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. 2016 Mar 18;44(5):2058-74.
doi: 10.1093/nar/gkw051. Epub 2016 Feb 3.

Transcription profile of Escherichia coli: genomic SELEX search for regulatory targets of transcription factors

Akira Ishihama  1 , Tomohiro Shimada  2 , Yukiko Yamazaki  3
Affiliations

Transcription profile of Escherichia coli: genomic SELEX search for regulatory targets of transcription factors

Akira Ishihama et al. Nucleic Acids Res. .

Abstract

Bacterial genomes are transcribed by DNA-dependent RNA polymerase (RNAP), which achieves gene selectivity through interaction with sigma factors that recognize promoters, and transcription factors (TFs) that control the activity and specificity of RNAP holoenzyme. To understand the molecular mechanisms of transcriptional regulation, the identification of regulatory targets is needed for all these factors. We then performed genomic SELEX screenings of targets under the control of each sigma factor and each TF. Here we describe the assembly of 156 SELEX patterns of a total of 116 TFs performed in the presence and absence of effector ligands. The results reveal several novel concepts: (i) each TF regulates more targets than hitherto recognized; (ii) each promoter is regulated by more TFs than hitherto recognized; and (iii) the binding sites of some TFs are located within operons and even inside open reading frames. The binding sites of a set of global regulators, including cAMP receptor protein, LeuO and Lrp, overlap with those of the silencer H-NS, suggesting that certain global regulators play an anti-silencing role. To facilitate sharing of these accumulated SELEX datasets with the research community, we compiled a database, 'Transcription Profile of Escherichia coli' (www.shigen.nig.ac.jp/ecoli/tec/).

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Figures

Figure 1.
Figure 1.
Outline of genomic SELEX screening system. (A) Functional differentiation of RNAP. The core enzyme, with subunit structure α2ββ′ω, has RNA polymerization activity but cannot recognize promoters. After binding one of the sigma (σ) subunits, the holoenzyme acquires the ability to initiate transcription from constitutive promoters. The promoter selectivity of RNAP is further modulated through interactions with DNA-binding TFs that bind to their target DNA near promoters. Each DNA-bound TF interacts with a subunit of promoter-bound RNAP. Based on the contact subunit, TFs are classified into four groups (1,2). (B) Genomic SELEX system for identification of regulatory targets of TFs. A plasmid library of Escherichia coli genomic DNA was constructed using collections of genomic DNA fragments 200–300 bp in length. The mixture of genome DNA segments can be regenerated by PCR. All 285 E. coli TFs were expressed in His-tagged form by addition of a His6 sequence to either the N- or C-terminus, and then affinity purified. Mapping of TF-bound DNA segments on the E. coli genome was carried out by either SELEX-clos (cloning-sequencing) or SELEX-chip (DNA tiling array analysis) method. (C) The binding sites of TFs on the genome were classified into four types: type-A, spacer of bidirectional transcriptional units; type-B, spacer upstream of one transcription unit but downstream of another transcription unit; type-C, spacer downstream of both transcription units; and type-D, inside open reading frames. Regulatory targets of TFs were predicted based on the locations of their binding sites.
Figure 2.
Figure 2.
Single-target TFs. After in vitro SELEX screening of DNA-binding sequences of purified TFs, their regulatory targets were predicted based on their binding sites, as noted in Figure 1C. Among 156 SELEX patterns examined for 116 TFs, a small number of TFs regulated only one specific target operon. Some representative SELEX patterns are shown: (A) BetI: (B) UlaR; and (C) NanR. For each SELEX pattern, the upper panel shows the genome-wide distribution of TF-binding sites, whereas the lower panel shows the expanded local region of a TF-binding site [note that the expanded patterns were retrieved from TEC (Transcription Profile of Escherichia coli) database]. The Y-axis indicates the fluorescent intensity of SELEX fragment binding to each probe relative to that of library DNA. The Y-axis in each expanded image indicates the level of each peak relative to that of the highest peak.
Figure 3.
Figure 3.
Multi-target TFs. SELEX screening of TF-binding sequences revealed that a set of the bifunctional nucleoid proteins with both architectural roles in genome folding and regulatory role in transcription bind up to ∼1000 sites along the entire Escherichia coli genome. The number of binding peaks was: 1269 for Fis (A); 987 for H-NS (B); and 813 for IHF (C). High-level binding sites of these nucleoid proteins within type-A and type-B spacers are shown under green background. In the case of bidirectional transcription units within type-A spacers, the first genes on both sides are shown, whereas in the case of type-B spacers, the first genes in one transcriptional direction are shown. The Y-axis indicates the fluorescent intensity of SELEX fragment binding to each probe relative to that of library DNA.
Figure 4.
Figure 4.
Multi-TF promoters. After SELEX screening of regulatory targets for a number of TFs, multiple TFs were shown to bind the same promoters. (A) Promoter of the csgDEFG operon, which encodes CsgD, the master regulator of biofilm formation and the biofilm components CsgEFG. (B) Promoter of the flhDC operon, which encodes the master regulator of flagella formation. (C) Promoter of the gadE-mdtEF operon, which encodes the acid-stress response regulator and multi-drug efflux system proteins. (D) Promoter of the fimBE operon, which encodes the recombinase for fimbriae switching. TFs shown in blue indicate those hitherto listed in RegulonDB (29,30) and EcoCyc (31,32) whereas TFs shown in green were identified by this research team and published, but not listed in these databases: H-NS, RcdA and RcdB for the csgG promoter (,,–77); and CpxR for the flhD promoter (,,–79). TFs shown in orange are those identified by the SELEX screening (Ishihama, A., unpublished).
Figure 5.
Figure 5.
Biding of TFs inside operons. After SELEX screening of DNA-binding sequences for a total of116 TFs, some operons were found to carry the binding sites for specific TFs. Some representative operons are shown: (A) hyfA operon (13 581 bp); (B) sdhC operon (9872 bp); (C) wcaC operon (13 390 bp); and (D) nuoA operon (15 024 bp). Some TFs with clear binding peaks are shown, but there were several small peaks within all four operons. The binding level of the general silencer H-NS is also shown (blue line) even though the peak height is low. The fluorescent intensity of SELEX fragment binding to each probe relative to that of library DNA was determined, and is shown as the relative value to that of the highest peak.
Figure 6.
Figure 6.
Network involving the silencer H-NS and the anti-silencers Crp, LeuO and Lrp. Among the 987 binding sites of the nucleoid protein H-NS (see Figure 3B), 431 are located near promoter regions within type-A and type-B spacers, where H-NS acts as a general silencer of gene expression. These H-NS binding sites overlap with binding sites of global regulators: Crp (73 sites), LeuO (87 sites) and Lrp (97 sites). High-level overlap of binding sites between the silencer H-NS and the global regulators Crp, LeuO and Lrp indicates that these global regulators act as anti-silencers, and that each is involved in derepression of a large set of target operons (for details see Table 3).
Figure 7.
Figure 7.
TEC database. All SELEX patterns described herein are compiled in the TEC database. Using this database, various types of analysis are possible: (A) search for the list of regulatory targets under direct control of each TF. (B) The location of binding peaks of a test TF along the entire E. coli genome can be visualized at various scales, from the whole genome to restricted areas. (C) The relative location of binding sites of different TFs can be visualized on various scales. (D) Heat map of binding intensity of test TFs along the entire E. coli genome. (E) Search for the consensus sequence of TF binding, using the whole set of TF-binding sequences.
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