Post-mitotic accumulation of histone variant H3.3 in new cortical neurons establishes neuronal transcriptome, identity, and connectivity

Histone variants, which can be expressed outside of S-phase and deposited DNA synthesis-independently, provide replacement histones in terminally post-mitotic cells, including neurons. Histone variants can also serve active roles in gene regulation by modulating chromatin states or enabling nucleosome turnover at regulatory regions. Here, we find that newborn cortical excitatory neurons substantially accumulate the histone H3 variant H3.3 immediately post-mitosis. Co-deletion of H3.3-encoding genes H3f3a and H3f3b from new neurons abrogates this accumulation, and causes widespread disruptions in the developmental establishment of the neuronal transcriptome. These broad transcriptomic changes coincide with neuronal maturation phenotypes in acquisition of distinct neuronal identities and formation of axon tracts. Stage-dependent deletion of H3f3a and H3f3b from ([1][1]) cycling neural progenitor cells, ([2][2]) neurons immediately after terminal mitosis, or ([3][3]) several days later, reveals the first post-mitotic days as a critical window for de novo H3.3. After H3.3 accumulation within this developmental window, co-deletion of H3f3a and H3f3b from neurons causes progressive H3.3 depletion over several months without widespread transcriptional disruptions. Our study thus uncovers a key role for H3.3 in establishing neuronal transcriptome, identity, and connectivity immediately post-mitosis that is distinct from its role in maintaining total histone H3 levels over the neuronal lifespan. Significance DNA is tightly packaged around histones into chromatin, which compacts the genome, but also restricts access to DNA. All transactions on DNA, notably gene transcription, require chromatin reorganization that is precisely regulated, including via the use of variant forms of histones. Here, we find that during a critical developmental window for establishing post-mitotic neuronal identity, newly generated cortical excitatory neurons substantially accumulate the histone H3 variant H3.3 immediately after terminal mitosis. Conditional deletion of H3.3-encoding genes from new neurons abrogates this accumulation, and disrupts neuronal gene expression, subtype identity, and axon projections. Thus, H3.3 plays important roles in establishing neuronal transcriptome, identity, and connectivity during post-mitotic development. These functions are distinct from long-term maintenance of histone levels in mature neurons. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3