Spatial reorganization of chromosome architecture in human stromal cells upon therapy-induced senescence

Cellular senescence is a basic biological process characterized by an essentially irreversible growth arrest. Senescent cells accumulate a variety of epigenetic alternations, but the three-dimensional (3D) organization of chromatin remains underexplored. We aim to characterize the 3D architecture of interphase chromosomes in proliferating, and therapy-induced senescent (TIS) cells of human stromal origin. Although the overall organization of chromatin into active (A) and repressive (B) compartments and topologically associated domains (TADs) is largely conserved between these conditions, a subset of TADs switches between compartments of proliferative versus TIS cells. On a global level, the Hi-C interaction matrices of senescent cells (TIS) are characterized by a relative gain of long-range, while a simultaneous loss of short-range interactions within chromosomes. Direct measurements of distances between genetic loci, chromosome volumes, and chromatin accessibility were performed. The structural model arising from this study provides a unique high-resolution view of the complex chromosomal architecture in human senescent cells. Overall design: Human stromal cells, either proliferating (CTRL), treated by the chemotherapeutic agent bleomycin (BLEO) or damaged by ionizing radiation (RAD), 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 et al., 2014) with minor modifications.