Abstract 324: Knockout of the Pro-apoptotic Er-stress Gene Chac1 in Mice Results in Embryonic Lethality and Activation of the Notch Pathway

Abstract

The ER-stress pathway is activated by oxidized phospholipids in human aortic endothelial cells (HAECs), and may play a role in the development of atherosclerosis. Systems genetics screens of transcriptome wide RNA co-expression in populations of human cells can broadly define novel functional gene-gene relationships. Using this method, we predicted a role for the human CHAC1 gene downstream of ATF4 in the ER-stress pathway in HAECs. Targeted cell culture and genetic perturbations refined the ATF4 dependent ER-stress induction of CHAC1 and a role in regulating apoptosis. Herein we report the generation of a mouse model with the Chac1 gene deleted (Chac1-KO), and heterozygous insufficiency (Chac1-Het). We obtained transgene positive C57BL6 founders that were bred to subsequent generations producing Chac1-Het mice. Intercrossing Chac1-Het mice revealed embryonic lethality, as no Chac1-KO progeny were generated from 25 litters. Chac1-KO embryos were produced at 13 days post coitus and used to make Mouse Embryonic Fibroblasts (MEFs), and the absence of Chac1 expression in these cells was validated. Since Chac1 is an ER-stress inducible gene activated by ATF4, we examined the expression of this pathway in Chac1-KO MEFs, versus controls. We noted increased basal and ER-stress induced expression of ATF4 and its target CHOP accompanying Chac1 knockout, highlighting a novel role for Chac1 in feedback regulation of the ATF4 branch of the ER-stress pathway. We also noted increased expressions of Parp1 and Caspase3, in Chac1-KO MEFs, supporting a role in the regulation of apoptosis pathways. Additionally, Chac1 cells had a higher proliferation rate, displayed a distinct morphology, and grew to a higher density. Since recent reports show a role for Chac1 in blocking Notch maturation, we examined expression of the Notch pathway in Chac1-KO MEFs, versus controls. We noted increased expression of the Notch target gene Hes1 in Chac1-KO MEFs at baseline. Following treatment with the proteasome inhibitor MG132, Chac1-KO MEFs had increased expressions of Hes1, Hey2 and Nrarp, target genes of the Notch pathway. These data reveal activation of the Notch pathway in Chac1-KO cells treated with MG132, a hyper proliferative phenotype and embryonic lethality at dpc 13.