Histone H3.3 fills gaps in heterochromatin generated by Smarcad1 in mouse embryonic stem cells (ESC)

Nucleosome turnover concomitant with incorporation of the replication-independent histone variant H3.3 is a hallmark of regulatory regions in the animal genome. In our current understanding, nucleosome turnover is universally linked to DNA accessibility and histone acetylation. In mouse embryonic stem cells, H3.3 is also highly enriched at interstitial heterochromatin, most prominently intracisternal-A particle endogenous retroviral elements. Interstitial heterochromatin is established over confined domains by the TRIM28/SETDB1 corepressor complex and has stereotypical features of repressive chromatin, such as H3K9me3 and recruitment of all HP1 isoforms. Here, we demonstrate that fast histone turnover and H3.3 incorporation is compatible with these hallmarks of heterochromatin. We identify Smarcad1 to be a chromatin remodeler that generates nucleosome-free regions which are subsequently filled with histone H3.3, generating a surprisingly dynamic heterochromatin state. Loss of histone H3.3 elicits a highly specific opening of interstitial heterochromatin demonstrating that in the wake of Smarcad1 remodeling, H3.3 is required to maintain minimal DNA accessibility by reassembling nucleosomes. Chromatin accessibility was assessed using Omni-ATAC (Corces et al. 2017). Wildtype and H3.3 knockout (H3.3KO) cell lines were compared to following rescue cell lines: H3.3 (H3.3KO+H3.3), H3.2 (H3.3KO+H3.2) or H3.3 L126A I130A (H3.3KO+H3.3LI->AA). Further, Smarcad1 or ATRX was knocked down using RNAi. ATAC-Seq experiments were performed in two batches as indicated. Histone H3.3 and H3K9me3 occupancy was assessed using a crosslinking ChIP-Seq protocol described below.