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Genomic mapping of cAMP receptor protein (CRP Mt) in Mycobacterium tuberculosis: relation to transcriptional start sites and the role of CRPMt as a transcription factor.

Kahramanoglou C, Cortes T, Matange N, Hunt DM, Visweswariah SS, Young DB, Buxton RS - Nucleic Acids Res. (2014)

Bottom Line: CRP(Mt) binding overlapped a palindromic consensus sequence.Analysis by RNA sequencing revealed widespread changes in transcriptional profile in a mutant strain lacking CRP(Mt) during exponential growth, and in response to nutrient starvation.The subset of genes that are differentially expressed in the deletion mutant under both culture conditions conformed to a pattern resembling canonical CRP regulation in Escherichia coli, with binding close to the transcriptional start site associated with repression and upstream binding with activation.

View Article: PubMed Central - PubMed

Affiliation: Division of Mycobacterial Research, MRC National Institute for Medical Research, Mill Hill, London, NW7 1AA, UK.

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ChIP-seq mapping of CRPMt-binding sites. (A) Sequence reads from ChIP are mapped onto the M. tuberculosis H37Rv genome and displayed using the Artemis browser. No peak enrichment was observed in a mock-IP sample (red trace). One hundred and ninety one peaks were identified in the IP sample using anti-CRPMt antibody (black trace). (B) A consensus motif resembling that defined for E. coli CRP was identified within 50 bp of the bound centre for 97% of the 191 of ChIP-seq peaks. (C) A clear correlation was observed between peak enrichment and match to the consensus motif. (D) Representative Artemis profiles of CBSs. CRPMt binding (i) to the divergent promoter region between Rv0078A and Rv0079; (ii) in the intragenic region within Rv1592c; and (iii) overlapping the sRNA ncRv13943. Red boxes denote the CBS and blue boxes denote the TSS.
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Figure 1: ChIP-seq mapping of CRPMt-binding sites. (A) Sequence reads from ChIP are mapped onto the M. tuberculosis H37Rv genome and displayed using the Artemis browser. No peak enrichment was observed in a mock-IP sample (red trace). One hundred and ninety one peaks were identified in the IP sample using anti-CRPMt antibody (black trace). (B) A consensus motif resembling that defined for E. coli CRP was identified within 50 bp of the bound centre for 97% of the 191 of ChIP-seq peaks. (C) A clear correlation was observed between peak enrichment and match to the consensus motif. (D) Representative Artemis profiles of CBSs. CRPMt binding (i) to the divergent promoter region between Rv0078A and Rv0079; (ii) in the intragenic region within Rv1592c; and (iii) overlapping the sRNA ncRv13943. Red boxes denote the CBS and blue boxes denote the TSS.

Mentions: We were able to map 98% of the sequences uniquely to the H37Rv genome (allowing for up to two mismatches per read) and achieved a near-complete representation of the entire genome (98% of the genome was mapped). The remaining 2% of the genome includes PE/PPE genes, which contain highly repetitive sequences that are poorly resolved by short-read sequencing. To visualize the genome coverage, the number of reads mapping to each position on the M. tuberculosis chromosome was calculated and the traces visualized in the Artemis genome browser. Peaks were called using the CisGenome software (33) to identify enriched regions in the CRPMt-IP compared to the mock-IP (performed in the absence of antibody) and input (sheared genomic DNA). To validate the results, the data were also analysed using the BayesPeak package in R/Bioconductor, and peaks called in only one of the two methods were discarded. As seen in Figure 1A, there was no significant enrichment of any regions of the M. tuberculosis chromosome in the mock-IP (or the input; data not shown) indicating negligible non-specific binding to the resin or the antibody. In the CRPMt-IP, however, 191 peaks were found, denoting CRPMt-binding sites (here abbreviated to CBSs) on the chromosome (Figure 1A). The average length of the CBSs was 276 bp, and in total this represents 0.7% of the entire M. tuberculosis genome. No differences were observed in CRPMt-DNA binding between cells grown in Dubos medium and cells grown in Middlebrook 7H9 medium (data not shown). No ChIP-seq signals were detected for the crp-deletion strain.


Genomic mapping of cAMP receptor protein (CRP Mt) in Mycobacterium tuberculosis: relation to transcriptional start sites and the role of CRPMt as a transcription factor.

Kahramanoglou C, Cortes T, Matange N, Hunt DM, Visweswariah SS, Young DB, Buxton RS - Nucleic Acids Res. (2014)

ChIP-seq mapping of CRPMt-binding sites. (A) Sequence reads from ChIP are mapped onto the M. tuberculosis H37Rv genome and displayed using the Artemis browser. No peak enrichment was observed in a mock-IP sample (red trace). One hundred and ninety one peaks were identified in the IP sample using anti-CRPMt antibody (black trace). (B) A consensus motif resembling that defined for E. coli CRP was identified within 50 bp of the bound centre for 97% of the 191 of ChIP-seq peaks. (C) A clear correlation was observed between peak enrichment and match to the consensus motif. (D) Representative Artemis profiles of CBSs. CRPMt binding (i) to the divergent promoter region between Rv0078A and Rv0079; (ii) in the intragenic region within Rv1592c; and (iii) overlapping the sRNA ncRv13943. Red boxes denote the CBS and blue boxes denote the TSS.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4117774&req=5

Figure 1: ChIP-seq mapping of CRPMt-binding sites. (A) Sequence reads from ChIP are mapped onto the M. tuberculosis H37Rv genome and displayed using the Artemis browser. No peak enrichment was observed in a mock-IP sample (red trace). One hundred and ninety one peaks were identified in the IP sample using anti-CRPMt antibody (black trace). (B) A consensus motif resembling that defined for E. coli CRP was identified within 50 bp of the bound centre for 97% of the 191 of ChIP-seq peaks. (C) A clear correlation was observed between peak enrichment and match to the consensus motif. (D) Representative Artemis profiles of CBSs. CRPMt binding (i) to the divergent promoter region between Rv0078A and Rv0079; (ii) in the intragenic region within Rv1592c; and (iii) overlapping the sRNA ncRv13943. Red boxes denote the CBS and blue boxes denote the TSS.
Mentions: We were able to map 98% of the sequences uniquely to the H37Rv genome (allowing for up to two mismatches per read) and achieved a near-complete representation of the entire genome (98% of the genome was mapped). The remaining 2% of the genome includes PE/PPE genes, which contain highly repetitive sequences that are poorly resolved by short-read sequencing. To visualize the genome coverage, the number of reads mapping to each position on the M. tuberculosis chromosome was calculated and the traces visualized in the Artemis genome browser. Peaks were called using the CisGenome software (33) to identify enriched regions in the CRPMt-IP compared to the mock-IP (performed in the absence of antibody) and input (sheared genomic DNA). To validate the results, the data were also analysed using the BayesPeak package in R/Bioconductor, and peaks called in only one of the two methods were discarded. As seen in Figure 1A, there was no significant enrichment of any regions of the M. tuberculosis chromosome in the mock-IP (or the input; data not shown) indicating negligible non-specific binding to the resin or the antibody. In the CRPMt-IP, however, 191 peaks were found, denoting CRPMt-binding sites (here abbreviated to CBSs) on the chromosome (Figure 1A). The average length of the CBSs was 276 bp, and in total this represents 0.7% of the entire M. tuberculosis genome. No differences were observed in CRPMt-DNA binding between cells grown in Dubos medium and cells grown in Middlebrook 7H9 medium (data not shown). No ChIP-seq signals were detected for the crp-deletion strain.

Bottom Line: CRP(Mt) binding overlapped a palindromic consensus sequence.Analysis by RNA sequencing revealed widespread changes in transcriptional profile in a mutant strain lacking CRP(Mt) during exponential growth, and in response to nutrient starvation.The subset of genes that are differentially expressed in the deletion mutant under both culture conditions conformed to a pattern resembling canonical CRP regulation in Escherichia coli, with binding close to the transcriptional start site associated with repression and upstream binding with activation.

View Article: PubMed Central - PubMed

Affiliation: Division of Mycobacterial Research, MRC National Institute for Medical Research, Mill Hill, London, NW7 1AA, UK.

Show MeSH
Related in: MedlinePlus