H3.3 deposition counteracts the replication-dependent enrichment of H3.1 at chromocenters in embryonic stem cells

Abstract

Chromocenters in mouse cells are membrane-less nuclear compartments that represent typical heterochromatin stably maintained during the cell cycle. Here, we explore how histone H3 variants, replicative H3.1/H3.2 or replacement H3.3, mark these domains during the cell cycle. In mouse embryonic stem cells (ESCs), neuronal precursor cells (NPCs) as well as immortalized 3T3 cells, we find a strong and distinct H3.1 enrichment at chromocenters, with some variation in ESCs. Mechanistically, this H3.1 selective enrichment depends on the DNA Synthesis Coupled (DSC) deposition pathway operating in S phase. Yet, this selective enrichment is challenged when we target H3.3 deposition through the DNA Synthesis Independent (DSI) deposition pathway mediated by HIRA. Altering the H3.1/H3.3 equilibrium at chromocenters in ESCs affects its heterochromatin properties leading to mitotic defects. We thus reveal opposing mechanisms for H3.1 and H3.3 deposition with different enforcement according to cell cycle and potency which determine their ratio at chromocenters and are critical for genome stability and cell survival.