Cloning Egr-1 Regulated Genes in Breast Cells

摘要

Egr1 transcription factor is an immediate early gene that is induced in response to many stimuli including stimuli that cause genotoxic stress and DNA damage. Egr1 plays a key role in stress responses that follow chemotherapy or irradiation treatments for breast cancer, by stimulating apoptosis. in addition, Egr1 and p53 both play roles in cell cycle arrest and DNA repair stress responses that can allow the cell to repair DNA damage before proliferating. We developed a protocol to determine which genes are regulated by Egr1 during these activities by subjecting cells to irradiation and a technique called chromatin immunoprecipitation (Chip). We collect the promoter sequences that were bound to Egr1 protein at the time of fixation, process the DNA to amplify and then label with %fluorescent dyes for hybridization on a microarray of identified promoter DNAs. Scanning and special software that normalizes and calculates the signal to control ratios followed by bioinformatic analyses indicates the regulated genes. Many genes important to breast cancer were revealed such as PTEN, p53 and the GADD45 which are all are transactivated by Egr1. With further work we can determine if any new genes are targets for cancer therapy.