Cleavage of histone H2A during embryonic stem cell differentiation destabilizes nucleosomes to counteract gene activation.

Histone proteolysis is an understudied phenomenon in which the N-terminal tails of histones are irreversibly cleaved by intracellular proteases. During development, histone post-translational modifications (PTMs) are known to orchestrate gene expression patterns that ultimately drive cell fate decisions. Therefore, deciphering the mechanisms of histone proteolysis is necessary to enhance the understanding of cellular differentiation. Here, we show that histone H2A is cleaved by the lysosomal protease Cathepsin L during mouse ESC differentiation. Using quantitative mass spectrometry (MS), we identified L23 to be the primary cleavage site that gives rise to the main clipped form of H2A (cH2A), which reaches a maximum level of ∼1% of total H2A after 4 days of ESC differentiation. Using ChIP-seq, we found that preventing proteolysis leads to an increase in acetylated H2A at promoter regions in differentiated ES cells. We also identified novel readers of different acetylated forms of H2A in pluripotent ES cells, such as members of the PBAF remodeling complex, and showed that H2A proteolysis abolishes this recognition. Analysis of the histone H3 PTM profiles of full-length (FL) H2A and cH2A containing nucleosomes demonstrates that cH2A is associated with marks found on active genes, consistent with ChIP-seq experiments. cH2A-containing nucleosomes are also less stable or turned over at faster rates than nucleosomes containing FL H2A. Altogether, our data suggest that proteolysis serves as an efficient mechanism to silence pluripotency genes and destabilize the nucleosome core particle.