Limits...
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.


Related in: MedlinePlus

Ncb2 exclusive occupancy in the AR isolate mostly leads to activated transcription of the target genes.(a) Semi-quantitative RT-PCR was used to study the effect of exclusive enrichment of Ncb2 and gene expression of selected genes in the AR isolate. Most of the exclusive target genes of Ncb2 in the AR isolate were found activated except for UTP22 which was repressed. CDR1 and CDR2 genes served as controls that over-expressed in the AR isolate. ACT1 expression level was used as control that equally expresses in both the isolates. RT-PCR was performed from c-DNA prepared from three different RNA preparations. For presentation cropped gels are shown. For complete gel see Supplementary Fig. S5. (b) Bar diagram demonstrating expression profile of AR exclusive enriched genes. Bars represent standard deviations observed for the replicate experiments. (c) ChIP-PCR analysis of Ncb2 exclusive occupancy in AR isolate. Chromatin immunoprecipitation was performed on cross linked chromatin isolated from AS and AR isolates, respectively. Control and test designate immunoprecipitations performed on cross-linked chromatin with pre-immune serum and anti-Ncb2 antibody, respectively. Amplification of ADH1 and ACT1 promoter regions were used as a positive and negative control for Ncb2 binding in both the isolates. Cropped gels are used for presentation, for complete gel see Supplementary Fig. S6. (d) Bar diagram showing the Ncb2 exclusive occupancy in AR isolate as compared to AS isolate. Bars represent the standard deviations observed for the replicate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5382705&req=5

f3: Ncb2 exclusive occupancy in the AR isolate mostly leads to activated transcription of the target genes.(a) Semi-quantitative RT-PCR was used to study the effect of exclusive enrichment of Ncb2 and gene expression of selected genes in the AR isolate. Most of the exclusive target genes of Ncb2 in the AR isolate were found activated except for UTP22 which was repressed. CDR1 and CDR2 genes served as controls that over-expressed in the AR isolate. ACT1 expression level was used as control that equally expresses in both the isolates. RT-PCR was performed from c-DNA prepared from three different RNA preparations. For presentation cropped gels are shown. For complete gel see Supplementary Fig. S5. (b) Bar diagram demonstrating expression profile of AR exclusive enriched genes. Bars represent standard deviations observed for the replicate experiments. (c) ChIP-PCR analysis of Ncb2 exclusive occupancy in AR isolate. Chromatin immunoprecipitation was performed on cross linked chromatin isolated from AS and AR isolates, respectively. Control and test designate immunoprecipitations performed on cross-linked chromatin with pre-immune serum and anti-Ncb2 antibody, respectively. Amplification of ADH1 and ACT1 promoter regions were used as a positive and negative control for Ncb2 binding in both the isolates. Cropped gels are used for presentation, for complete gel see Supplementary Fig. S6. (d) Bar diagram showing the Ncb2 exclusive occupancy in AR isolate as compared to AS isolate. Bars represent the standard deviations observed for the replicate experiments.

Mentions: The analysis of the genome-wide Ncb2 recruitment dynamics revealed 25 gene promoters which displayed an exclusive occupancy of Ncb2 in the AR isolate whereas its binding was absent in AS isolate (Supplementary Table S5). In this category, for validation, we carried out expression analysis for the genes which included orf19.1698, CHK1, orf19.1434, orf19.7063, PDC12, ARO80 and UTP22 with different enrichment ratios and P-values and performed end point semi-quantitative RT-PCR analysis (Fig. 3a,b; Supplementary Fig. S5). Remarkably, with the exception of UTP22, most of the genes showing selective enrichment in the AR isolate were accompanied by an increased expression. This implies that the exclusive enrichment of Ncb2 in AR isolate mainly result in transcriptional activation (Fig. 3a,b; Supplementary Fig. S5). Subsequent ChIP-coupled PCR on ChIPed DNA obtained from AS and AR isolates validated the exclusive recruitment dynamics of Ncb2 (Fig. 3c,d; Supplementary Fig. S6). These results revealed that Ncb2 binding was exclusively present only in the ChIPed DNA isolated from the AR isolate (Fig. 3c,d; Supplementary Fig. S6). Therefore, it can be concluded reasonably that Ncb2 exclusively occupies a subset of genes in the AR isolate and is primarily involved in the positive regulation of gene expression (Fig. 3a,b,c,d; Supplementary Figs S5 and S6).


The global regulator Ncb2 escapes from the core promoter and impacts transcription in response to drug stress in Candida albicans
Ncb2 exclusive occupancy in the AR isolate mostly leads to activated transcription of the target genes.(a) Semi-quantitative RT-PCR was used to study the effect of exclusive enrichment of Ncb2 and gene expression of selected genes in the AR isolate. Most of the exclusive target genes of Ncb2 in the AR isolate were found activated except for UTP22 which was repressed. CDR1 and CDR2 genes served as controls that over-expressed in the AR isolate. ACT1 expression level was used as control that equally expresses in both the isolates. RT-PCR was performed from c-DNA prepared from three different RNA preparations. For presentation cropped gels are shown. For complete gel see Supplementary Fig. S5. (b) Bar diagram demonstrating expression profile of AR exclusive enriched genes. Bars represent standard deviations observed for the replicate experiments. (c) ChIP-PCR analysis of Ncb2 exclusive occupancy in AR isolate. Chromatin immunoprecipitation was performed on cross linked chromatin isolated from AS and AR isolates, respectively. Control and test designate immunoprecipitations performed on cross-linked chromatin with pre-immune serum and anti-Ncb2 antibody, respectively. Amplification of ADH1 and ACT1 promoter regions were used as a positive and negative control for Ncb2 binding in both the isolates. Cropped gels are used for presentation, for complete gel see Supplementary Fig. S6. (d) Bar diagram showing the Ncb2 exclusive occupancy in AR isolate as compared to AS isolate. Bars represent the standard deviations observed for the replicate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5382705&req=5

f3: Ncb2 exclusive occupancy in the AR isolate mostly leads to activated transcription of the target genes.(a) Semi-quantitative RT-PCR was used to study the effect of exclusive enrichment of Ncb2 and gene expression of selected genes in the AR isolate. Most of the exclusive target genes of Ncb2 in the AR isolate were found activated except for UTP22 which was repressed. CDR1 and CDR2 genes served as controls that over-expressed in the AR isolate. ACT1 expression level was used as control that equally expresses in both the isolates. RT-PCR was performed from c-DNA prepared from three different RNA preparations. For presentation cropped gels are shown. For complete gel see Supplementary Fig. S5. (b) Bar diagram demonstrating expression profile of AR exclusive enriched genes. Bars represent standard deviations observed for the replicate experiments. (c) ChIP-PCR analysis of Ncb2 exclusive occupancy in AR isolate. Chromatin immunoprecipitation was performed on cross linked chromatin isolated from AS and AR isolates, respectively. Control and test designate immunoprecipitations performed on cross-linked chromatin with pre-immune serum and anti-Ncb2 antibody, respectively. Amplification of ADH1 and ACT1 promoter regions were used as a positive and negative control for Ncb2 binding in both the isolates. Cropped gels are used for presentation, for complete gel see Supplementary Fig. S6. (d) Bar diagram showing the Ncb2 exclusive occupancy in AR isolate as compared to AS isolate. Bars represent the standard deviations observed for the replicate experiments.
Mentions: The analysis of the genome-wide Ncb2 recruitment dynamics revealed 25 gene promoters which displayed an exclusive occupancy of Ncb2 in the AR isolate whereas its binding was absent in AS isolate (Supplementary Table S5). In this category, for validation, we carried out expression analysis for the genes which included orf19.1698, CHK1, orf19.1434, orf19.7063, PDC12, ARO80 and UTP22 with different enrichment ratios and P-values and performed end point semi-quantitative RT-PCR analysis (Fig. 3a,b; Supplementary Fig. S5). Remarkably, with the exception of UTP22, most of the genes showing selective enrichment in the AR isolate were accompanied by an increased expression. This implies that the exclusive enrichment of Ncb2 in AR isolate mainly result in transcriptional activation (Fig. 3a,b; Supplementary Fig. S5). Subsequent ChIP-coupled PCR on ChIPed DNA obtained from AS and AR isolates validated the exclusive recruitment dynamics of Ncb2 (Fig. 3c,d; Supplementary Fig. S6). These results revealed that Ncb2 binding was exclusively present only in the ChIPed DNA isolated from the AR isolate (Fig. 3c,d; Supplementary Fig. S6). Therefore, it can be concluded reasonably that Ncb2 exclusively occupies a subset of genes in the AR isolate and is primarily involved in the positive regulation of gene expression (Fig. 3a,b,c,d; Supplementary Figs S5 and S6).

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.


Related in: MedlinePlus