Help for the TB Regulatory Network
Binding sites for a TF are any location were the TF has been shown to bind via ChIP-Seq. Binding sites need to have a minimum coverage and pass several filters as described in the following publications. One important filter was the lsr2 filter described below.
- Galagan, J.E., Minch, K., Peterson, M., Lyubetskaya, A., Azizi, E., Sweet, L., Gomes, A., Rustad, T., Dolganov, G., Glotova, I., Abeel, T., Mahwinney, C., Kennedy, A.D., Allard, R., Brabant, W., Krueger, A., Jaini, S., Honda, B., Yu, W.H., Hickey, M.J., Zucker, J., Garay, C., Weiner, B., Sisk, P., Stolte, C., Winkler, J.K., Van de Peer, Y., Iazzetti, P., Camacho, D., Dreyfuss, J., Liu, Y., Dorhoi, A., Mollenkopf, H.J., Drogaris, P., Lamontagne, J., Zhou, Y., Piquenot, J., Park, S.T., Raman, S., Kaufmann, S.H., Mohney, R.P., Chelsky, D., Moody, D.B., Sherman, D.R., Schoolnik, G.K. (2013) The Mycobacterium tuberculosis regulatory network and hypoxia. Nature.499(7457):178-83
- Galagan, J., Lyubetskaya, A., Gomes, A. (2013) ChIP-Seq and the Complexity of Bacterial Transcriptional Regulation. Current topics in microbiology and immunology.363:43-68
- Jaini, S., Lyubetskaya, A., Gomes, A., Peterson, M., Park, S.T., Raman, S., Schoolnik, G., Galagan, J.E. Transcription Factor Binding Site Mapping Using ChIP-Seq. In: Hatfull G, Jacobs WR, Jr., editors. Molecular Genetics of Mycobacteria, 2nd Edition: ASM Press; In Press.
- Direct Interactions
- A binding site in an upstream intergenic region is predicted to regulate the downstream gene. One binding site can give rise to two interactions in this manner in the case of a divergent intergenic region (a region region with two genes pointing away from the region)
- A binding site upstream and within 500bp of the start codon of a gene is predicted to regulate the downstream gene.
- A binding site upstream and within 1000bp of the start codon of a gene is predicted to regulate the downstream gene.if induction of the TF results in a significant fold change in the expression of the gene (zscore>1)
- A binding site downstream and within 4000bp of the start codon of a gene is predicted to regulate the downstream gene.if induction of the TF results in a highly significant fold change in the expression of the gene (zscore>2)
- Indirect Interactions
- If a binding site is predicted to regulate a gene, it is also predicted to regulate any gene downstream in the same predicted operon
ChIP-Seq mapping of a large number of MTB TFs revealed certain genomic regions bound by the majority of TFs (often >20 TFs). Binding by other TFs in these regions most often occurred at almost precisely the same location as binding by the nucleoid associated protein and TF lsr2. Whereas binding by most TFs in these regions was weak, lsr2 binding was strong. Our analysis suggested that such binding was likely an artifact, perhaps driven by interactions with lsr2 or related to the AT rich regions bound by lsr2.
On the other hand, several known TF binding sites do overlap weak lsr2 binding. In these cases, the TF binding site was always highly enriched over the lsr2 binding site.
To remove these likely artifacts, TF binding sites that overlap lsr2 binding sites are filtered out unless the coverage for the TF binding site is at least 2-fold greater than the coverage for the lsr2 binding site.
Binding site distances are the distance from the position of maximum coverage for a binding site and the start codon of the gene. For a predicted interaction between a TF and target gene, the distances for all corresponding peaks for the TF are shown as a comma seperated list.
Coverage is the maximum read coverage for a binding site in a ChIP-Seq experiment normalized to the mean coverage across the genome for the experiment. Normalization by mean coverage facilitates comparison of peak heights between experiments with different numbers of sequencing reads.
Interaction fold change is the fold change in the expression of a target gene when the corresponding TF is induced. TFs were induced using an inducible promoter system and microarray expression analysis performed as described in Galagan (2013) Nature. Fold change can be positive or negative and is calculated relative to control experiments in which the TF was not artificially induced. For each target gene, the expression after TF induction was also compared to a distribution of expression for target gene over all TF induction experiments to calculate a zscore for the fold change.
Binding types are either genic or intergenic based on the location of maximum coverage for the binding site relative to predicted genes.
Interactions can have the following types:
- upstream: an interaction resulting from a binding site upstream of the target gene - the site can be either intergenic or genic
- in-gene: an interaction resulting from a binding site inside the target gene
- downstream: an interaction resulting from a binding site downstream of the target gene - the site can be either intergenic or genic