Abstract
Recurrent somatic mutations of H3F3A in aggressive pediatric high-grade gliomas generate K27M or G34R/V mutant histone H3.3. H3.3-G34R/V mutants are common in tumors with mutations in p53 and ATRX, an H3.3-specific chromatin remodeler. To gain insight into the role of H3-G34R, we generated fission yeast that express only the mutant histone H3. H3-G34R specifically reduces H3K36 tri-methylation and H3K36 acetylation, and mutants show partial transcriptional overlap with set2 deletions. H3-G34R mutants exhibit genomic instability and increased replication stress, including slowed replication fork restart, although DNA replication checkpoints are functional. H3-G34R mutants are defective for DNA damage repair by homologous recombination (HR), and have altered HR protein dynamics in both damaged and untreated cells. These data suggest H3-G34R slows resolution of HR-mediated repair and that unresolved replication intermediates impair chromosome segregation. This analysis of H3-G34R mutant fission yeast provides mechanistic insight into how G34R mutation may promote genomic instability in glioma.
Highlights
The genomic DNA of eukaryotes is packaged into chromatin, which regulates all DNA transactions including transcription, replication and repair
In contrast to the complexity of mammals, where fifteen genes encode three different histone H3 proteins (H3.1, H3.2, H3.3), fission yeast have three genes that code for a single histone H3 protein (Figure 1a) (Matsumoto and Yanagida, 1985)
Counting only late anaphase cells, we found that chromosomes mis-segregated in 7.4% of H3-Glycine 34 to Arginine (G34R) cells compared to 0.9% in H3-WT, 1.3% in set2D and 25.6% in clr4D cells that lack heterochromatin
Summary
The genomic DNA of eukaryotes is packaged into chromatin, which regulates all DNA transactions including transcription, replication and repair. Proteins that regulate chromatin dynamics are frequently mutated in cancer, and recently mutations in the histone genes themselves have been discovered (Schwartzentruber et al, 2012; Wu et al, 2012; Behjati et al, 2013). One such mutant is the Glycine 34 to Arginine (G34R) mutant of histone H3.3, identified as a frequent somatic mutation in pediatric high-grade cortical glioma (pHGG). G34R mutations are only found in H3.3, primarily in one of two H3.3 genes (H3F3A) and not in the
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