A Simple Method to Detect the Inhibition of Transcription Factor-DNA Binding Due to Protein–Protein Interactions In Vivo

摘要

Binding of transcription factors (TFs) to cis-regulatory elements (DNA) could modulate the expression of downstream genes, while interactions between TFs and other proteins might inhibit them binding to DNA. Nowadays, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) approaches are usually employed to detect the inhibitory effect. However, EMSA might not reflect the inhibitory effect in vivo. ChIP requires preparation of specific antibody or stable genetic transformation and complicated experimental steps, making it laborious and time-consuming. Here, based on the yeast one-hybrid (Y1H) system, we present a simple method to detect the inhibition of TF–DNA binding due to protein–protein interactions in vivo. When interactions between TFs and other proteins inhibit TFs binding to DNA, the reporter (Aureobasidin A resistance) gene is not activated, thereby inhibiting yeast growth on media containing the AbA antibiotic. Two examples were tested with the newly developed method to demonstrate its feasibility. In conclusion, this method provides an alternative strategy for detecting the inhibition of DNA-binding of TFs due to their interactions with other proteins in vivo.