Local nuclear to cytoplasmic ratio regulates H3.3 incorporation via cell cycle state during zygotic genome activation

Early embryos often have unique chromatin states prior to zygotic genome
activation (ZGA). In Drosophila, ZGA occurs after 13 reductive
nuclear divisions during which the nuclear to cytoplasmic (N/C) ratio
grows exponentially. Previous work found that histone H3 chromatin
incorporation decreases while its variant H3.3 increases leading up to
ZGA. In other cell types, H3.3 is associated with sites of active
transcription and heterochromatin, suggesting a link between H3.3 and ZGA.
Here, we test what factors regulate H3.3 incorporation at ZGA. We find
that H3 nuclear availability falls more rapidly than H3.3 leading up to
ZGA. We generate H3/H3.3 chimeric proteins at the endogenous H3.3A locus
and observe that chaperone binding, but not gene structure, regulates H3.3
behavior. We identify the N/C ratio as a major determinant of H3.3
incorporation. To isolate how the N/C ratio regulates H3.3 incorporation
we test the roles of genomic content, zygotic transcription, and cell
cycle state. We determine that cell cycle regulation, but not H3
availability or transcription, controls H3.3 incorporation. Overall, we
propose that local N/C ratios control histone variant usage via cell cycle
state during ZGA.