The histone H3.3 K27M mutation suppresses Ser31phosphorylation and mitotic fidelity, which can directly drive gliomagenesis.

Serine 31 is a phospho-site unique to the histone H3.3 variant; mitotic phospho-Ser31 is restricted to pericentromeric heterochromatin, and disruption of phospho-Ser31 results in chromosome segregation defects and loss of p53-dependant G cell-cycle arrest. Ser31 is proximal to the H3.3 lysine 27-to-methionine (K27M) mutation that drives ∼80% of pediatric diffuse midline gliomas. Here, we show that expression of the H3.3 K27M mutant in normal, diploid cells results in increased chromosome missegregation and failure to arrest in the following G. Expression of a non-phosphorylatable S31A mutant also drives chromosome missegregation, while the expression of a double K27M + phosphomimetic S31E mutant restores mitotic fidelity and the p53 response to chromosome missegregation. We show that patient-derived H3.3 K27M tumor cells have decreased mitotic Ser31 phosphorylation and increased frequency of chromosome missegregation. CRISPR reversion of the K27M mutation to wild type (WT) restores phospho-Ser31 levels and results in a decrease in chromosome missegregation. However, inserting an S31A mutation by CRISPR into these revertant cells disrupts mitotic fidelity. In vitro and in vivo analyses reveal that Chk1-the mitotic Ser31 kinase-is preferentially retained at pericentromeres in K27M-expressing tumor cells, compared with MLysine27-to-methionine mutation (M27K) isogenic revertants, correlating with both diminished phospho-Ser31 and mitotic defects. Interestingly, whereas M27K revertant cells do not form xenograft tumors in mice, H3.3 S31A cells do, similar to those formed by H3.3 K27M cells. Replication-competent avian leukosis virus splice-acceptor (RCAS)/cellular receptor for subgroup A avian sarcoma and leukosis virus (TVA) mice expressing S31A also form diffuse midline gliomas morphologically indistinguishable from K27M tumors. Together, our results reveal that the H3.3 K27M mutant alters H3.3 Ser31 phosphorylation, which, in turn, has profound impacts on chromosome segregation/cell-cycle regulation.