Effect of mutation at the K202 site of the HDAC8 on gene expression in HeLa cells

HDAC8, a member of class I HDACs, plays a pivotal role in cell cycle regulation by deacetylating the cohesin subunit SMC3. While cyclins and CDKs are well-established cell cycle regulators, our knowledge of other regulators remains limited. Here we reveal acetylated K202 in HDAC8 as a key cell cycle regulator responsive to stress. K202 acetylation in HDAC8, primarily catalyzed by Tip60, negatively modulates HDAC8 activity, leading to increased SMC3 acetylation and cell cycle arrest. Furthermore, cells mimicking K202 acetylation display significant alterations in gene expression, potentially linked to changes in 3D genome structure, including enhanced chromatid loop interactions. K202 acetylation negatively impacts cell cycle progression by disrupting the expression of cell cycle-related genes and sister chromatid cohesion, resulting in G2/M phase arrest. These findings illuminate the reversible acetylation of HDAC8 as a novel cell cycle regulator, expanding our understanding of stress-responsive cell cycle dynamics. Overall design: To directly examine the influence of K202 acetylation on cellular processes, we harnessed CRISPR/Cas9 technology to modify the HDAC8 gene in HeLa cells. We generated the K202Q missense mutation, which mimics the acetylated state, and concurrently, we also generated the K202R mutant. Monoclonal cells were screened and confirmed for successful mutation of the HDAC8 gene through DNA sequence analysis.