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Genome-Scale Mapping of Escherichia coli σ54 Reveals Widespread, Conserved Intragenic Binding.

Bonocora RP, Smith C, Lapierre P, Wade JT - PLoS Genet. (2015)

Bottom Line: Strikingly, the majority of σ54 binding sites are located inside genes.We conclude that many intragenic σ54 binding sites are likely to be functional.Consistent with this assertion, we identify three conserved, intragenic σ54 promoters that drive transcription of mRNAs with unusually long 5' UTRs.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.

ABSTRACT
Bacterial RNA polymerases must associate with a σ factor to bind promoter DNA and initiate transcription. There are two families of σ factor: the σ70 family and the σ54 family. Members of the σ54 family are distinct in their ability to bind promoter DNA sequences, in the context of RNA polymerase holoenzyme, in a transcriptionally inactive state. Here, we map the genome-wide association of Escherichia coli σ54, the archetypal member of the σ54 family. Thus, we vastly expand the list of known σ54 binding sites to 135. Moreover, we estimate that there are more than 250 σ54 sites in total. Strikingly, the majority of σ54 binding sites are located inside genes. The location and orientation of intragenic σ54 binding sites is non-random, and many intragenic σ54 binding sites are conserved. We conclude that many intragenic σ54 binding sites are likely to be functional. Consistent with this assertion, we identify three conserved, intragenic σ54 promoters that drive transcription of mRNAs with unusually long 5' UTRs.

No MeSH data available.


Related in: MedlinePlus

ChIP-seq enrichment represents genuine σ54 binding.(A) Targeted validation of σ54 binding sites. ChIP-qPCR measurement of σ54 binding at putative sites identified by ChIP-seq in wild-type (MG1655; black bars) and ΔrpoN (RPB146; white bars) E. coli strains. The cognate promoter IDs and the fold above threshold (FAT; see Methods) scores (Tables 1 & 2) are indicated in parentheses and below the gene name, respectively. Gene names for “outside sense” (OS) and “outside antisense” (OA) binding sites correspond to the first gene downstream of the binding site (downstream relative to the orientation of the binding site). Gene names for “inside sense” (IS) and “inside antisense” (IA) binding sites correspond to the gene that contains the binding site. Occupancy units represent background-subtracted enrichment of target regions relative to a control region within the transcriptionally silent gene bglB. Error bars represent the standard deviation from three independent biological replicates. Significant differences between wild-type and ΔrpoN values are indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). (B) Correlation of ChIP-qPCR and ChIP-seq data. Values obtained from ChIP-qPCR (occupancy units) and ChIP-seq (FAT) using the σ54 antibody in E. coli were compared.
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pgen.1005552.g002: ChIP-seq enrichment represents genuine σ54 binding.(A) Targeted validation of σ54 binding sites. ChIP-qPCR measurement of σ54 binding at putative sites identified by ChIP-seq in wild-type (MG1655; black bars) and ΔrpoN (RPB146; white bars) E. coli strains. The cognate promoter IDs and the fold above threshold (FAT; see Methods) scores (Tables 1 & 2) are indicated in parentheses and below the gene name, respectively. Gene names for “outside sense” (OS) and “outside antisense” (OA) binding sites correspond to the first gene downstream of the binding site (downstream relative to the orientation of the binding site). Gene names for “inside sense” (IS) and “inside antisense” (IA) binding sites correspond to the gene that contains the binding site. Occupancy units represent background-subtracted enrichment of target regions relative to a control region within the transcriptionally silent gene bglB. Error bars represent the standard deviation from three independent biological replicates. Significant differences between wild-type and ΔrpoN values are indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). (B) Correlation of ChIP-qPCR and ChIP-seq data. Values obtained from ChIP-qPCR (occupancy units) and ChIP-seq (FAT) using the σ54 antibody in E. coli were compared.

Mentions: We predicted that all σ54-transcribed promoters would be bound by RNAP:σ54 under all growth conditions in which σ54 is expressed. Therefore, we mapped the binding of σ54 using ChIP-seq for cells grown to mid-logarithmic phase in M9 minimal medium. Using a high stringency analysis, we identified 145 ChIP-seq peaks that correspond to putative sites of σ54 binding (Tables 1 and 2 and S1 Table). The identified peaks have a bimodal shape, typical of ChIP-seq data (Fig 1A) [21]. We used MEME [22] to identify enriched sequence motifs in the 150 bp regions surrounding each of the putative σ54 binding sites. A motif closely resembling the known σ54–24/-12 promoter elements [6] was identified in 135 of the regions (Fig 1B). This represents a highly significant enrichment for the motif within these sequences (MEME E-value 1.8e-213). Furthermore, the distribution of motif positions within the ChIP-based query sequences was non-random: motifs were far more likely to be located at the center of the query sequence than expected by chance (p = 1.1e-61; Fig 1C), as would be expected for genuine σ54 binding sites. ChIP-seq peaks without an associated motif are listed in S1 Table. We selected 13 putative σ54 binding sites for validation. All of these sites are associated with a motif identified by MEME. We used quantitative PCR to measure enrichment of these sites by ChIP (ChIP-qPCR) of σ54 in wild type cells or cells in which rpoN is deleted. In all cases, we detected robust enrichment in wild type cells that was significantly higher than that in ΔrpoN cells (Fig 2A). Moreover, ChIP-qPCR enrichment scores correlated well with enrichment measured by ChIP-seq (R2 = 0.71; Fig 2B). We also selected five of the ten regions for which we detected a ChIP-seq peak but no motif. We suspected that these were false positives that are commonly found in ChIP-seq datasets for regions that are highly transcribed [12,23,24,25]. We used ChIP-qPCR of σ54 in wild type cells and ΔrpoN cells. We observed no significant difference in enrichment levels between the wild type and ΔrpoN cells (S1 Fig), consistent with these sites being false positives. We conclude that nearly all of the binding sites identified by ChIP-seq, for which we also detected a motif, represent genuine sites of σ54 binding. For all further analyses, we required that the ChIP-enriched sequences include a σ54 promoter motif identified by MEME to be called as a genuine σ54 binding site (Tables 1 and 2). We refer to these 135 binding sites as “high stringency sites”. Note that we use the term “binding site” rather than “promoter” because we do not know which sites represent functional promoters, as opposed to σ54 binding sites that never lead to productive transcription.


Genome-Scale Mapping of Escherichia coli σ54 Reveals Widespread, Conserved Intragenic Binding.

Bonocora RP, Smith C, Lapierre P, Wade JT - PLoS Genet. (2015)

ChIP-seq enrichment represents genuine σ54 binding.(A) Targeted validation of σ54 binding sites. ChIP-qPCR measurement of σ54 binding at putative sites identified by ChIP-seq in wild-type (MG1655; black bars) and ΔrpoN (RPB146; white bars) E. coli strains. The cognate promoter IDs and the fold above threshold (FAT; see Methods) scores (Tables 1 & 2) are indicated in parentheses and below the gene name, respectively. Gene names for “outside sense” (OS) and “outside antisense” (OA) binding sites correspond to the first gene downstream of the binding site (downstream relative to the orientation of the binding site). Gene names for “inside sense” (IS) and “inside antisense” (IA) binding sites correspond to the gene that contains the binding site. Occupancy units represent background-subtracted enrichment of target regions relative to a control region within the transcriptionally silent gene bglB. Error bars represent the standard deviation from three independent biological replicates. Significant differences between wild-type and ΔrpoN values are indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). (B) Correlation of ChIP-qPCR and ChIP-seq data. Values obtained from ChIP-qPCR (occupancy units) and ChIP-seq (FAT) using the σ54 antibody in E. coli were compared.
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pgen.1005552.g002: ChIP-seq enrichment represents genuine σ54 binding.(A) Targeted validation of σ54 binding sites. ChIP-qPCR measurement of σ54 binding at putative sites identified by ChIP-seq in wild-type (MG1655; black bars) and ΔrpoN (RPB146; white bars) E. coli strains. The cognate promoter IDs and the fold above threshold (FAT; see Methods) scores (Tables 1 & 2) are indicated in parentheses and below the gene name, respectively. Gene names for “outside sense” (OS) and “outside antisense” (OA) binding sites correspond to the first gene downstream of the binding site (downstream relative to the orientation of the binding site). Gene names for “inside sense” (IS) and “inside antisense” (IA) binding sites correspond to the gene that contains the binding site. Occupancy units represent background-subtracted enrichment of target regions relative to a control region within the transcriptionally silent gene bglB. Error bars represent the standard deviation from three independent biological replicates. Significant differences between wild-type and ΔrpoN values are indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). (B) Correlation of ChIP-qPCR and ChIP-seq data. Values obtained from ChIP-qPCR (occupancy units) and ChIP-seq (FAT) using the σ54 antibody in E. coli were compared.
Mentions: We predicted that all σ54-transcribed promoters would be bound by RNAP:σ54 under all growth conditions in which σ54 is expressed. Therefore, we mapped the binding of σ54 using ChIP-seq for cells grown to mid-logarithmic phase in M9 minimal medium. Using a high stringency analysis, we identified 145 ChIP-seq peaks that correspond to putative sites of σ54 binding (Tables 1 and 2 and S1 Table). The identified peaks have a bimodal shape, typical of ChIP-seq data (Fig 1A) [21]. We used MEME [22] to identify enriched sequence motifs in the 150 bp regions surrounding each of the putative σ54 binding sites. A motif closely resembling the known σ54–24/-12 promoter elements [6] was identified in 135 of the regions (Fig 1B). This represents a highly significant enrichment for the motif within these sequences (MEME E-value 1.8e-213). Furthermore, the distribution of motif positions within the ChIP-based query sequences was non-random: motifs were far more likely to be located at the center of the query sequence than expected by chance (p = 1.1e-61; Fig 1C), as would be expected for genuine σ54 binding sites. ChIP-seq peaks without an associated motif are listed in S1 Table. We selected 13 putative σ54 binding sites for validation. All of these sites are associated with a motif identified by MEME. We used quantitative PCR to measure enrichment of these sites by ChIP (ChIP-qPCR) of σ54 in wild type cells or cells in which rpoN is deleted. In all cases, we detected robust enrichment in wild type cells that was significantly higher than that in ΔrpoN cells (Fig 2A). Moreover, ChIP-qPCR enrichment scores correlated well with enrichment measured by ChIP-seq (R2 = 0.71; Fig 2B). We also selected five of the ten regions for which we detected a ChIP-seq peak but no motif. We suspected that these were false positives that are commonly found in ChIP-seq datasets for regions that are highly transcribed [12,23,24,25]. We used ChIP-qPCR of σ54 in wild type cells and ΔrpoN cells. We observed no significant difference in enrichment levels between the wild type and ΔrpoN cells (S1 Fig), consistent with these sites being false positives. We conclude that nearly all of the binding sites identified by ChIP-seq, for which we also detected a motif, represent genuine sites of σ54 binding. For all further analyses, we required that the ChIP-enriched sequences include a σ54 promoter motif identified by MEME to be called as a genuine σ54 binding site (Tables 1 and 2). We refer to these 135 binding sites as “high stringency sites”. Note that we use the term “binding site” rather than “promoter” because we do not know which sites represent functional promoters, as opposed to σ54 binding sites that never lead to productive transcription.

Bottom Line: Strikingly, the majority of σ54 binding sites are located inside genes.We conclude that many intragenic σ54 binding sites are likely to be functional.Consistent with this assertion, we identify three conserved, intragenic σ54 promoters that drive transcription of mRNAs with unusually long 5' UTRs.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Center, New York State Department of Health, Albany, New York, United States of America.

ABSTRACT
Bacterial RNA polymerases must associate with a σ factor to bind promoter DNA and initiate transcription. There are two families of σ factor: the σ70 family and the σ54 family. Members of the σ54 family are distinct in their ability to bind promoter DNA sequences, in the context of RNA polymerase holoenzyme, in a transcriptionally inactive state. Here, we map the genome-wide association of Escherichia coli σ54, the archetypal member of the σ54 family. Thus, we vastly expand the list of known σ54 binding sites to 135. Moreover, we estimate that there are more than 250 σ54 sites in total. Strikingly, the majority of σ54 binding sites are located inside genes. The location and orientation of intragenic σ54 binding sites is non-random, and many intragenic σ54 binding sites are conserved. We conclude that many intragenic σ54 binding sites are likely to be functional. Consistent with this assertion, we identify three conserved, intragenic σ54 promoters that drive transcription of mRNAs with unusually long 5' UTRs.

No MeSH data available.


Related in: MedlinePlus