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A novel high-throughput B1H-ChIP method for efficiently validating and screening specific regulator–target promoter interactions

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Abstract

Confident protein–DNA interaction (PDI) data could significantly improve our understanding of transcriptional regulation network in both prokaryotes and eukaryotes. New methods need to be established for validating and mining the protein–DNA interaction data produced by bioinformatic tools and large-scale screening assays. In this study, we integrated bacterial one-hybrid technique (B1H) with classical chromatin immunoprecipitation (ChIP) assay to develop an innovative B1H-ChIP method, which has the advantages of being high-throughput, low cost, and easy-to-perform. Using this method, we validated two pairs of previously reported PDIs and further successfully discovered five novel target genes for Mce2R and four novel regulators of the gene dnaA in the human pathogen Mycobacterium tuberculosis. New PDI data suggest that Mce2R may play novel roles in the regulation of multi-drug resistance, cell wall synthesis, and intracellular growth of M. tuberculosis, and there exists a probable selective regulation of dnaA under different host conditions. Our findings provide important new information for understanding unique regulatory mechanisms in the pathogen. The B1H-ChIP approach has wide applications both in validating and discovering PDIs and in unraveling transcriptional regulatory network in prokaryotes and eukaryotes.

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Acknowledgment

This work was supported by the National Natural Science Foundation of China (30930003, 31025002), the Fundamental Research Funds for the Central Universities (2011PY140) and Hubei Chutian Scholar Program (To He Z-G).

Conflicts of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. National Key Laboratory of Agricultural Microbiology, Center for Proteomics Research, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China

    Jumei Zeng, Yuqing Li, Shuguang Zhang & Zheng-Guo He

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  1. Jumei Zeng
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  2. Yuqing Li
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  3. Shuguang Zhang
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  4. Zheng-Guo He
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Correspondence to Zheng-Guo He.

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Fig. S1

The effect of regulators on the dnaA gene expression was assayed by constructing a series of lacZ alone or promoter–lacZ co-expression plasmids. The activity of β-galactosidase was further examined and presented as Miller units. Left column: schematic representation of each clone used to generate strains. Null promoter-lacZ, hsp60-lacZ were used as controls. Middle column: exponentially growing cultures of corresponding strains were scribed onto 7H10 plates containing 30-mg/ml kan and 50-mg/ml X-gal. The plates were incubated subsequently for 37 °C overnight. Right column: β-galactosidase activity was expressed as Miller units. The values presented were the averages of three independent experiments. For statistical analysis, two-way analysis of variance with Bonferroni multiple comparison tests were performed using a P value of ≤0.05. a The effect of KmtR on the dnaA gene expression was assayed by β-galactosidase activity assays. b The effect of SmtB on the dnaA gene expression was assayed by β-galactosidase activity assays. c The effect of IdeR on the dnaA gene expression was assayed by β-galactosidase activity assays. d The effect of Rv3295 on the dnaA gene expression was assayed by β-galactosidase activity assays (JPEG 55 kb)

High resolution image (TIFF 637 kb)

Fig S2

The effect of Mce2R on the gene expression was assayed by constructing a series of lacZ alone or promoter–lacZ co-expression plasmids. The activity of β-galactosidase was further examined and presented as Miller units. Left column: schematic representation of each clone used to generate strains. Null promoter–lacZ, hsp60-lacZ were used as controls. Middle column: exponentially growing cultures of corresponding strains were scribed onto 7H10 plates containing 30-mg/ml kan and 50-mg/ml X-gal. The plates were incubated subsequently for 37 °C overnight. Right column: β-galactosidase activity was expressed as Miller units. The values presented were the averages of three independent experiments. For statistical analysis, two-way analysis of variance with Bonferroni multiple comparison tests were performed using a P value of ≤0.05. a The effect of Mce2R on Rv1375 gene expression was assayed by β-galactosidase activity assays. b The effect of Mce2R on Rv2011c gene expression was assayed by β-galactosidase activity assays. c The effect of Mce2R on Rv3161c gene expression was assayed by β-galactosidase activity assays. d The effect of Mce2R on lsr2 gene expression was assayed by β-galactosidase activity assays (JPEG 59 kb)

High resolution image (TIFF 699 kb)

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Zeng, J., Li, Y., Zhang, S. et al. A novel high-throughput B1H-ChIP method for efficiently validating and screening specific regulator–target promoter interactions. Appl Microbiol Biotechnol 93, 1257–1269 (2012). https://doi.org/10.1007/s00253-011-3748-7

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