MacroH2A-Mediated Gene Repression through Nucleosome Compaction and Remodeling Inhibition

MacroH2A (mH2A) is a histone variant primarily implicated in heterochromatin maintenance and transcriptional repression, yet how its conserved histone-fold and its variant-specific domains reshape chromatin to enforce gene silencing remains poorly understood. Here, we dissect the domain-specific contributions of mH2A to nucleosome dynamics and chromatin remodeling. We show that, in addition to its linker region, the C-terminal tail of mH2A histone-fold also stabilizes nucleosome entry/exit DNA. Cryo-EM analysis reveals that this C-terminus tracks along nucleosomal DNA toward the dyad, adopting an on-dyad binding mode reminiscent of linker histone H1. Additionally, the mH2A linker potently inhibits DNA translocation by both the INO80 and Chd1 chromatin remodelers, while the histone fold selectively suppresses INO80 activity. In contrast, the macro domain has no detectable impact on terminal DNA accessibility or remodeling. Together, our results uncover a previously unappreciated architectural role for the mH2A histone fold and establish the linker domain as a dominant regulator of nucleosome dynamics and chromatin remodeling, providing a mechanistic framework for how mH2A enforces transcriptional repression.