Zhang, HaoyueHutchinson–Gilford progeria syndrome (HGPS) is a severe human premature aging disorder caused by a lamin A mutant named progerin. Death occurs at a mean age of 13 y from cardiovascular problems. Previous studies revealed loss of vascular smooth muscle cells (SMCs) from large arteries in HGPS patient and mouse models, suggesting a causal connection between SMC loss and cardiovascular malfunction. The primary aim of this dissertation is to elucidate the molecular mechanisms underlying the massive SMC loss in HGPS. To study this, I develop an in vitro differentiation protocol to generate HGPS SMCs from induced pluripotent stem cells (iPSCs). My results indicate that HGPS SMCs exhibit a profound cell death phenotype, potentially recapitulating the in vivo SMC loss. Mechanistically, I find that HGPS SMCs bear deficient homologous recombination (HR). In addition, progerin accumulation strongly suppresses PARP1 and consequently triggers an activation of the error-prone non-homologous end joining (NHEJ) response during S/G2 phase. As a result, HGPS SMCs exhibit prolonged mitosis and mitotic catastrophe. Mis-regulated DNA damage response (DDR) is proposed to induce genome instability and various cellular phenotypes in HGPS, including HGPS SMC cell death. To better understand HGPS DDR misregulation, I examine HR and NHEJ in HGPS fibroblasts at different cell cycle phases. My analysis indicates that HR is deficient in S/G2 phase, whereas NHEJ, the dominant G0/G1 phase DDR pathway, is impaired in G0/G1 phase but active in S/G2 phase HGPS fibroblasts. The mis-regulation of HR and NHEJ may jeopardize genome integrity in both G0/G1 and S/G2 phase HGPS cells. Mechanistic study reveals that H2AX, a crucial upstream DDR signal, is reduced in G0/G1 but normal in S/G2 phase HGPS cells, implicating a potential cause of the cell cycle-dependent NHEJ mis-regulation. Furthermore, this reduction is correlated with impaired ATM activation and loss of H3K9me3 in HGPS. Restoration of H3K9me3 by methylene blue treatment can stimulate ATM activity, improve H2AX signaling and rescue NHEJ in G0/G1 phase HGPS cells. This dissertation not only is the first mechanistic study on HGPS SMC loss but also provides a molecular basis and therapeutic approach for the HGPS DDR deficiencies.enDissertationBiologyAging