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The DNA-binding network of Mycobacterium tuberculosis

Authors:
Kyle Minch at Intellectual Ventures
Kyle Minch
Tige Rustad at Center for Infectious Disease Research
Tige Rustad
Eliza Peterson at Institute for Systems Biology
Eliza Peterson
Jessica K Winkler at Center for Infectious Disease Research
Jessica K Winkler
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Abstract and Figures

Mycobacterium tuberculosis (MTB) infects 30% of all humans and kills someone every 20-30 s. Here we report genome-wide binding for ~80% of all predicted MTB transcription factors (TFs), and assayed global expression following induction of each TF. The MTB DNA-binding network consists of ~16,000 binding events from 154 TFs. We identify >50 TF-DNA consensus motifs and >1,150 promoter-binding events directly associated with proximal gene regulation. An additional ~4,200 binding events are in promoter windows and represent strong candidates for direct transcriptional regulation under appropriate environmental conditions. However, we also identify >10,000 'dormant' DNA-binding events that cannot be linked directly with proximal transcriptional control, suggesting that widespread DNA binding may be a common feature that should be considered when developing global models of coordinated gene expression.
A global view of DNA binding. (a) TF-binding sites identified by ChIP-seq plotted with Circos59. Sense (blue) and antisense (orange) CDS and operon boundaries illustrated with black edges. The 4.4-Mb H37Rv chromosome is divided into nonoverlapping 50-bp windows, and green spikes represent the total number of TF-binding events within each window. (b) Histogram of number of TF-binding events per 50-bp window. (c) Number of ChIP-binding events (out-degree) for each of the 156 DNA-binding proteins with at least one binding site.
A global view of DNA binding. (a) TF-binding sites identified by ChIP-seq plotted with Circos59. Sense (blue) and antisense (orange) CDS and operon boundaries illustrated with black edges. The 4.4-Mb H37Rv chromosome is divided into nonoverlapping 50-bp windows, and green spikes represent the total number of TF-binding events within each window. (b) Histogram of number of TF-binding events per 50-bp window. (c) Number of ChIP-binding events (out-degree) for each of the 156 DNA-binding proteins with at least one binding site.
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Global view of DNA-binding and transcriptional regulation in MTB. (a) Plot of binding distribution in the 1-kb nucleotide window (−500 to +500) surrounding CDS or transcription start sites. ROC/AUC analysis indicated that the optimal promoter window size is −150 to +70 nucleotides (indicated by vertical dashed lines and shading of histogram). Binding events correlated with 1.5-fold induction or repression in the overexpression data set24 are depicted in red and green, respectively. (b) The relationship between the number of binding events detected versus the number of transcriptional changes associated with induction of each TF in this study. ​Rv0494 (orange triangle) is an example of a TF with prolific binding but limited differential gene expression. Additional genes discussed in text highlighted in yellow with symbols as follows: ​Rv1657/​ArgR (downward triangle), Rv1846v/​BlaI (square), ​Rv3133c/​DosR (octagon), and ​Rv3849/​EspR (diamond).
Global view of DNA-binding and transcriptional regulation in MTB. (a) Plot of binding distribution in the 1-kb nucleotide window (−500 to +500) surrounding CDS or transcription start sites. ROC/AUC analysis indicated that the optimal promoter window size is −150 to +70 nucleotides (indicated by vertical dashed lines and shading of histogram). Binding events correlated with 1.5-fold induction or repression in the overexpression data set24 are depicted in red and green, respectively. (b) The relationship between the number of binding events detected versus the number of transcriptional changes associated with induction of each TF in this study. ​Rv0494 (orange triangle) is an example of a TF with prolific binding but limited differential gene expression. Additional genes discussed in text highlighted in yellow with symbols as follows: ​Rv1657/​ArgR (downward triangle), Rv1846v/​BlaI (square), ​Rv3133c/​DosR (octagon), and ​Rv3849/​EspR (diamond).
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Rv0494 as an example of widespread binding and local gene regulation. ​Rv0494 binding and regulation of gene expression was plotted using Circos59, with sense (blue) and antisense (orange) CDS and operon boundaries illustrated with black edges. The ​Rv0494 gene locus is positioned at ~2 o’clock, and ChIP-binding sites are denoted at the terminus of each edge radiating out from ​Rv0494 (peaks of P<0.001 indicated by purple lines, 0.001<P<0.01 indicated by yellow lines). ​Rv0494 consensus motifs corresponding to peak significance thresholds are indicated by the colour-matched ribbons. Genes that exhibit significant differential regulation (more than twofold change with empirical Bayes method P<0.01 from five biological replicate microarrays) upon induction of ​Rv0494 are indicated by blue (gene repression) and red (gene induction) bars. The ​Rv0494 ChIP-binding site between regulated genes ​Rv3094c–​Rv3095 is shown connected by a grey ribbon.
Rv0494 as an example of widespread binding and local gene regulation. ​Rv0494 binding and regulation of gene expression was plotted using Circos59, with sense (blue) and antisense (orange) CDS and operon boundaries illustrated with black edges. The ​Rv0494 gene locus is positioned at ~2 o’clock, and ChIP-binding sites are denoted at the terminus of each edge radiating out from ​Rv0494 (peaks of P<0.001 indicated by purple lines, 0.001<P<0.01 indicated by yellow lines). ​Rv0494 consensus motifs corresponding to peak significance thresholds are indicated by the colour-matched ribbons. Genes that exhibit significant differential regulation (more than twofold change with empirical Bayes method P<0.01 from five biological replicate microarrays) upon induction of ​Rv0494 are indicated by blue (gene repression) and red (gene induction) bars. The ​Rv0494 ChIP-binding site between regulated genes ​Rv3094c–​Rv3095 is shown connected by a grey ribbon.
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Independent validation of ​Rv0494 binding. (a) Schematic of DNAs used in these experiments. Three DNAs are annealed to form a single dsDNA product: a specific query sequence (orange box) is annealed in a 3-piece dsDNA fragment to a unique 12-mer sequence covalently coupled to a reporter dye. In these experiments, the specific query DNA was labelled with IR680 (red) and specific or nonspecific competitor DNAs were labelled with IR800 (green). (b) Purified recombinant ​Rv0494 binds specifically to ChIP-identified wild-type sequence (left panel), the 17-mer consensus motif (middle panel) and the 9-mer consensus motif (right panel). In the absence of protein, dye-coupled DNA does not shift (lane 1); however, the protein–DNA complex runs at a higher molecular weight (lane 2). This protein–DNA complex persists in the face of 20 × molar excess green-labelled nonspecific competitor DNA (lane 3), but can be outcompeted by the addition of 20 × molar excess green-labelled specific competitor DNA (lane 4).
Independent validation of ​Rv0494 binding. (a) Schematic of DNAs used in these experiments. Three DNAs are annealed to form a single dsDNA product: a specific query sequence (orange box) is annealed in a 3-piece dsDNA fragment to a unique 12-mer sequence covalently coupled to a reporter dye. In these experiments, the specific query DNA was labelled with IR680 (red) and specific or nonspecific competitor DNAs were labelled with IR800 (green). (b) Purified recombinant ​Rv0494 binds specifically to ChIP-identified wild-type sequence (left panel), the 17-mer consensus motif (middle panel) and the 9-mer consensus motif (right panel). In the absence of protein, dye-coupled DNA does not shift (lane 1); however, the protein–DNA complex runs at a higher molecular weight (lane 2). This protein–DNA complex persists in the face of 20 × molar excess green-labelled nonspecific competitor DNA (lane 3), but can be outcompeted by the addition of 20 × molar excess green-labelled specific competitor DNA (lane 4).
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The DNA-binding network of Mycobacterium tuberculos
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Control site location and transcriptional regulation in Escherichia coli
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  • Sep 1991
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The regulatory regions for 119 Escherichia coli promoters have been analyzed, and the locations of the regulatory sites have been cataloged. The following observations emerge. (i) More than 95% of promoters are coregulated with at least one other promoter. (ii) Virtually all sigma 70 promoters contain at least one regulatory site in a proximal position, touching at least position -65 with respect to the start point of transcription. There are not yet clear examples of upstream regulation in the absence of a proximal site. (iii) Operators within regulons appear in very variable proximal positions. By contrast, the proximal activation sites of regulons are much more fixed. (iv) There is a forbidden zone for activation elements downstream from approximately position -20 with respect to the start of transcription. By contrast, operators can occur throughout the proximal region. When activation elements appear in the forbidden zone, they repress. These latter examples usually involve autoregulation. (v) Approximately 40% of repressible promoters contain operator duplications. These occur either in certain regulons where duplication appears to be a requirement for repressor action or in promoters subject to complex regulation. (vi) Remote operator duplications occur in approximately 10% of repressible promoters. They generally appear when a multiple promoter region is coregulated by cyclic AMP receptor protein. (vii) Sigma 54 promoters do not require proximal or precisely positioned activator elements and are not generally subject to negative regulation. Rationales are presented for all of the above observations.
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