The histone chaperone ANP32B regulates chromatin incorporation of the atypical human histone variant macroH2A
Eukaryotic chromatin structure is highly conserved, with the canonical histone proteins revealing only small sequence changes across species. Yet, all vertebrates exhibit three much larger histone H2A variants, macroH2A. A distinctive feature of these atypical histones is the globular macrodomain module, which can bind metabolites and is connected to the histone fold through a flexible linker. MacroH2A variants impact heterochromatin organization, transcription regulation and establish a barrier for cellular reprogramming. However, the mechanisms of how these large H2A variants are incorporated into chromatin and the identity of any chaperones required for histone deposition have remained elusive. Here, we developed a split-GFP-based cellular readout for histone incorporation and conducted a genome-wide mutagenesis screen in haploid human cells to identify proteins that regulate macroH2A dynamics. We identified and validated the histone chaperone ANP32B as a regulator of macroH2A chromatin deposition. ANP32B associates with macroH2A in cells and in vitro binds to histones with low nanomolar affinity. In vitro nucleosome assembly assays show that ANP32B stimulates deposition of macroH2A-H2B and not of H2A-H2B onto tetraso me. In cells, depletion of ANP32B in cells strongly affects global macroH2A deposition, revealing ANP32B as a macroH2A chaperone. Our study highlights the power of haploid cell functional genomics coupled with cellular imaging to identify factors that are required for chromatin plasticity and diversity. Overall design: CUT&RUN with non-targeting IgG antibodies and anti-macroH2A1 antibodies was performed on HepG2 cells transfected with either non-targeting control siRNAs or ANP32B-targeting siRNAs.