-DIPG and suggest a possibility to target this pathway for therapeutic interventions.

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