An E2 ubiquitin-conjugating enzyme links diubiquitinated H2B to H3K27M oncohistone function

The H3K27M oncogenic histone (oncohistone) mutation drives ~80% of incurable childhood brain tumors known as diffuse midline gliomas (DMGs). The major molecular feature of H3K27M mutant DMGs is a global loss of H3K27 trimethylation (H3K27me3), a phenotype conserved in <i>Caenorhabditis elegans</i> (<i>C. elegans</i>). Here, we perform unbiased genome-wide suppressor screens in <i>C. elegans</i> expressing H3K27M and isolate 20 suppressors, all of which at least partially restore H3K27me3. 19/20 suppressor mutations map to the same histone H3.3 gene in which the K27M mutation was originally introduced. Most of these create single amino acid substitutions between residues R26-Y54, which do not disrupt oncohistone expression. Rather, they are predicted to impair interactions with the Polycomb Repressive Complex 2 (PRC2) and are functionally conserved in human cells. Further, we mapped a single extragenic H3K27M suppressor to <i>ubc-20</i>, an E2 ubiquitin-conjugating enzyme, whose loss rescued H3K27me3 to nearly 50% wild-type levels despite continued oncohistone expression and chromatin incorporation. We demonstrate that <i>ubc-20</i> is the major enzyme responsible for generating diubiquitinated histone H2B. Our study provides in vivo support for existing models of PRC2 inhibition via direct oncohistone contact and suggests that the effects of H3K27M may be modulated by H2B ubiquitination.