Postmitotic accumulation of histone variant H3.3 in new cortical neurons establishes neuronal chromatin, transcriptome, and identity

Histone variants, which can be expressed outside of S-phase and deposited DNA synthesis-independently, provide long-term histone replacement in postmitotic cells, including neurons. Beyond replenishment, histone variants also play active roles in gene regulation by modulating chromatin states or enabling nucleosome turnover. Here, we uncover crucial roles for the histone H3 variant H3.3 in neuronal development. We find that newborn cortical excitatory neurons, which have only just completed replication-coupled deposition of canonical H3.1 and H3.2, substantially accumulate H3.3 immediately post mitosis. Co-deletion of H3.3-encoding genes H3f3a and H3f3b from newly postmitotic neurons abrogates H3.3 accumulation, markedly alters the histone posttranslational modification (PTM) landscape, and causes widespread disruptions to the establishment of the neuronal transcriptome. These changes coincide with developmental phenotypes in neuronal identities and axon projections. Thus, preexisting, replication-dependent histones are insufficient for establishing neuronal chromatin and transcriptome; de novo H3.3 is required. Stage-dependent deletion of H3f3a and H3f3b from (1) cycling neural progenitor cells, (2) neurons immediately post mitosis, or (3) several days later, reveals the first postmitotic days to be a critical window for de novo H3.3. After H3.3 accumulation within this developmental window, co-deletion of H3f3a and H3f3b does not lead to immediate H3.3 loss, but causes progressive H3.3 depletion over several months without widespread transcriptional disruptions or cellular phenotypes. Our study thus uncovers key developmental roles for de novo H3.3 in establishing neuronal chromatin, transcriptome, identity, and connectivity immediately post mitosis that are distinct from its role in maintaining total histone H3 levels over the neuronal lifespan. Overall design: We analyzed a total of 12 samples of rRNA-depleted RNA from P0 mouse cortex using UMI RNA-seq. Libraries were generated using Click-seq. 4 samples were controls, 4 samples were H3f3a/H3f3b Neurod6-Cre conditional knockouts (dKO-N), and 4 samples were H3f3a/H3f3b Emx1-Cre conditional knockouts (dKO-E). We analyzed a total of 5 samples P0 mouse cortex using H3K4me3 CUT&Tag. 3 samples were controls, 2 samples were H3f3a/H3f3b Neurod6-Cre conditional knockouts (dKO-N). We analyzed a total of 5 samples P0 mouse cortex using H3K27me3 CUT&Tag, 3 controls and 2 were H3f3a/H3f3b Neurod6-Cre conditional knockouts (dKO-N). We analyzed 6 samples of P0 mouse cortex using ATAC-seq, 3 controls and 3 H3f3a/H3f3b Neurod6-Cre conditional knockouts (dKO-N). For snRNAseq, corticies from embroynic or adult mice were dissected and flash frozen, followed by nuclei dissociation and processing on the 10x Genomics Chromium controller.