AIDmut-Seq: a Three-Step Method for Detecting Protein-DNA Binding Specificity
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. AIDmu...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2023-02-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.03783-22 |
| _version_ | 1811164409600409600 |
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| author | Feixuan Li Xiao-Yu Liu Lei Ni Fan Jin |
| author_facet | Feixuan Li Xiao-Yu Liu Lei Ni Fan Jin |
| author_sort | Feixuan Li |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-10T15:22:08Z |
| format | Article |
| id | doaj.art-f5ec34b338014ed7acbb35732a4b3698 |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-04-10T15:22:08Z |
| publishDate | 2023-02-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-f5ec34b338014ed7acbb35732a4b36982023-02-14T14:15:49ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972023-02-0111110.1128/spectrum.03783-22AIDmut-Seq: a Three-Step Method for Detecting Protein-DNA Binding SpecificityFeixuan Li0Xiao-Yu Liu1Lei Ni2Fan Jin3Hefei National Research Center for Physical Sciences at the Microscale, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, People’s Republic of ChinaSchool of Medicine, Southern University of Science and Technology, Shenzhen, People’s Republic of ChinaCAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, People’s Republic of ChinaCAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, People’s Republic of ChinaABSTRACT 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.https://journals.asm.org/doi/10.1128/spectrum.03783-22activation induced cytidine deaminasePseudomonas aeruginosaprotein-DNA interactions |
| spellingShingle | Feixuan Li Xiao-Yu Liu Lei Ni Fan Jin AIDmut-Seq: a Three-Step Method for Detecting Protein-DNA Binding Specificity Microbiology Spectrum activation induced cytidine deaminase Pseudomonas aeruginosa protein-DNA interactions |
| title | AIDmut-Seq: a Three-Step Method for Detecting Protein-DNA Binding Specificity |
| title_full | AIDmut-Seq: a Three-Step Method for Detecting Protein-DNA Binding Specificity |
| title_fullStr | AIDmut-Seq: a Three-Step Method for Detecting Protein-DNA Binding Specificity |
| title_full_unstemmed | AIDmut-Seq: a Three-Step Method for Detecting Protein-DNA Binding Specificity |
| title_short | AIDmut-Seq: a Three-Step Method for Detecting Protein-DNA Binding Specificity |
| title_sort | aidmut seq a three step method for detecting protein dna binding specificity |
| topic | activation induced cytidine deaminase Pseudomonas aeruginosa protein-DNA interactions |
| url | https://journals.asm.org/doi/10.1128/spectrum.03783-22 |
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