Abstract 11113: A Novel ATF4 Dependent ER-Stress Induced Transcriptional Unit Within the CHAC1 Promoter
A systems genetics screen of transcriptome wide RNA co-expression in a population of human cell cultures can broadly define novel functional gene-gene relationships. Using this method, we predicted a role for the human gene, CHAC1, within the ER-stress pathway functionally linked to ATF4. Targeted cell culture and genetic perturbations refined a role for CHAC1 as an ER-stress gene regulated by ATF4. ATF4 siRNAs blocked induction of CHAC1 mRNA by ER-stress, and ATF4 over-expression induced CHAC1 mRNA expression. ATF4 is an important gene involved in ER-stress and apoptotic signaling, and we demonstrated the pro-apoptotic potential for CHAC1. ATF4 plays important roles in osteoblast development, metabolic disease and atherosclerosis. Herein a direct transcriptional link between ATF4 and the CHAC1 promoter by ATF4 is defined in the setting of ER-stress. We cloned the CHAC1 promoter upstream of luciferase reporter system to define functional elements by deletion analysis. A highly conserved bipartite DNA element dubbed the ATF/CRE and ACM were the major regulators of basal CHAC1 expression, induction by ER-stress agonists or ATF4 co-expression. We compared the CHAC1 promoter -luciferase reporter to well-characterized ATF4 target genes CHOP and ASNS, using parallel luciferase assays. Notably, the CHAC1 promoter-reporter was induced to the highest extent following ATF4 co-expression compared to CHOP and ASNS promoters. Using a novel Immunoblot-EMSA assay we determined binding of ATF4 and ATF3, but not CHOP to CHAC1 ATF/CRE and ACM oligos. We also used the CHip method to query binding of ATF3 and ATF4 at the CHAC1 promoter under control and ER-stress conditions. We noted significant binding of ATF3 to the CHAC1 promoter under control and ER-stress treatment. Interestingly, we noted a dramatic increase in ATF4 occupancy at the CHAC1 promoter accompanying ER-stress. The ATF4 responsive ATF/CRE and ACM of the CHAC1 promoter represent a novel target for ATF4, and may reveal important contributions to disease processes involving ATF4. These data also implicate CHAC1 as an important target gene of ATF4, which may mediate important biological effects in the setting of ER-stress, and might have implications for cardiovascular and metabolic disease.
Author Disclosures: R.R. Crawford: None. E.T. Prescott: None. I.N. Mungrue: None.
- © 2014 by American Heart Association, Inc.