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The global regulator Ncb2 escapes from the core promoter and impacts transcription in response to drug stress in Candida albicans

View Article: PubMed Central - PubMed

ABSTRACT

Ncb2, the β subunit of NC2 complex, a heterodimeric regulator of transcription was earlier shown to be involved in the activated transcription of CDR1 gene in azole resistant isolate (AR) of Candida albicans. This study examines its genome-wide role by profiling Ncb2 occupancy between genetically matched pair of azole sensitive (AS) and AR clinical isolates. A comparison of Ncb2 recruitment between the two isolates displayed that 29 genes had higher promoter occupancy of Ncb2 in the AR isolate. Additionally, a host of genes exhibited exclusive occupancy of Ncb2 at promoters of either AR or AS isolate. The analysis also divulged new actors of multi-drug resistance, whose transcription was activated owing to the differential occupancy of Ncb2. The conditional, sequence-specific positional escape of Ncb2 from the core promoter in AS isolate and its preferential recruitment to the core promoter of certain genes in AR isolates was most noteworthy means of transcription regulation. Together, we show that positional rearrangement of Ncb2 resulting in either activation or repression of gene expression in response to drug-induced stress, represents a novel regulatory mechanism that opens new opportunities for therapeutic intervention to prevent development of drug tolerance in C. albicans cells.

No MeSH data available.


Confirmation of the relation of Ncb2 higher occupancy and gene expression in AR isolate by semi-quantitative RT-PCR and ChIP-PCR.(a) End point semi-quantitative RT-PCR was used to validate some of the genes found to be highly enriched by Ncb2 in the AR isolate and their relation to gene expression. RT-PCR was performed in triplicate and a representative figure (cropped gel pictures) is shown. For full gel see Supplementary Fig. S3. CDR1 and CDR2 genes were used as positive controls for the gene over-expressed in the AR isolate. ACT1 expression level was used as a control for isoexpression between AR and AS isolates. (b) Bar diagram showing the expression profile of genes highly enriched in AR isolate as compared to AS isolate by Ncb2. Bars represent the standard deviations observed for the replicate experiments. (c) Recruitment profiles of Ncb2 in AR isolate as compared to AS isolate determined by ChIP-PCR. For presentation cropped gels are shown. For complete gel see Supplementary Fig. S4. IN represents amplification observed in input sample, control (C) and test (T) are immunoprecipitations carried out on cross-linked chromatin using pre-immune serum and anti-Ncb2 antibody, respectively. Amplification of CDR2 promoter region was used as a positive control. ADH1 and ACT1 promoter regions amplification were used as positive and negative control for Ncb2 binding. (d) Bar diagram representing the Ncb2 enrichments of AR over AS isolate. Bars represent standard deviations for the replicate experiments.
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f2: Confirmation of the relation of Ncb2 higher occupancy and gene expression in AR isolate by semi-quantitative RT-PCR and ChIP-PCR.(a) End point semi-quantitative RT-PCR was used to validate some of the genes found to be highly enriched by Ncb2 in the AR isolate and their relation to gene expression. RT-PCR was performed in triplicate and a representative figure (cropped gel pictures) is shown. For full gel see Supplementary Fig. S3. CDR1 and CDR2 genes were used as positive controls for the gene over-expressed in the AR isolate. ACT1 expression level was used as a control for isoexpression between AR and AS isolates. (b) Bar diagram showing the expression profile of genes highly enriched in AR isolate as compared to AS isolate by Ncb2. Bars represent the standard deviations observed for the replicate experiments. (c) Recruitment profiles of Ncb2 in AR isolate as compared to AS isolate determined by ChIP-PCR. For presentation cropped gels are shown. For complete gel see Supplementary Fig. S4. IN represents amplification observed in input sample, control (C) and test (T) are immunoprecipitations carried out on cross-linked chromatin using pre-immune serum and anti-Ncb2 antibody, respectively. Amplification of CDR2 promoter region was used as a positive control. ADH1 and ACT1 promoter regions amplification were used as positive and negative control for Ncb2 binding. (d) Bar diagram representing the Ncb2 enrichments of AR over AS isolate. Bars represent standard deviations for the replicate experiments.

Mentions: We compared the genome-wide recruitment data of Ncb2 between AS and AR isolates and thereby, identified genes that showed distinct occupancy of Ncb2 at the promoter regions. The analysis revealed that a number of genes have higher enrichment of Ncb2 occupancy in AR isolate (Supplementary Table S3). This included genes implicated in drug response, filamentous growth, various stresses, pathogenesis, transport, cell wall organization, signal transduction, and biofilm formation (Supplementary Table S4). To establish a relation between the higher occupancy of Ncb2 in AR isolate and gene expression, we randomly selected 6 genes (PMT1, orf19.4601, PGA17, RTA4, ERG11 and GSC1) of interest with different normalized log ratios (enrichments) and P-values. Their expression was checked by semi-quantitative end-point RT (reverse transcription) PCR. CDR1 and CDR2 were used as positive controls. Inspite of the higher Ncb2 occupancy, the expression data showed a mixed response of gene expression. For instance, three genes [orf19.4601 (Putative RNA polymerase III transcription initiation factor complex (TFIIIC) subunit), PGA17 (Putative GPI-anchored protein) and GSC1)] were up-regulated in AR isolate whereas PMT1 (Protein mannosyltransferase), RTA4 (Protein similar to S. cerevisiae Rsb1p, involved in fatty acid transport) and ERG11 were down regulated in AR cells (Fig. 2a,b; Supplementary Fig. S3). The validation though limited, points that the higher occupancy of Ncb2 in AR isolate does not always result in transcriptional activation. Nevertheless, Ncb2 probably manifests dual effect on transcriptional control mechanisms. Since genes that showing higher enrichment in ChIP-on-chip data displayed differential gene expression pattern in AS and AR isolates hence we analyzed the relative recruitment dynamics of Ncb2 in both the isolates. For this, we used ChIPed DNA from AS and AR isolates and subjected to ChIP-PCR analysis which revealed that Ncb2 binding correlates well with the ChIP-on-chip data (Fig. 2c,d; Supplementary Fig. S4).


The global regulator Ncb2 escapes from the core promoter and impacts transcription in response to drug stress in Candida albicans
Confirmation of the relation of Ncb2 higher occupancy and gene expression in AR isolate by semi-quantitative RT-PCR and ChIP-PCR.(a) End point semi-quantitative RT-PCR was used to validate some of the genes found to be highly enriched by Ncb2 in the AR isolate and their relation to gene expression. RT-PCR was performed in triplicate and a representative figure (cropped gel pictures) is shown. For full gel see Supplementary Fig. S3. CDR1 and CDR2 genes were used as positive controls for the gene over-expressed in the AR isolate. ACT1 expression level was used as a control for isoexpression between AR and AS isolates. (b) Bar diagram showing the expression profile of genes highly enriched in AR isolate as compared to AS isolate by Ncb2. Bars represent the standard deviations observed for the replicate experiments. (c) Recruitment profiles of Ncb2 in AR isolate as compared to AS isolate determined by ChIP-PCR. For presentation cropped gels are shown. For complete gel see Supplementary Fig. S4. IN represents amplification observed in input sample, control (C) and test (T) are immunoprecipitations carried out on cross-linked chromatin using pre-immune serum and anti-Ncb2 antibody, respectively. Amplification of CDR2 promoter region was used as a positive control. ADH1 and ACT1 promoter regions amplification were used as positive and negative control for Ncb2 binding. (d) Bar diagram representing the Ncb2 enrichments of AR over AS isolate. Bars represent standard deviations for the replicate experiments.
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f2: Confirmation of the relation of Ncb2 higher occupancy and gene expression in AR isolate by semi-quantitative RT-PCR and ChIP-PCR.(a) End point semi-quantitative RT-PCR was used to validate some of the genes found to be highly enriched by Ncb2 in the AR isolate and their relation to gene expression. RT-PCR was performed in triplicate and a representative figure (cropped gel pictures) is shown. For full gel see Supplementary Fig. S3. CDR1 and CDR2 genes were used as positive controls for the gene over-expressed in the AR isolate. ACT1 expression level was used as a control for isoexpression between AR and AS isolates. (b) Bar diagram showing the expression profile of genes highly enriched in AR isolate as compared to AS isolate by Ncb2. Bars represent the standard deviations observed for the replicate experiments. (c) Recruitment profiles of Ncb2 in AR isolate as compared to AS isolate determined by ChIP-PCR. For presentation cropped gels are shown. For complete gel see Supplementary Fig. S4. IN represents amplification observed in input sample, control (C) and test (T) are immunoprecipitations carried out on cross-linked chromatin using pre-immune serum and anti-Ncb2 antibody, respectively. Amplification of CDR2 promoter region was used as a positive control. ADH1 and ACT1 promoter regions amplification were used as positive and negative control for Ncb2 binding. (d) Bar diagram representing the Ncb2 enrichments of AR over AS isolate. Bars represent standard deviations for the replicate experiments.
Mentions: We compared the genome-wide recruitment data of Ncb2 between AS and AR isolates and thereby, identified genes that showed distinct occupancy of Ncb2 at the promoter regions. The analysis revealed that a number of genes have higher enrichment of Ncb2 occupancy in AR isolate (Supplementary Table S3). This included genes implicated in drug response, filamentous growth, various stresses, pathogenesis, transport, cell wall organization, signal transduction, and biofilm formation (Supplementary Table S4). To establish a relation between the higher occupancy of Ncb2 in AR isolate and gene expression, we randomly selected 6 genes (PMT1, orf19.4601, PGA17, RTA4, ERG11 and GSC1) of interest with different normalized log ratios (enrichments) and P-values. Their expression was checked by semi-quantitative end-point RT (reverse transcription) PCR. CDR1 and CDR2 were used as positive controls. Inspite of the higher Ncb2 occupancy, the expression data showed a mixed response of gene expression. For instance, three genes [orf19.4601 (Putative RNA polymerase III transcription initiation factor complex (TFIIIC) subunit), PGA17 (Putative GPI-anchored protein) and GSC1)] were up-regulated in AR isolate whereas PMT1 (Protein mannosyltransferase), RTA4 (Protein similar to S. cerevisiae Rsb1p, involved in fatty acid transport) and ERG11 were down regulated in AR cells (Fig. 2a,b; Supplementary Fig. S3). The validation though limited, points that the higher occupancy of Ncb2 in AR isolate does not always result in transcriptional activation. Nevertheless, Ncb2 probably manifests dual effect on transcriptional control mechanisms. Since genes that showing higher enrichment in ChIP-on-chip data displayed differential gene expression pattern in AS and AR isolates hence we analyzed the relative recruitment dynamics of Ncb2 in both the isolates. For this, we used ChIPed DNA from AS and AR isolates and subjected to ChIP-PCR analysis which revealed that Ncb2 binding correlates well with the ChIP-on-chip data (Fig. 2c,d; Supplementary Fig. S4).

View Article: PubMed Central - PubMed

ABSTRACT

Ncb2, the β subunit of NC2 complex, a heterodimeric regulator of transcription was earlier shown to be involved in the activated transcription of CDR1 gene in azole resistant isolate (AR) of Candida albicans. This study examines its genome-wide role by profiling Ncb2 occupancy between genetically matched pair of azole sensitive (AS) and AR clinical isolates. A comparison of Ncb2 recruitment between the two isolates displayed that 29 genes had higher promoter occupancy of Ncb2 in the AR isolate. Additionally, a host of genes exhibited exclusive occupancy of Ncb2 at promoters of either AR or AS isolate. The analysis also divulged new actors of multi-drug resistance, whose transcription was activated owing to the differential occupancy of Ncb2. The conditional, sequence-specific positional escape of Ncb2 from the core promoter in AS isolate and its preferential recruitment to the core promoter of certain genes in AR isolates was most noteworthy means of transcription regulation. Together, we show that positional rearrangement of Ncb2 resulting in either activation or repression of gene expression in response to drug-induced stress, represents a novel regulatory mechanism that opens new opportunities for therapeutic intervention to prevent development of drug tolerance in C. albicans cells.

No MeSH data available.