Validating CREB3L1 Binding Promotor Sequences

Triple negative breast cancer (TNBC) is known for its high mortality rate and risk of relapse, which is often accredited to a lack of targeted therapies. Previous work in the Anderson lab showed that cAMP-responsive element-binding protein 3-like protein 1 (CREB3L1), an anti-metastatic transcription factor, is downregulated in metastatic TNBC cells. CREB3L1 is found in the endoplasmic reticulum and is transported to the Golgi to be cleaved into active CREB3L1 (CREB3L1*) upon exposure to stress. CREB3L1* then enters the nucleus, binding to various promoters and regulating their expression. Previously, ChIP-seq identified genes directly regulated by CREB3L1*. This experiment aimed to validate these ChIP-seq findings and determine if CREB3L1* positively or negatively regulates gene expression. The genes of interest were isolated and amplified from gDNA, then validated via RT-qPCR and luciferase dual-reporter assays. Out of the six genes tested via RT-qPCR, three (SND1, YIPF5, EDEM3) showed results consistent with ChIP-seq findings, while the other three (TUBA1B, ARF1, AKR1A1) did not. For the luciferase dual-reporter assay, two genes were examined: YIPF5, which validated the ChIP-seq result, and TUBA1B, which did not.