Regional identity of human neural stem cells determines oncogenic responses to histone H3.3 mutants [RNA-Seq]

Point mutations within the histone H3.3 are frequent in aggressive childhood brain tumours known as paediatric high-grade gliomas (pHGGs). Intriguingly, different mutations arise in discrete anatomical regions: H3.3-G34R within the forebrain and H3.3-K27M preferentially within the hindbrain. The reasons for this contrasting aetiology are unknown. By engineering human foetal neural stem cell cultures from distinct regions, we demonstrate here that cell-intrinsic regional identity provides differential responsiveness to each mutant that mirrors the origins of pHGGs. Focusing on H3.3-G34R, we find that the oncohistone supports proliferation of forebrain cells, while inducing a cytostatic response in the hindbrain. Mechanistically, H3.3-G34R does not impose widespread transcriptional or epigenetic changes, but impairs recruitment of ZMYND11, a transcriptional repressor of highly expressed genes. We therefore propose that H3.3-G34R promotes tumorigenesis by stabilising the expression of key progenitor genes and, thus, locking initiating forebrain cells into their preexisting immature state. Fresh human foetal CNS tissue was obtained following elective termination of pregnancy at 13.5, 15.0 and 19.3 weeks of gestational age (WGA) (specimens GCGR-NS13, GCGR-NS15 and GCGR-NS19, respectively). The regions of interest were dissected under a stereoscope in ice-cold PBS and one half was directly lysed in Qiagen RLT plus buffer for RNA extraction. NSCs lines were established from the respective second halves as previously described (Conti et al., 2005 PloS Biology). Engineered WT or G34R lines overexpressed v5-tagged wild-type or G34R-mutant H3.3. PP5W and PP5G lines were engineered to overexpress wild-type and G34R-mutant H3.3 in combination with PDGFRa overexpression and knockout of endogenous TP53 gene.