Role of Chromatin Components in Replication Stress Response

Recent emerging evidence, including our preliminary data, suggests that KMT2D plays a role in the DNA damage response (DDR) and the maintenance of genome integrity (GI). Despite being one of the most frequently mutated genes in cancer, little is known about its biological roles beyond its enhancer-driven function. Therefore, the present study investigates the interplay between the described gene expression function of KMT2D and its emerging role in the DDR. By exploiting siRNA-mediated knockdown of KMT2D in human cells, we aimed to study (1) the relationship between the dysregulated enhancer function and the observed DDR defect; and (2) the gene expression changes that may relate to the observed DDR defect. First, we validated our knockdown system by assessing enhancer function by analyzing enhancer-specific histone post-translational modification (PTMs, H3K4me1 and H3K27ac). Subsequently, we set up the rescue of KMT2D enhancer function through an HDAC inhibitor (HDACi) treatment known to act on p300 and shown to rescue enhancer function, including in KMT2D knockdown cells. The HDACi treatment successfully restored enhancer function with little effect on the DDR. Ongoing work assesses whether enhancer function restoration through HDACi treatment impacts the defective DDR of KMT2D knockdown cells. In parallel, we are finalizing RNA-Seq analysis in knockdown cells to investigate the transcriptional effects of KMT2D in our cellular model.