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

Guangzhe Yang,Dong Chao,Zhenhua Ming,Jixing Xia

doi:10.3390/genes10090684

Abstract

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.

Highlights

Transcriptional regulation is an important way in the regulation of gene expression
electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) approaches are usually employed to detect whether interactions between transcription factors (TFs) and other proteins inhibited them binding to cis-elements [1,2,3,4,5]
Based on the observation that the reporter gene is expressed or not, we can determine whether TF–protein interaction inhibits TF binding to its target DNA

Summary

Introduction

Transcriptional regulation is an important way in the regulation of gene expression. Besides RNA polymerases, multiple proteins or transcription factors (TFs) are required to orchestrate the whole transcription event, especially in eukaryotic gene transcription. Transcription is modulated through the binding of TFs to cis-regulatory elements usually located upstream of genes. The binding of TFs to cis-elements may be regulated by other proteins. Some proteins could interact with TFs, which inhibit them binding to cis-elements, thereby affecting the transcription of downstream genes. EMSA (electrophoretic mobility shift assay) and ChIP (chromatin immunoprecipitation) approaches are usually employed to detect whether interactions between TFs and other proteins inhibited them binding to cis-elements [1,2,3,4,5]. The bHLH-type TF PIF1 regulated the DELLA gene

Methods

Results

Conclusion