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Driver mutations in genes encoding histone H3 proteins resulting in p.Lys27Met substitutions (H3-K27M) are frequent in pediatric midline brain tumors. However, the precise mechanisms by which H3-K27M causes tumor initiation remain unclear. Here, we use human hindbrain neural stem cells to model the consequences of H3.3-K27M on the epigenomic landscape in a relevant developmental context. Genome-wide mapping of epitope-tagged histone H3.3 revealed that both the wild type and the K27M mutant incorporate abundantly at pre-existing active enhancers and promoters, and to a lesser extent at Polycomb repressive complex 2 (PRC2)-bound regions. At active enhancers, H3.3-K27M leads to focal H3K27ac loss, decreased chromatin accessibility and reduced transcriptional expression of nearby neurodevelopmental genes. In addition, H3.3-K27M deposition at a subset of PRC2 target genes leads to increased PRC2 and PRC1 binding and augmented transcriptional repression that can be partially reversed by PRC2 inhibitors. Our work suggests that, rather than imposing de novo transcriptional circuits, H3.3-K27M drives tumorigenesis by locking initiating cells in their pre-existing, immature epigenomic state, via disruption of PRC2 and enhancer functions.

Polycomb repressive complex 2 (PRC2) is a conserved chromatin regulator that is responsible for the methylation of histone H3 lysine 27 (H3K27). PRC2 is essential for normal development and its loss of function thus results in a range of developmental phenotypes. Here, we review the latest advances in our understanding of mammalian PRC2 activity and present an updated summary of the phenotypes associated with its loss of function in mice. We then discuss recent studies that have highlighted regulatory interplay between the modifications laid down by PRC2 and other chromatin modifiers, including NSD1 and DNMT3A. Finally, we propose a model in which the dysregulation of these modifications at intergenic regions is a shared molecular feature of genetically distinct but highly phenotypically similar overgrowth syndromes in humans., Summary: This Review discusses the molecular biology of PRC2, the developmental phenotypes associated with its loss of function in mice, and its crosstalk with other chromatin regulators in the context of human overgrowth syndromes.

ATRX alterations occur at high frequency in neuroblastoma of adolescents and young adults. Particularly intriguing are the large N-terminal deletions of ATRX (Alpha Thalassemia/Mental Retardation, X-linked) that generate in-frame fusion (IFF) proteins devoid of key chromatin interaction domains, while retaining the SWI/SNF-like helicase region. We demonstrate that ATRX IFF proteins are redistributed from H3K9me3-enriched chromatin to promoters of active genes and identify REST as an ATRX IFF target whose activation promotes silencing of neuronal differentiation genes. We further show that ATRX IFF cells display sensitivity to EZH2 inhibitors, due to derepression of neurogenesis genes, including a subset of REST targets. Taken together, we demonstrate that ATRX structural alterations are not loss-of-function and put forward EZH2 inhibitors as a potential therapy for ATRX IFF neuroblastoma.

Mutations and structural alterations of the SWI/SNF-like chromatin remodeler ATRX (Alpha Thalassemia/Mental Retardation, X-linked) have been reported at high frequency in a number of adult and pediatric tumors. However, the consequences of ATRX mutations in cancer and their impact on the epigenome remain ill defined. Particularly intriguing are the large N-terminal deletions of ATRX in neuroblastoma that generate in-frame fusion (IFF) proteins devoid of key chromatin interaction domains, while retaining the SWI/SNF-like helicase domain. We hereby demonstrate that ATRX IFF proteins have distinct genomic distribution compared to wild type (WT) ATRX and are absent from H3K9me3-enriched chromatin, yet bound to active promoters. We find REST (RE-1 Silencing Transcription Factor) as one such ATRX IFF target that is activated and promotes silencing of neuronal differentiation genes. Notably, we uncover that ATRX IFF cells display exquisite sensitivity to EZH2 inhibitors (EZH2i) in vitro and in vivo, due in part to derepression of neurogenesis genes, including a subset of REST targets. Examination of the epigenomic landscape of neuroblastoma tumor specimens harboring ATRX IFFs reveals that H3K27me3 occupies a subset of neurogenesis genes that are transcriptionally silenced and sensitive to EZH2i in our cell-based assays. In summary, this study demonstrates that ATRX structural alterations are not loss-of-function as predicted, and supports EZH2i as a key therapeutic strategy for ATRX IFF neuroblastoma.