Functional redundancy of variant and canonical histone H3K9 modification in Drosophila

To determine the relative functions of K9 modification on variant and canonical H3, we compared the phenotypes caused by engineering H3.3K9R and H3K9R mutant genotypes in Drosophila melanogaster. Whereas most H3.3K9R and a small number of H3K9R mutant animals are capable of completing development and do not have substantially altered protein coding transcriptomes, all H3.3K9R H3K9R combined mutants die soon after embryogenesis and display decreased expression of genes enriched for K9ac. These data indicate that the role of K9ac in gene activation during development can be provided by either H3 or H3.3. Conversely, we found that H3.3K9 is methylated at telomeric transposons, and this mark contributes to repressive chromatin architecture, supporting a role for H3.3 in heterochromatin that is distinct from that of H3. Thus, our genetic and molecular analyses demonstrate that K9 modification of variant and canonical H3 have overlapping roles in development and transcriptional regulation, though to differing extents in euchromatin and heterochromatin. Overall design: FAIRE-seq was performed from imaginal wing discs from 3rd instar Drosophila larvae in triplicate on yw (WT) and three H3.3 mutants: 1) H3.3A null (H3.3A-null), 2) H3.3B with an endogenous K9R mutation (H3.3B-K9R), 3) and the double mutant of both 1 and 2 (H3.3-K9R). RNA-seq was performed on whole 1st instar larvae in triplicate on four genotypes: 1) endogenous histon_1e deletion mutant rescued by a 12x array of wild-type canonical histone genes (HWT), 2) endogenous histone deletion mutant rescued by a 12x array of canonical histone genes with a K9R mutation (H3.2-K9R), 3) H3.3B K9R mutation in an H3.3A null and HWT background (H3.3-K9R_H3.2-HWT), 4) H3.3B K9R mutation in an H3.3A null and H3.2-K9R background (H3.3-K9R_H3.2-K9R).