Tutorial: Discovering the DosR regulon using TBDB

Motivation

Recreate the identification of the ~50 genes in the DosR (also called DevR) regulon. Do so by clustering expression data in which DosR is upregulated. See what other genes cluster with it. Recreate the main result of Voskuil et al.'s 2003 paper in just five minutes.

1. Search for DosR from the TBDB main page using the Quick Search box.

2. Click on 'Samples and Conditions' in Expression Data section of Quick Search results table.

3. This leads to Gene Expression Samples and Conditions histogram. This histogram by default shows significance value/absolute value (z-score) of DosR on the x-axis and the number of samples within a gene expression significance value range on the y-axis.

4. Select samples above certain gene expression significance value by moving the expandable sliding bar. Select samples above significance value of 3 as shown below.

Selected samples will appear in a table on the right side of histogram. Notice that hypoxia, Non-replicating persistence and Diethylenetriamine/nitric oxide adduct (a nitric oxide donor) are well represented among the conditions under which DosR showed significance gene expression.

5. Click on the cluster icon above the table to generate and view a cluster of selected samples using default values.

6. Visualize the clustered data. On the cluster radar image there should be a large red cluster of upregulated genes. Click on that area to select and zoom. This cluster contains DosR (Rv3133c) and HspX (alpha crystallin, Rvc2031c).

7. This cluster consists of about ~50 genes and contains many DosR regulon members. The cluster more or less completely recapitulates the 48 genes found in Schoolnik's paper (Voskuil et al., 2003). The cluster also includes data from other similar studies from different labs (Voskuil's, Sherman's), so they aren't simply a reclustering of the same data from the paper.

8. A similar result can be obtained by clustering the VISCONTI datasets (which looked at hypoxic response). So, different data, same result.

9. Look at DosR (Rv3133c) in the operon browser. Notice that Rv3126c through Rv3134c (also present in the cluster) are also clearly visualized as a regulon.

10. Other parts of the DosR regulon are easy to visualize in the browser, for example Rv2623-Rv2631. Also Rv2028c-Rv2032, which includes HspX.

11. It should be noted that the expression correlations in the Operon Browser came from many experiments from different labs, testing hypotheses unrelated to hypoxia and persistence, showing the general use of the tool for non-hypothesis-driven queries.

12. Look at the gene family associated with the DosR gene by clicking on Gene Family link on Gene Detail page. Notice how M. smegmatis, and the MCS and KMS strains cluster outside the M. tuberculosis and M. bovis strains. Interestingly M. ulcerans clusters more closely.

References

Voskuil, M. I., Schnappinger, D., Visconti, K. C., Harrell, M. I., Dolganov, G. M., Sherman, D. R., et al. (2003). Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J Exp Med, 198(5), 705-713.

Voskuil, M.I., Visconti, K.C. and Schoolnik, G.K. Mycobacterium tuberculosis gene expression during adaptation to stationary phase and low-oxygen dormancy. Tuberculosis, 84(3-4):218-27.