Surprising Phenotypic Diversity of Cancer-associated mutations at Gly 34 in the Histone H3 tail

Deep sequencing of cancer genomes has identified frequent mutation of core histones, termed oncohistones, but the role of some mutants is poorly understood. Here, using fission yeast as a model, we determine the consequences of mutation of histone H3.3 at Gly 34, a site frequently mutated in pediatric cortical high-grade glioma (G34R/V), and in virtually all giant cell tumors of bone (G34W). H3-G34 mutations cause a surprising diversity of outcomes, differentially affecting modification of the nearby H3K36 residue, subtelomeric silencing, genomic stability, sensitivity to irradiation, alkylating agents, hydroxyurea and influencing DNA repair. Since fifteen genes encode H3 isoforms, and only one allele is targeted in cancer, G34R/V/W H3 likely represents a small portion of total cellular H3. Whilst the presence of wild type H3 rescues most G34 mutant phenotypes, hydroxyurea sensitivity, subtelomeric silencing and homologous recombination defects dominate in cells expressing G34R and wild type H3. Together, these studies demonstrate the complexity associated with different substitutions at even a single residue in histone H3 and highlight the utility of genetically tractable systems for their analysis. Overall design: Examination of total RNA in S.pombe with single copy H3 and H4 histone genes (hht2 and hhf2) bearing H3 WT, H3 G34R, H3 G34V, H3 G34K mutations or deleted for set2. Also examination of H3 G34R and H3 G34K mutants compared to WT H3 in strains retaining 2 wild type copies of H3 and H4.