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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.
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This content will become publicly available on November 22, 2025
Design of a Bioluminescent Assay Platform for Quantitative Measurement of Histone Acetyltransferase Enzymatic Activity
Abstract Protein acetylation and acylation are widespread post‐translational modifications (PTMs) in eukaryotic and prokaryotic organisms. Histone acetyltransferase (HATs) enzymes catalyze the addition of short‐chain acyl moieties to lysine residues on cellular proteins. Many HAT members are found to be dysregulated in human diseases, especially oncological processes. Screening potent and selective HAT inhibitors has promising application for therapeutic innovation. A biochemical assay for quantification of HAT activity utilizing luminescent output is highly desirable to improve upon limitations associated with the classic radiometric assay formats. Here we report the design of a bioluminescent technological platform for robust and sensitive quantification of HAT activity. This platform utilizes the metabolic enzyme acetyl‐CoA synthetase 1 (ACS1) for a coupled reaction with firefly luciferase to generate luminescent signal relative to the HAT‐catalyzed acetylation reaction. The biochemical assay was implemented in microtiter plate format and our results showed this assay sensitively detected catalytic activity of HAT enzyme p300, accurately measured its steady‐state kinetic parameters of histone acetylation and measured the inhibitory potency of HAT inhibitor. This platform demonstrated excellent robustness, reproducibility, and signal‐to‐background ratios, with a screening window Z’=0.79. Our new bioluminescent design provides an alternative means for HAT enzymatic activity quantitation and HAT inhibitor screening.
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- Award ID(s):
- 2203942
- PAR ID:
- 10557188
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- ChemBioChem
- Volume:
- 26
- Issue:
- 1
- ISSN:
- 1439-4227
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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