The H3K27M mutation alters stem cell growth, epigenetic regulation, and differentiation potential [MeDIP-Seq]

Lysine 27 to methionine mutation (H3K27M) of the H3F3A gene, which encodes the variant histone H3.3, is found in the majority of Diffuse Intrinsic Pontine Gliomas (DIPGs). DIPGs are the most aggressive form of pediatric gliomas and have a median survival of <1 year from diagnosis. As H3K27M mutation is necessary but not sufficient to cause DIPGs, it is accompanied by several other mutations in tumors. However, the mechanisms by which H3K27M increases vulnerability to DIPG tumorigenesis, while expected to involve altered epigenetic regulation is unclear. Thus, in this work we built pairs of isogenic human embryonic stem cell lines with versus without this mutation, in the absence of other DIPG contributory mutations, to investigate mechanisms by which H3K27M mutation could affect cellular proliferation and differentiation and how these were related to alterations in the transcriptome, H3K27me3, and the DNA methylome. We found that H3K27M increased stem cell proliferation and interfered with differentiation, resulting in loss of most H3K27me3 and resulting in anomalous onset of expression of developmental genes during multilineage or directed differentiation. This work suggests mechanisms by which H3K27M mutation influences stem cell properties, contributing to DIPG tumorigenesis. Overall design: The H3F3a-K28M-Puro clonal cell lines (mutant clone 1 and clone 9) were generated from H1 embryonic stem cell (H1ESC) line (obtained from WiCell) by the Genome Engineering and iPSC Center (GEiC), Washington University in St. Louis. These lines were used for derivation of neural progenitor cells, as described below, and comparisons of gene expression in the wild type versus mutant hESCs and neural progenitor cells were performed. MeDIP was performed on four biological replicates of WT and H3K27M mutant undifferentiated H1 hESCs and their 5-day differentiated neural EB derivatives cultured in parallel to those used for RNA-sequencing.