Abstract
The incorporation of the histone H3 variant, H3.3, into chromatin by the H3.3-specific chaperone DAXX and the ATP-dependent chromatin remodeling factor ATRX is a critical mechanism for silencing repetitive DNA. DAXX and ATRX are also components of promyelocytic nuclear bodies (PML-NBs), which have been identified as sites of H3.3 chromatin assembly. Here, we use a transgene array that can be visualized in single living cells to investigate the mechanisms that recruit PML-NB proteins (i.e. PML, DAXX, ATRX, and SUMO-1, SUMO-2 and SUMO-3) to heterochromatin and their functions in H3.3 chromatin assembly. We show that DAXX and PML are recruited to the array through distinct SUMOylation-dependent mechanisms. Additionally, PML is recruited during S phase and its depletion increases H3.3 deposition. Since this effect is abrogated when PML and DAXX are co-depleted, it is likely that PML represses DAXX-mediated H3.3 chromatin assembly. Taken together, these results suggest that, at heterochromatin, PML-NBs coordinate H3.3 chromatin assembly with DNA replication, which has important implications for understanding how transcriptional silencing is established and maintained.