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AIDmut-Seq: a Three-Step Method for Detecting Protein-DNA Binding Specificity

Authors :
Feixuan Li
Xiao-Yu Liu
Lei Ni
Fan Jin
Source :
Microbiology Spectrum, Vol 11, Iss 1 (2023)
Publication Year :
2023
Publisher :
American Society for Microbiology, 2023.

Abstract

ABSTRACT Transcriptional factors (TFs) and their regulons make up the gene regulatory networks. Here, we developed a method based on TF-directed activation-induced cytidine deaminase (AID) mutagenesis in combination with genome sequencing, called AIDmut-Seq, to detect TF targets on the genome. AIDmut-Seq involves only three simple steps, including the expression of the AID-TF fusion protein, whole-genome sequencing, and single nucleotide polymorphism (SNP) profiling, making it easy for junior and interdisciplinary researchers to use. Using AIDmut-Seq for the major quorum sensing regulator LasR in Pseudomonas aeruginosa, we confirmed that a few TF-guided C-T (or G-A) conversions occurred near their binding boxes on the genome, and a number of previously characterized and uncharacterized LasR-binding sites were detected. Further verification of AIDmut-Seq using various transcriptional regulators demonstrated its high efficiency for most transcriptional activators (FleQ, ErdR, GacA, ExsA). We confirmed the binding of LasR, FleQ, and ErdR to 100%, 50%, and 86% of their newly identified promoters by using in vitro protein-DNA binding assay. And real-time RT-PCR data validated the intracellular activity of these TFs to regulate the transcription of those newly found target promoters. However, AIDmut-Seq exhibited low efficiency for some small transcriptional repressors such as RsaL and AmrZ, with possible reasons involving fusion-induced TF dysfunction as well as low transcription rates of target promoters. Although there are false-positive and false-negative results in the AIDmut-Seq data, preliminary results have demonstrated the value of AIDmut-Seq to act as a complementary tool for existing methods. IMPORTANCE Protein-DNA interactions (PDI) play a central role in gene regulatory networks (GRNs). However, current techniques for studying genome-wide PDI usually involve complex experimental procedures, which prevent their broad use by scientific researchers. In this study, we provide a in vivo method called AIDmut-Seq. AIDmut-Seq involves only three simple steps that are easy to operate for researchers with basic skills in molecular biology. The efficiency of AIDmut-Seq was tested and confirmed using multiple transcription factors in Pseudomonas aeruginosa. Although there are still some defects regarding false-positive and false-negative results, AIDmut-Seq will be a good choice in the early stage of PDI study.

Details

Language :
English
ISSN :
21650497
Volume :
11
Issue :
1
Database :
Directory of Open Access Journals
Journal :
Microbiology Spectrum
Publication Type :
Academic Journal
Accession number :
edsdoj.f5ec34b338014ed7acbb35732a4b3698
Document Type :
article
Full Text :
https://doi.org/10.1128/spectrum.03783-22