Probing the Function of Metazoan Histones with a Systematic Library of H3 and H4 Mutants

Replication-dependent histone genes often reside in tandemly arrayed gene clusters, hindering systematic loss-of function analyses. Here we used CRISPR/Cas9 and the attP/attB double-integration system to alter the numbers and sequences of the histone genes in their original genomic context in Drosophila. As few as 8 copies of histone gene suffices to support embryo development and adult viability, whereas flies with 20 copies are indistinguishable from WT flies. Using a hierarchical assembly method, 40 alanine substitution mutations, covering all known modified residues on histone H3 and H4, were introduced and characterized. Mutations at multiple residues compromise viability, fertility, and DNA damage responses. In particular, H4K16 is necessary for expression of male X-linked genes, male viability and maintenance of ovarian germline stem cells, whereas H3K27 is essential for late embryogenesis. Finally, using simplified mosaic analysis, we found that H3R26 is required for H3K27 trimethylation. Taken together, we have developed a powerful strategy and valuable reagents for systematically probing histone functions in the fly.