Histone H3 availability is more important for development than H3.2 versus H3.3 subtype identity.

The distinct contributions of replication-dependent and replication-independent histones to development and genome function remain unclear. In this study, we investigate how the distinct protein identities of the histone H3.2 and H3.3 subtypes contribute to development and gene regulation in Drosophila. Comparing animals in which the replication-independent H3.3 genes were mutated to produce the replication-dependent H3.2 protein with those carrying deletions of the replication-independent H3.3 genes revealed that replication-independent H3.3 is essential for fertility, adult locomotor behavior, and normal longevity. However, development to adulthood does not depend on which replication-independent H3 subtype is expressed from the H3.3 loci. Moreover, replication-independent H3.3 is not required to establish or maintain global patterns of chromatin accessibility or gene expression in the adult brain. Surprisingly, we find that expression of H3.2 from the replication-dependent HisC locus is essential in post-replicative cells in the absence of replication-independent H3.3, and we uncover a critical role for the HIRA histone chaperone complex in preserving genome function when replication-independent H3.3 is deleted. We conclude that an available pool of H3 is more critical than the specific identity of H3 in the pool.