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ChIP-exo analysis reveals novel uncharacterized transcription factors involved in the diverse regulatory functions of Escherichia coli

ABSTRACT: ChIP-exo analysis reveals novel uncharacterized transcription factors involved in the diverse regulatory functions of Escherichia coli

PROVIDER: PRJNA670504 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12867600.fastq.gz	Fastqsanger.gz
	SRR12867601.fastq.gz	Fastqsanger.gz
	SRR12867602.fastq.gz	Fastqsanger.gz
	SRR12867603.fastq.gz	Fastqsanger.gz
	SRR12867604.fastq.gz	Fastqsanger.gz

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ChIP-exo analysis reveals novel uncharacterized transcription factors involved in the diverse regulatory functions of Escherichia coli

Project description:Bacteria regulate gene expression to adapt to changing environments through transcriptional regulatory networks (TRNs). Although extensively studied, TRNs are not fully characterized since the identity of all the transcriptional regulators that comprise a TRN are not known. Here, we experimentally evaluate 40 uncharacterized proteins in the model organism Escherichia coli K-12 MG1655, which were previously predicted to be transcription factors (TFs). First, we used myc-tagging and a multiplexed ChIP-exo assay to characterize genome-wide binding sites for these candidate TFs. We then compared those binding sites with those of RNA polymerase and sigma factors to increase the confidence in the measured DNA-binding events. Finally, we used these data to infer potential functional annotations for 13 candidate TFs. Our results revealed that these candidate TFs have a varied number of regulatory genes and are involved in E. coli’s primary cellular processes. Taken together, thes results: (1) expand the number of confirmed TFs from 249 to 276, close to the estimated total of 280 TFs in E. coli K-12 MG1655; (2) provide a roadmap for the functional characterization of the newly discovered TFs; and (3) enable the elucidation of the first comprehensive TRN in this model organism.

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Project description:Bacteria regulate gene expression to adapt to changing environments through transcriptional regulatory networks (TRNs). Although extensively studied, no TRN is fully characterized since the identity and activity of all the transcriptional regulators comprising a TRN are not known. Here, we experimentally evaluate 40 uncharacterized proteins in Escherichia coli K-12 MG1655, which were computationally predicted to be transcription factors (TFs). First, we used a multiplexed chromatin immunoprecipitation method combined with lambda exonuclease digestion (multiplexed ChIP-exo) assay to characterize binding sites for these candidate TFs; 34 of them were found to be DNA-binding proteins. We then compared the relative location between binding sites and RNA polymerase (RNAP). We found 48% (283/588) overlap between the TFs and RNAP. Finally, we used these data to infer potential functions for 10 of the 34 TFs with validated DNA binding sites and consensus binding motifs. Taken together, this study: (i) significantly expands the number of confirmed TFs to 276, close to the estimated total of about 280 TFs; (ii) provides putative functions for the newly discovered TFs and (iii) confirms the functions of four representative TFs through mutant phenotypes.

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Systematic discovery of uncharacterized transcription factors in Escherichia coli K-12 MG1655 (ChIP-exo data set)

Project description:Transcriptional regulation enables cells to respond to environmental changes. Yet, among the estimated 304 candidate transcription factors (TFs) in Escherichia coli K-12 MG1655, only 185 have been experimentally characterized. Here we developed an integrated workflow that contains the prediction of TFs using machine learning and comprehensive experimental validation using a suite of genome-wide experiments. Applying this workflow we: 1) computationally identified 16 candidate genes encoding uncharacterized TFs; 2) confirmed that ten of these 16 are TF candidates that showed 255 DNA binding sites; 3) found high-confidence TF-binding sequence motifs for six of the ten TFs; 4) reconstructed the regulons of the ten TFs by determining gene expression change upon deletion of each TF; and 5) further determined the regulatory roles of three TFs (YdcI, YeiE and YiaJ) to be regulating acetate metabolism, iron homeostasis under iron limited condition, and utilization of L-ascorbate, respectively. Together, these results demonstrate how the integrated workflow can be used to discover, characterize, and elucidate regulatory functions of uncharacterized TFs. This workflow can be applied to less studied bacteria for systematic discovery and characterization of their transcriptional regulatory networks.

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Project description:Bacteria regulate gene expression to adapt to changing environments through transcriptional regulatory networks (TRNs). Although extensively studied, TRNs are not fully characterized since we do not know the identity of all the transcriptional regulators that comprise a TRN. Here, we experimentally evaluate 40 uncharacterized proteins in the model organisms Escherichia coli K-12 MG1655, which were previously predicted to be transcription factors (TFs). First, we used myc-tagging and a multiplexed ChIP-exo assay to characterize genome-wide binding sites for the 40 candidate TFs. Then, we compared those binding sites with those of RNA polymerase and sigma factors to increase the confidence in the measured DNA-binding events. Finally, we used these data to infer potential functional annotation for 13 candidate TFs. As a result, we characterized 570 new binding sites for 34 of 40 candidate TFs. Taken together, the study; 1) expands the number of confirmed TFs from 249 to 278, that is close to the estimated total of about 280 TFs in E. coli K-12 MG1655, 2) it provides a roadmap for functional characterization of the 34 discovered TFs, and 3) it enables the elucidation of the first comprehensive TRN.

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