MacroH2A2-Enriched Domains Are Largely Stable Across the Cell Cycle but Focally Displaced at Mitotic Regulatory Elements.

The macroH2A variants mH2A1 and mH2A2 are structurally similar but not identical. Our previous study demonstrated that mH2A1 is reloaded during cell-cycle progression, but whether mH2A2 follows similar dynamics has remained unclear. Here, we used native ChIP-seq in synchronized Huh-7 cells to profile both variants at G1/S and G2/M. Although mH2A1- and mH2A2-enriched domains overlapped extensively, mH2A2 domains were largely stable across the cell cycle, in sharp contrast to the dynamic reloading of mH2A1. Only a small subset of mH2A2 domains showed phase-specific deposition or displacement. Among these, G1/S-unique mH2A2 domains were preferentially located in the active A compartment and coincided with reduced chromatin accessibility at binding sites for cell-cycle regulators. These G1/S-unique domains co-localize with genes involved in mitotic progression within the same A compartment, suggesting potential regulatory roles in both chromatin organization and transcriptional regulation. These findings refine the classical view of macroH2A variants as static repressive marks: mH2A2 is not entirely static, but its cell-cycle dynamics are far more restricted than those of its paralog mH2A1, occurring only at a small subset of genomic loci, with a regulatory logic distinct from that of mH2A1.