Analyzing CREB binding motif and sites in the mouse lens epithelial cells alpha-TN4 overexpressing Vector, WT-CREB, S133A-CREB, R314A-CREB and S133A/R314A –CREB.
Transcription factor CREB regulates lens Epithelial-Mesenchymal Transition(EMT)in both S133 phosphorylation dependent and independent manners. We found that S133A-CREB can activate lens EMT, while R314A-CREB attenuated it. To understand the mechanism by which S133A-CREB and R314A-CREB modulates EMT gene expression. ChIP assay using CREB antibody in the in the mouse lens epithelial cells alpha-TN4 overexpressing Vector, WT-CREB, S133A-CREB, R314A-CREB and S133A/R314A-CREB, and ChIP-seq was conducted on the ChIP DNA. We found that WT-CREB, S133A-CREB as well as R314A-CREB binds to the promoter regions of mesenchymal genes, and S133A-CREB retained canonical CREB binding motif, while R314A-CREB exhibited shifted binding motif preference to ATF1. Overall design: Chromatin immunoprecipitation-DNA sequencing for CREB in in the mouse lens epithelial cells alpha-TN4 overexpressing Vector, WT-CREB, S133A-CREB, R314A-CREB and S133A/R314A –CREB.