Genome-wide investigation of chromatin features in human senescent cells and characterization of genome-transcriptome interplays by CUT&Tag profiling

Significant alterations in epigenetic landscapes can affect chromatin accessibility and genome-wide transcriptional expression. Cellular senescence, a stable state of growth arrest, is functionally engaged in numerous physiological and pathophysiological processes, and can promote organismal aging, restrain life quality and compromise healthspan. Mounting evidence suggests that senescent cells experience profound chromatin remodeling, but the underlying mechanisms linking epigenetic reorganization and gene expression profile remain less clear. In this study, we aimed to delineate the genome-wide redistribution of accessible chromatin regions leading to broad transcriptome expression changes during human diploid fibroblast (HDF) senescence, especially upon therapy-induced senescence (TIS). We report the distinct senescence-activated accessibility regions (SAARs) that tend to be distributed in H3K9me3-, H3K36me3- and H3K27ac-decorated enhancer regions, where the SAARs are responsible for increased SASP expression and multiple signaling events associated with SASP component secretion into the extracellular space. Mechanistically, a number of factors are involved in the activities of CCCTC-binding factor (CTCF), a master regulator of genome architecture facilitating establishment of conserved chromatin loops by cooperating with cohesin and other partners. Taken together, results of this study help identify key transcription factors regulating the senescence-specific program with outputs from multi-omics analysis, and provide potential therapeutic targets for future pipelines of anti-aging industry. Overall design: Primary human diploid fibroblasts (HDFs), namely PSC27, either proliferating (CTRL), exposed to ionizing radiation (RAD) or treated by the chemotherapeutic agent bleomycin (BLEO), were chemically dissociated, and fixed at room temperature. Cells were permeabilized and concentration was adjusted to 1x106 cells/ml. An fxCycle far red dye and RNase A were added and samples were incubated prior to resuspension at a concentration of 107 cells/ml and immediately processed for FACS. FACS was performed using BD FACSAria and appropriate gates were set based on the relative levels of fxCycle in order to isolate G0-G1 cells. After FACS sorting, cell were pelleted before frozen in liquid nitrogen and stored at -80°C. Hi-C library preparation was carried out using the in situ method described previously (Rao etal., 2014) with minor modifications.