MacroH2A-mediated gene repression through nucleosome compaction and remodeling inhibition

MacroH2A (mH2A) is a histone variant primarily associated with heterochromatin maintenance and transcriptional repression. However, how its individual domains contribute to chromatin regulation and modulate nucleosome structure and function remains incompletely understood. Here, we show that in addition to the linker region, the mH2A histone fold plays a modest but significant role in restricting entry/exit DNA dynamics. Mutagenesis studies identify the C-terminal tail of the histone fold as an important determinant of ATP-dependent chromatin remodeling inhibition. Consistent with these findings, cryo-electron microscopy (cryo-EM) study reveals that the mH2A C-terminal tail tracks along the entry/exit DNA toward the dyad. Biochemical and biophysical analyses further demonstrate that the mH2A linker synergizes with linker histone H1 to promote chromatin compaction through a non-competing interaction near the proximal linker DNA region. Together, these findings define previously unrecognized structural and regulatory roles for the mH2A histone fold and provide a mechanistic framework for how it corroborates with mH2A-linker to enforces chromatin compaction and transcriptional repression. We investigated how the individual domains of the histone variant macroH2A regulate nucleosome structure, chromatin remodelling, and chromatin compaction using biochemical, biophysical, mutagenesis, and cryo-EM approaches. We found that the macroH2A histone fold, particularly its C-terminal tail, restricts entry/exit DNA dynamics and inhibits ATP-dependent chromatin remodeling, while the linker region synergizes with linker histone H1 to promote chromatin compaction, revealing complementary mechanisms by which macroH2A reinforces repressive chromatin.