Project description:Diffuse Intrinsic Pontine Glioma (DIPG) is a universally fatal childhood brain tumour which frequently carries a mutation in genes encoding histone H3. These mutations lead to a lysine-to-methionine (K-to-M) substitution at position 27 (H3K27M) within the histone H3 tail and initiate global shifts in the abundance of Polycomb Repressive Complex 2 (PRC2) mediated H3K27 methylation (me) and P300/CBP mediated acetylation (ac). Acquisition of the H3K27M mutation is the founding genetic event in DIPG tumours, making it likely that these global shifts in H3K27me/ac are instrumental in promoting the early steps toward gliomagenesis. Here, we show that the H3.3K27M oncogene directly disrupts the function of tissue specific gene enhancers and increases Polycomb target gene repression limiting developmental potential of neural stem cells (NSCs). To study the initial functional consequences of H3K27M in glioma, we developed an isogenic human model system. To do this, we modelled the earliest stages of DIPG development by introducing either K27M or wildtype (WT) H3.3 into hindbrain derived NSC cultures. Importantly, this model faithfully recapitulated the global H3K27ac/me dynamics observed in patient samples. Moreover, epitope tagging of the K27M and WT histones allowed us to globally assess the localisation of K27M and WT H3.3. This for the first time facilitated a global analysis of H3.3K27M distribution, as it relates to the WT histone in a biologically relevant context. We found that the presence of the K27M mutation does not alter the localisation of the mutant histone, which is most abundant at enhancer elements with lower levels at gene promoters. Remarkably, the H3.3K27M oncogene limits the developmental potential of NSCs by directly impeding tissue specific enhancer function. Moreover, we found that PRC2 function is primarily altered outside of stably bound Polycomb target sites, with the majority of H3K27me3 lost outside of these regions. Finally, we provide in vivo evidence for sequestration of the PRC2 complex at a subset of Polycomb target promoters. Taken together, these findings provide important new mechanistic insights on the initial steps of DIPG development.

Project description:Diffuse intrinsic pontine glioma (DIPG) is an aggressive childhood tumor of the brainstem with currently no curative treatment available. The vast majority of DIPGs carry a mutation in histone H3 leading to a lysine 27-to-methionine exchange (H3K27M). The H3K27M mutation has been shown to inhibit EZH2, the catalytic subunit of the polycomb repressive complex 2, which presumably leads to global reduction of H3K27 trimethylation and upregulation of tumor-driving genes. However, the exact pathomechanism and cellular context remain elusive. Recent single cell transcriptome data suggested the DIPG cell of origin to be of oligodendroglial lineage, whereas in vivo studies demonstrated neural stem or progenitor cells (NPCs, NSCs) to be susceptible for malignant transformation upon H3K27M expression. Here, we used targeted human induced pluripotent stem cells (iPSCs) carrying an inducible H3.3-K27M allele in the endogenous H3F3A locus together with specific differentiation methods to study the effects of the mutation in disease-relevant cell identities of the neural lineage. Phenotypic, transcriptomic, and chromatin immunoprecipitation sequencing (ChIP-seq) analyses revealed a distinct role of H3.3-K27M for deregulating bivalent promoter-associated developmental genes producing diverse outcomes in different cell types. While being fatal for iPSCs, H3.3-K27M increased proliferation in NSCs and to a lesser extent in oligodendroglial progenitor cells (OPCs) but not in astroglial-restricted precursors. Importantly, we demonstrated that only NSCs but not OPCs gave rise to tumors upon induction of the H3.3-K27M and TP53 inactivation in an orthotopic xenograft model faithfully recapitulating human DIPGs. Thus, we provide evidence that indeed only NSCs can develop H3.3-K27M-driven DIPG-like tumors but that the mutation actively drives acquisition of oligodendroglial characteristics, thus explaining the different observations of cell identity. Identification of the originating cell type may help to determine specific vulnerabilities of this tumor for developing targeted therapies.

Dataset Information

Histone H3.3 K27M chromatin functions implicate a network of neurodevelopmental factors including ASCL1 and NEUROD1 in DIPG

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