The effects of distinct classes of peroxisome proliferator-activated receptor ␥ (PPAR␥) ligands on myogenesis and MyoD gene expression were examined in mouse skeletal muscle C2C12 myoblasts. Treatment of C2C12 cells with the PPAR␥ ligand, 15-deoxy-⌬ 12,14-prostaglandin J2 (15d-PGJ2), repressed morphologically defined myogenesis and reduced endogenous mRNA levels of the myogenic differentiation markers MyoD, myogenin, and ␣-actin. In contrast, two synthetic PPAR␥ ligands, L-805645 and ciglitazone, exhibited no effects. In transient transfection assays, 15d-PGJ2 specifically inhibited the expression of a MyoD promoter-luciferase reporter gene (MyoDLuc) in a cell type-and promoterspecific manner, indicating that 15d-PGJ2 functions in part by repressing MyoD gene transcription. The inhibition of MyoD gene expression by 15d-PGJ2 is mediated by the distal region of the MyoD gene promoter. PPAR␥ on its own also inhibited MyoDLuc expression and further augmented the 15d-PGJ2 response. In contrast, L-805645 and ciglitazone did not inhibit MyoDLuc expression on their own but did so in the presence of ectopically expressed PPAR␥. Interestingly, a transdominant inhibitor of PPAR␥ (hPPAR␥2⌬500) had no effect on the 15d-PGJ2-dependent repression of MyoD-Luc expression but overcame L-805645/PPAR␥-dependent repression. Finally, saturating concentrations of L-805645, which did not affect myogenesis, failed to ablate 15d-PGJ2-mediated repression of the myogenic program. Thus, distinct PPAR␥ ligands may repress MyoD gene expression through PPAR␥-dependent and-independent pathways, and 15d-PGJ2 can inhibit the myogenic program independent of its cognate receptor, PPAR␥.

Intrinsic apoptosis is principally governed by the BCL-2 family of proteins, but some non-BCL-2 proteins are also critical to control this process. To identify novel apoptosis regulators, we performed a genome-wide CRISPR-Cas9 library screen, and identified the mitochondrial E3 ubiquitin ligase MARCHF5/MITOL/RNF153 as an important regulator of BAK apoptotic function. Deleting MARCHF5 in diverse cell lines dependent on BAK conferred profound resistance to BH3-mimetic drugs. The loss of MARCHF5 or its E3 ubiquitin ligase activity surprisingly drove BAK to adopt an activated conformation, with resistance to BH3-mimetics afforded by the formation of inhibitory complexes with pro-survival proteins MCL-1 and BCL-XL. Importantly, these changes to BAK conformation and pro-survival association occurred independently of BH3-only proteins and influence on pro-survival proteins. This study identifies a new mechanism by which MARCHF5 regulates apoptotic cell death and provides new insight into how cancer cells respond to BH3-mimetic drugs. These data also highlight the emerging role of ubiquitin signalling in apoptosis that may be exploited therapeutically.

1289 Hematopoietic stem cells due to their life-long function should protect genome integrity to avoid accumulation of genetic aberrations leading to malignant transformation or bone marrow failure. We reported recently that human HSC of cord blood origin are exquisitely sensitive to DNA damage-induced apoptosis. Thus, following 3Gy of ionizing radiation HSC show evidence of persistent DNA damage response and greater p53-dependent apoptosis in comparison with commited myeloid progenitors. To elucidate the molecular basis of these observations we carried out a genome-wide loss-of-function genetic screen using a library of 80 000 shRNA vectors targeting more than 16 000 human genes. A screen was performed on immortalized (by TLS-ERG infection) cord blood cells (TEX), which radio-sensitivity is similar to early hematopoietic cells. To find out the radio-protective hits we exposed infected cells to four rounds of 4Gy irradiation in three independent experiments. TEX cells exhibited steady increase of their proliferative potential after each irradiation exposure. Cells were gathered after each irradiation round and their DNA was subjected to sequencing to determine protective hits. Upon the analysis we recognized known regulators of DNA damage response (e.g. p53) and identified many genes that previously were not connected to genotoxic stress response. Thus, the knockdown of these genes was at least as effective as p53 knockdown in protection of TEX cells against gamma-irradiation. The validation of the chosen hits on TEX cells showed that about half of them indeed mediate the protection against irradiation. Further validation included real-time PCR of infected cells to ensure the absence of off-target effect along with Western blot analysis of affected protein. We followed up the investigation of chosen hits on primary human lineage-negative cord blood cells and observed that only few candidates were mediating the same effect on HSC/progenitors cells as on TEX cells. To further validate the effect of the most prominently effective hits we employed several shRNA vectors for the same target gene. Following this step of validation, we choose to investigate the knockdown of CHEK2, the gene with reported role in DNA damage response in several murine tissues. Our preliminary studies in long-term cytokine-supplemented cultures demonstrated that CHEK2 is involved in DNA damage response of human HSC and progenitors. The further investigation of the effect of CHEK2 knockdown on repopulating human HSC is currently underway. An integrated analysis of our observations revealed several putative CHEK2-centered molecular networks, which connect DNA damage response to HSC function. Disclosures: No relevant conflicts of interest to declare.