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Reconstruction of the core and extended regulons of global transcription factors.

Dufour YS, Kiley PJ, Donohue TJ - PLoS Genet. (2010)

Bottom Line: Our results show that this approach correctly predicted many regulon members, provided new insights into the biological functions of the respective regulons for these regulators, and suggested models for the evolution of the corresponding transcriptional networks.In addition, the members of the so-called extended regulons for the FNR-type regulators vary even among closely related species, possibly reflecting species-specific adaptation to environmental and other factors.The comparative genomics approach we developed is readily applicable to other regulatory networks.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

ABSTRACT
The processes underlying the evolution of regulatory networks are unclear. To address this question, we used a comparative genomics approach that takes advantage of the large number of sequenced bacterial genomes to predict conserved and variable members of transcriptional regulatory networks across phylogenetically related organisms. Specifically, we developed a computational method to predict the conserved regulons of transcription factors across alpha-proteobacteria. We focused on the CRP/FNR super-family of transcription factors because it contains several well-characterized members, such as FNR, FixK, and DNR. While FNR, FixK, and DNR are each proposed to regulate different aspects of anaerobic metabolism, they are predicted to recognize very similar DNA target sequences, and they occur in various combinations among individual alpha-proteobacterial species. In this study, the composition of the respective FNR, FixK, or DNR conserved regulons across 87 alpha-proteobacterial species was predicted by comparing the phylogenetic profiles of the regulators with the profiles of putative target genes. The utility of our predictions was evaluated by experimentally characterizing the FnrL regulon (a FNR-type regulator) in the alpha-proteobacterium Rhodobacter sphaeroides. Our results show that this approach correctly predicted many regulon members, provided new insights into the biological functions of the respective regulons for these regulators, and suggested models for the evolution of the corresponding transcriptional networks. Our findings also predict that, at least for the FNR-type regulators, there is a core set of target genes conserved across many species. In addition, the members of the so-called extended regulons for the FNR-type regulators vary even among closely related species, possibly reflecting species-specific adaptation to environmental and other factors. The comparative genomics approach we developed is readily applicable to other regulatory networks.

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Identification of FnrL binding sites in the R. sphaeroides genome by ChIP–chip assays.A representative region of the R. sphaeroides genome showing profiles resulting from the enrichment of DNA fragments by immuno-precipitation of the β′ subunit (blue) or σ70 (red) subunit of RNA polymerase or FnrL (green) is plotted along the indicated genomic coordinates. The data plot the log2 of the ratio of the immunoprecipitated sample to the control sample as a function of probe location along the genome (coordinates are indicated in base pairs). DNA regions significantly enriched (p-value ≤0.01) by FnrL immuno-precipitation (green boxes), positions of sequences matching the FnrL consensus binding site (green tick mark) and the coordinates of annotated genes (black boxes). The data were plotted using SignalMap 1.9 (NimbleGen Systems).
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pgen-1001027-g005: Identification of FnrL binding sites in the R. sphaeroides genome by ChIP–chip assays.A representative region of the R. sphaeroides genome showing profiles resulting from the enrichment of DNA fragments by immuno-precipitation of the β′ subunit (blue) or σ70 (red) subunit of RNA polymerase or FnrL (green) is plotted along the indicated genomic coordinates. The data plot the log2 of the ratio of the immunoprecipitated sample to the control sample as a function of probe location along the genome (coordinates are indicated in base pairs). DNA regions significantly enriched (p-value ≤0.01) by FnrL immuno-precipitation (green boxes), positions of sequences matching the FnrL consensus binding site (green tick mark) and the coordinates of annotated genes (black boxes). The data were plotted using SignalMap 1.9 (NimbleGen Systems).

Mentions: To evaluate our predictions, we directly identified members of the R. sphaeroides FnrL regulon using chromatin immuno-precipitation on a chip (ChIP-chip) assays, DNA target sequence analysis, and expression profile clustering. FnrL is a member of the FNR sub-family and contains an O2-labile [4Fe-4S] cluster (T. Patschkowski and PJ. Kiley, unpublished data), like its homolog FNR in E. coli [4]. To probe genome-wide interactions of FnrL with DNA in vivo, we used antibodies to FnrL for ChIP-chip assays [12]. FnrL activity is high in the absence of O2 [28], so we analyzed these interactions in wild-type R. sphaeroides growing under anaerobic conditions in the presence of light (photosynthetic growth conditions). By identifying regions of the genome that were significantly enriched by immuno-precipitation with antibodies against FnrL (p-value ≤0.01) in three biological replicates, we found 27 sites bound by FnrL (Table 2, Figure 5). Of these 27 sites, 6 were in the promoter regions of genes previously shown to require FnrL for increased activity in the absence of O2 [28]–[32], illustrating that this assay identifies bona fide FnrL binding sites.


Reconstruction of the core and extended regulons of global transcription factors.

Dufour YS, Kiley PJ, Donohue TJ - PLoS Genet. (2010)

Identification of FnrL binding sites in the R. sphaeroides genome by ChIP–chip assays.A representative region of the R. sphaeroides genome showing profiles resulting from the enrichment of DNA fragments by immuno-precipitation of the β′ subunit (blue) or σ70 (red) subunit of RNA polymerase or FnrL (green) is plotted along the indicated genomic coordinates. The data plot the log2 of the ratio of the immunoprecipitated sample to the control sample as a function of probe location along the genome (coordinates are indicated in base pairs). DNA regions significantly enriched (p-value ≤0.01) by FnrL immuno-precipitation (green boxes), positions of sequences matching the FnrL consensus binding site (green tick mark) and the coordinates of annotated genes (black boxes). The data were plotted using SignalMap 1.9 (NimbleGen Systems).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908626&req=5

pgen-1001027-g005: Identification of FnrL binding sites in the R. sphaeroides genome by ChIP–chip assays.A representative region of the R. sphaeroides genome showing profiles resulting from the enrichment of DNA fragments by immuno-precipitation of the β′ subunit (blue) or σ70 (red) subunit of RNA polymerase or FnrL (green) is plotted along the indicated genomic coordinates. The data plot the log2 of the ratio of the immunoprecipitated sample to the control sample as a function of probe location along the genome (coordinates are indicated in base pairs). DNA regions significantly enriched (p-value ≤0.01) by FnrL immuno-precipitation (green boxes), positions of sequences matching the FnrL consensus binding site (green tick mark) and the coordinates of annotated genes (black boxes). The data were plotted using SignalMap 1.9 (NimbleGen Systems).
Mentions: To evaluate our predictions, we directly identified members of the R. sphaeroides FnrL regulon using chromatin immuno-precipitation on a chip (ChIP-chip) assays, DNA target sequence analysis, and expression profile clustering. FnrL is a member of the FNR sub-family and contains an O2-labile [4Fe-4S] cluster (T. Patschkowski and PJ. Kiley, unpublished data), like its homolog FNR in E. coli [4]. To probe genome-wide interactions of FnrL with DNA in vivo, we used antibodies to FnrL for ChIP-chip assays [12]. FnrL activity is high in the absence of O2 [28], so we analyzed these interactions in wild-type R. sphaeroides growing under anaerobic conditions in the presence of light (photosynthetic growth conditions). By identifying regions of the genome that were significantly enriched by immuno-precipitation with antibodies against FnrL (p-value ≤0.01) in three biological replicates, we found 27 sites bound by FnrL (Table 2, Figure 5). Of these 27 sites, 6 were in the promoter regions of genes previously shown to require FnrL for increased activity in the absence of O2 [28]–[32], illustrating that this assay identifies bona fide FnrL binding sites.

Bottom Line: Our results show that this approach correctly predicted many regulon members, provided new insights into the biological functions of the respective regulons for these regulators, and suggested models for the evolution of the corresponding transcriptional networks.In addition, the members of the so-called extended regulons for the FNR-type regulators vary even among closely related species, possibly reflecting species-specific adaptation to environmental and other factors.The comparative genomics approach we developed is readily applicable to other regulatory networks.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

ABSTRACT
The processes underlying the evolution of regulatory networks are unclear. To address this question, we used a comparative genomics approach that takes advantage of the large number of sequenced bacterial genomes to predict conserved and variable members of transcriptional regulatory networks across phylogenetically related organisms. Specifically, we developed a computational method to predict the conserved regulons of transcription factors across alpha-proteobacteria. We focused on the CRP/FNR super-family of transcription factors because it contains several well-characterized members, such as FNR, FixK, and DNR. While FNR, FixK, and DNR are each proposed to regulate different aspects of anaerobic metabolism, they are predicted to recognize very similar DNA target sequences, and they occur in various combinations among individual alpha-proteobacterial species. In this study, the composition of the respective FNR, FixK, or DNR conserved regulons across 87 alpha-proteobacterial species was predicted by comparing the phylogenetic profiles of the regulators with the profiles of putative target genes. The utility of our predictions was evaluated by experimentally characterizing the FnrL regulon (a FNR-type regulator) in the alpha-proteobacterium Rhodobacter sphaeroides. Our results show that this approach correctly predicted many regulon members, provided new insights into the biological functions of the respective regulons for these regulators, and suggested models for the evolution of the corresponding transcriptional networks. Our findings also predict that, at least for the FNR-type regulators, there is a core set of target genes conserved across many species. In addition, the members of the so-called extended regulons for the FNR-type regulators vary even among closely related species, possibly reflecting species-specific adaptation to environmental and other factors. The comparative genomics approach we developed is readily applicable to other regulatory networks.

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