Histone acetylation levels are reduced during mitosis. To study the mitotic regulation of H3K9ac, we used an array of inhibitors targeting specific histone deacetylases. We evaluated the involvement of the targeted enzymes in regulating H3K9ac during all mitotic stages by immunofluorescence and immunoblots. We identified HDAC2, HDAC3, and SIRT1 as modulators of H3K9ac mitotic levels. HDAC2 inhibition increased H3K9ac levels in prophase, whereas HDAC3 or SIRT1 inhibition increased H3K9ac levels in metaphase. Next, we performed ChIP-seq on mitotic-arrested cells following targeted inhibition of these histone deacetylases. We found that both HDAC2 and HDAC3 have a similar impact on H3K9ac, and inhibiting either of these two HDACs substantially increases the levels of this histone acetylation in promoters, enhancers, and insulators. Altogether, our results support a model in which H3K9 deacetylation is a stepwise process—at prophase, HDAC2 modulates most transcription-associated H3K9ac-marked loci, and at metaphase, HDAC3 maintains the reduced acetylation, whereas SIRT1 potentially regulates H3K9ac by impacting HAT activity.

Mitosis comprises multiple changes, including chromatin condensation and transcription reduction. Intriguingly, while histone acetylation levels are reduced during mitosis, the mechanism of this reduction is unclear. We studied the mitotic regulation of H3K9ac by using inhibitors of histone deacetylases. We evaluated the involvement of the targeted enzymes in regulating H3K9ac during mitotic stages and cytokinesis by immunofluorescence and immunoblots. We identified HDAC2, HDAC3 and SIRT1 as modulators of the mitotic levels of H3K9ac. HDAC2 inhibition increased H3K9ac levels in prophase, whereas HDAC3 or SIRT1 inhibition, increased H3K9ac levels in metaphase. Next, we performed ChIP-seq in mitotic cells following targeted inhibition of these histone deacetylases. While the genomic areas impacted by HDAC2 and HDAC3 were mostly concordant, a subset of loci were unique to each enzyme. Interestingly, HDAC3-specific targets were enriched for genes involved in mitosis regulation. Our results support a model in which H3K9 deacetylation is a stepwise process - at prophase HDAC2 modulates most transcription-associated H3K9ac-marked loci and at metaphase HDAC3 maintains the reduced acetylation, whereas SIRT1 potentially regulates H3K9ac by impacting HAT activity.