PML is recruited to heterochromatin during S phase and represses DAXX-mediated histone H3.3 chromatin assembly

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.

PML Bodies in Mitosis

“…Nuclear PML bodies generally distribute to the interchromatin space, and a number of reports show that their periphery frequently associate with specific chromatin elements or genomic loci [34,52,53,54,55,56,57]. These observations are in agreement with the fact that a large number of proteins that are recruited by PML bodies participate in chromatin metabolic processes, such as genome maintenance, DNA replication and gene expression [58,59,60]. In some extreme cases PML bodies appear to completely engulf certain chromatin elements.…”

Nuclear body phase separation drives telomere clustering in ALT cancer cells

“…As PARylation is one of the earliest events during DNA damage recognition, it is possible that a temporal order of signals beginning with PARP activity and culminating in SUMO–SIM interactions is responsible for phase separation of DNA damage foci. Furthermore, PML bodies associate with genomic loci other than telomeres in non-ALT cells to regulate multiple functions including DNA repair, transcription, viral genome replication, and heterochromatin domain formation ( Dellaire and Bazett-Jones, 2004 ; Eskiw et al , 2004 ; Ching et al , 2005 ; Luciani et al , 2006 ; Shastrula et al , 2019 ). Our work demonstrates local sumoylation as a mechanism for generating telomere association of PML bodies by either directly nucleating PML bodies or enabling sumoylated telomeres to fuse with existing PML bodies to form APBs.…”

DAXX in cancer: phenomena, processes, mechanisms and regulation

“…In osteosarcoma Saos-2 cells, the colocalization of a DAXX double SIM mutant and a PML mutant lacking SUMOylation sites was severely impaired (6), further supporting the importance of PML SUMOylation and the SIM–SUMO interaction in tethering DAXX to PML-NBs. Notably, SIM2 has been shown to play a dominant role, while SIM1 appears dispensable, for DAXX’s recruitment to PML-NBs in several cell lines such as COS-1 and HeLa (14,44). The intramolecular SIM1–4HB interaction in DAXX (16) could mask SIM1, which potentially renders SIM1 unavailable for binding SUMOylated PML.…”

“…The structural basis of the SIM-facilitated SUMOylation and chain formation was recently reviewed (46). Notably, SIM2 is critical for targeting DAXX to heterochromatin sites for depositing H3.3, which, interestingly, depends on UBC9, while SIM1 appears to strengthen DAXX's recruitment to chromatin sites (44). Shastrula et al proposed that UBC9-mediated SUMOylation of unknown heterochromatin proteins mediates DAXX recruitment (44).…”