Transcription facilitated genome-wide recruitment of topoisomerase I and DNA gyrase

Abstract

Movement of the transcription machinery along a template alters DNA topology resulting in the accumulation of supercoils in DNA. The positive supercoils generated ahead of transcribing RNA polymerase (RNAP) and the negative supercoils accumulating behind impose severe topological constraints impeding transcription process. Previous studies have implied the role of topoisomerases in the removal of torsional stress and the maintenance of template topology but the in vivo interaction of functionally distinct topoisomerases with heterogeneous chromosomal territories is not deciphered. Moreover, how the transcription-induced supercoils influence the genome-wide recruitment of DNA topoisomerases remains to be explored in bacteria. Using ChIP-Seq, we show the genome-wide occupancy profile of both topoisomerase I and DNA gyrase in conjunction with RNAP in Mycobacterium tuberculosis taking advantage of minimal topoisomerase representation in the organism. The study unveils the first in vivo genome-wide interaction of both the topoisomerases with the genomic regions and establishes that transcription-induced supercoils govern their recruitment at genomic sites. Distribution profiles revealed co-localization of RNAP and the two topoisomerases on the active transcriptional units (TUs). At a given locus, topoisomerase I and DNA gyrase were localized behind and ahead of RNAP, respectively, correlating with the twin-supercoiled domains generated. The recruitment of topoisomerases was higher at the genomic loci with higher transcriptional activity and/or at regions under high torsional stress compared to silent genomic loci. Importantly, the occupancy of DNA gyrase, sole type II topoisomerase in Mtb, near the Ter domain of the Mtb chromosome validates its function as a decatenase.

Fig 1. Topoisomerases occupancy on Mtb genome.

(A) Twin supercoiled domain model. The movement of RNA polymerase machine on the genome/TUs generates wave of negative supercoils upstream and positive supercoils downstream to the transcription machine. The supercoils have to be removed by the action of topoisomerases allowing the unobstructed translocation of RNAP. The in vivo interaction of topoisomerases with the twin supercoiled domains is yet to be demonstrated. (B) ChIP-Seq analysis of RNA polymerase (RNAPCS), DNA gyrase (GyrCS) and Topoisomerase I (Topo ICS) occupancy on Mtb genome. UCSC genome browser view of Topo I, RNAP and DNA gyrase occupancy across the Mtb genome (0.2 Mb-4.1 Mb representative region of Mtb genome). The peak height correspond to the signal intensity (IP/Mock ratio of RNAP, Gyrase and Topo I IPs) of protein binding on the particular site.

Fig 2. Topo I, RNAP and DNA gyrase co-localize on TUs.

(A) TopoI, gyrase and RNAP association. Enrichment ratio (ER) for Topo I, Gyrase and RNAP was calculated on protein coding genes as described earlier [22] and in Materials and Methods. RNAP and topoisomerase enriched genes (ER>2) were monitored for the enrichment of Topo I/DNA gyrase and RNAP respectively. The number of genes enriched with both RNAP and topoisomerases are represented at the intesection of venn diagram (B) Distribution profile of Topo I, RNAP and DNA gyrase across the TUs. Read counts from transcriptionally active (RPM>1) protein-coding TUs >1 kb in length (N = 541) were taken with reference to the TSS and mean read counts were calculated in 50 bp sized bins. Data were normalized to the input samples followed by removal of background using the read counts associated with the genes with no expression (RPM<1). Data were normalized with the maxima to generate the pattern of distribution on TUs.

Fig 3. Topoisomerase occupancy varies on genes with different expression levels.

The genes were segregated based on the available RNA-Seq data [22] under highly expressed (HE) and lowly expressed (LE) category (N = 342). The HE and LE class of gene co-ordinates were analyzed for the occupancy of topoisomerases. TSS (0) was taken as a reference point and mean read count was plotted around it to generate occupancy profile. Mean read counts corresponding to gene co-ordinates were plotted at a single nucleotide resolution.

Fig 4. Transcription induction recruits topoisomerases to TU.

Determination of the occupancy of RNAP, Topo I and DNA gyrase on activated TU. (A) Schematic of the experimental set up. Induction of transcription introduces supercoils on the template which recruits topoisomerases. Construct pJam-Rv3852 was electroporated into M. smegmatis and the transformants were grown up to the exponential phase. Cultures were induced with acetamide for 6 h to activate the transcription of rv3852 cloned under the acetamidase promoter. Arrows (red) indicate the position of primers for the amplification of target region (B) Induced (I) and uninduced (UI) cultures were processed for ChIP and enrichment of RNAP, Topo I and DNA gyrase was monitored by the qPCR using rv3852 specific primers. Unrelated IgG antibody was used as a negative control. (C) Depiction of the architecture of TU and the positions of the primers used for monitoring the gyrase binding on promoter region (P) and gene body of Rv1303 and rrS TUs. Mtb cells were treated with Moxifloxacin (Moxi) and gyrase-DNA clevage complexes around promoter and gene body were detected by qPCR. (D) Effect of transcription inhibition on topoisomerase activity. Mtb cells were treated with or without Rifampicin (Rif) followed by treatment with Moxifloxacin to induce gyrase-DNA cleavage complex formation. Promoter regions of Rv1303 and rrS were monitored for the formation of gyrase-DNA cleavage complex in the presence and absence of Rif. Error bars represent the SD obtained from three independent experiments. Significance of the observations was assessed by applying unpaired t-test (* = P<0.05; *** = P<0.001; **< P<0.01; ns = not significant).

Fig 5. Transcription induced supercoils recruit topoisomerases.

(A) and (B) Occupancy of topoisomerases on convergent and divergent genes respectively. The convergent (N = 68) and divergent (N = 128) gene pairs were extracted (from NCBI) and the intergenic region between the gene pairs was divided into 50 equal sized bins and the number of reads in every bin were averaged and mean values were plotted to monitor the occupancy of Topo I and DNA gyrase. Significance of the observations was tested by applying unpaired t-test (*** = P<0.0001) (C) The occupancy of topoisomerases downstream to the transcription termination sites (TTS) of HE and LE genes (N = 342). Based on the RNA-Seq profile, genes with Low (LE) and High expression (HE) were segregated and their predicted TTS was used as a reference point for the analysis. The mean read counts were calculated around the TTS (-200 bp to +200 bp) and plotted at a single nucleotide resoltion to generate the occupancy profile of DNA gyrase and Topo I.

Fig 6. Genome-wide distribution profile of Topo I, DNA gyrase, RNA polymerase and transcript abundance.

Mtb genome was divided into 9 bins (~ 500 kb) and mean read counts in each bin were calculated. The data were normalized to the input and plotted. The plots represent the linear genome expanding from left ori (O) to the right ori. (A) Relative localization of ori and ter (T) region on circular and linear genome. Occupancy profile of (B) Topo I (C) DNA gyrase (D) Transcript abundance across the genome of Mtb [22] (E) Overlay of Topo I and DNA gyrase (F) Occupancy profile of E. coli DNA gyrase derived from the ChIP-ChIP data [17] (G) Depiction of putative Mtb dif site sequence derived from the gyrase peak at Ter domain.

Fig 7. DNA topoisomerases interact with the supercoiled domain on transcriptionally active TUs.

Advancement of RNA polymerase on DNA template generates waves of positive supercoils ahead and negative supercoils behind (Twin-supercoiled domain). Topo I is recruited to sites where negative supercoils are prominent while DNA gyrase occupies the region with positive supercoils. Together these enzymes maintain the transcription template topology for optimal transcription.