The histone H3.3 K27M mutation found in diffuse midline gliomas coordinately disrupts adjacent H3.3 Ser31 phosphorylation and the fidelity of chromosome segregation

Day, C. A.; Grigore, F.; Hakkim, F. L.; Langfald, A.; Fadness, S.; Schwab, P.; Sepaniac, L.; Stumpff, J.; Daniels, D. J.; Vaughan, K. T.; Robinson, J. P.; Hinchcliffe, E. H.

doi:10.1101/2022.05.27.493485

bioRxiv · 10.1101/2022.05.27.493485

The histone H3.3 K27M mutation found in diffuse midline gliomas coordinately disrupts adjacent H3.3 Ser31 phosphorylation and the fidelity of chromosome segregation

Day, C. A.·Grigore, F.·Hakkim, F. L.·Langfald, A.·Fadness, S.·Schwab, P.·Sepaniac, L.·Stumpff, J.·Daniels, D. J.·Vaughan, K. T.·Robinson, J. P.·Hinchcliffe, E. H.

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Abstract

During the cell cycle, differential phosphorylation of select histone H3 serine/threonine residues regulates chromatin structure, necessary for both dynamic transcriptional control and proper chromosome segregation1-2. Histone H3.3 contains a highly conserved serine residue (Ser31) within its N-terminal tail that is unique to this variant. During interphase phosphorylation of Ser31 amplifies stimulation-induced transcription and is required for early metazoan development3-6. During mitosis Ser31 phosphorylation at the pericentromere supports proper chromosome segregation, albeit by unknown mechanisms7-10. H3.3 Ser31 is flanked by mutational sites that drive several human cancers, including pediatric gliomas5-8. This is typified by the H3.3K27M mutation found in [~]80% of diffuse midline gliomas, which undergo epigenetic reprogramming in proliferative cells coordinate with loss of global H3 lysine 27 trimethylation (H3K27Me3)11-14. However, whether the K27M mutation influences the neighboring Ser31 phosphorylation and whether disrupting Ser31 phosphorylation plays a distinct role in driving gliomagenesis has not been tested. Here we show that H3.3K27M mutant cells have reduced capacity for H3.3 Ser31 phosphorylation at the mitotic pericentromere, increased rates of chromosome missegregation, and impaired G1 checkpoint responses to chromosome instability. CRISPR-reversion of K27M to wild-type restores phospho-Ser31 levels and suppresses chromosome segregation defects. CRISPR editing to introduce a non-phosphorylatable H3.3S31A alone is sufficient to increase the frequency of chromosome missegregations. Finally, expression of H3.3S31A in a PDGF{beta}-driven RCAS/TVA mouse model is sufficient to drive high grade gliomagenesis, generating diffuse tumors morphologically indistinguishable from those generated by H3.3K27M expression. Importantly, this occurs without the loss of H3K27 triple methylation that is the hallmark of K27M tumors. Our results reveal that the H3.3 K27M mutation alters the neighboring Ser31 phosphorylation, and loss of proper H3.3 Ser31 phosphorylation contributes to the formation of diffuse midline gliomas.

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Day, C. A., Grigore, F., Hakkim, F. L., Langfald, A., Fadness, S., Schwab, P., Sepaniac, L., Stumpff, J., Daniels, D. J., Vaughan, K. T., Robinson, J. P., Hinchcliffe, E. H.. 2022-05-28. The histone H3.3 K27M mutation found in diffuse midline gliomas coordinately disrupts adjacent H3.3 Ser31 phosphorylation and the fidelity of chromosome segregation. https://doi.org/10.1101/2022.05.27.493485

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