Integrated Regulatory Map of the Genome of Mycobacterium Tuberculosis
In order to gain insights into growth-phase dependent gene expression control in Mycobacterium tuberculosis (M. tb), we developed two complementary single-nucleotide resolution approaches, strand-specific RNA-seq and ChIP-seq, and studied the global transcriptome and the genome-wide dynamics of RNA polymerase (RNAP) and NusA in exponential and stationary phase cultures. NusA is an essential regulator of bacterial transcriptional elongation, pausing, termination and anti-termination. ChIP-seq data revealed that RNAP and NusA were bound ubiquitously across the genome and that the NusA profile mirrored RNAP distribution in both phases of growth. Interestingly, the differential binding of RNAP and NusA in the two conditions correlated with transcriptional activity as demonstrated by RNA-seq studies. The unprecedented wealth of information obtained by integrating the two approaches was exploited for the identification of transcriptional units and for mapping putative promoters. In addition, the high-resolution and accuracy offered by RNA-seq combined with ChIP-seq allowed us to refine the existing annotation of the M. tb genome by identifying anti-sense and intergenic transcripts corresponding to previously unknown features.
In addition to studying components of the M. tb transcriptional machinery, we are studying transcriptional regulators in M. tb such as EspR, PhoP, OxyS, etc using ChIP-seq technology in order to identify potential regulatory-protein binding sites and regulons. We are also involved in sequencing whole genomes of M. tb strains and drug-resistant mutants of M. tb and M. smegmatis to characterize new M. tb strains identify sequence polymorphisms that may contribute to resistance to different drugs.
Keywords: Bacterial Whole-genome Sequencing, ChIP-seq, RNA-seq, Tuberculosisback