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

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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.

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Ncb2 acts as a positive as well as negative regulator of the gene expression of AS exclusive target genes.(a) To establish a relation between Ncb2 AS-specific enrichment and gene transcription, we employed semi-quantitative RT-PCR analysis for selected genes. Expression data shows that it acts to control gene expression positively and negatively in the AS isolate. CDR1 expression level was used as a control for genes induced in AR strain. ACT1 expression level was used for the normalization of the data. RT-PCR was performed from RNA isolated from three different preparations. Cropped gels were used for presentation. Complete gels are shown in Supplementary Fig. S7. (b) Bar diagram was used to find out the differences in the gene expression of genes exclusively occupied by Ncb2 in the AS isolate. Bars represent the standard deviation observed for the replicate experiments. (c) ChIP analysis of the promoter regions of selected genes of Ncb2 exclusive occupancy in AS isolate. Control and test represents experiment performed on cross-linked chromatin with pre-immune and anti-Ncb2 antibody. Precipitation of ADH1 and ACT1 promoter regions were used as positive and negative control for Ncb2 binding. For presentation purpose cropped gels are shown. For full gel see Supplementary Fig. S8. (d) Bar diagram showing the exclusive occupancy of Ncb2 in AS isolate as compared to AR isolate. Bars represent the standard deviation observed for the replicate experiments.
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f4: Ncb2 acts as a positive as well as negative regulator of the gene expression of AS exclusive target genes.(a) To establish a relation between Ncb2 AS-specific enrichment and gene transcription, we employed semi-quantitative RT-PCR analysis for selected genes. Expression data shows that it acts to control gene expression positively and negatively in the AS isolate. CDR1 expression level was used as a control for genes induced in AR strain. ACT1 expression level was used for the normalization of the data. RT-PCR was performed from RNA isolated from three different preparations. Cropped gels were used for presentation. Complete gels are shown in Supplementary Fig. S7. (b) Bar diagram was used to find out the differences in the gene expression of genes exclusively occupied by Ncb2 in the AS isolate. Bars represent the standard deviation observed for the replicate experiments. (c) ChIP analysis of the promoter regions of selected genes of Ncb2 exclusive occupancy in AS isolate. Control and test represents experiment performed on cross-linked chromatin with pre-immune and anti-Ncb2 antibody. Precipitation of ADH1 and ACT1 promoter regions were used as positive and negative control for Ncb2 binding. For presentation purpose cropped gels are shown. For full gel see Supplementary Fig. S8. (d) Bar diagram showing the exclusive occupancy of Ncb2 in AS isolate as compared to AR isolate. Bars represent the standard deviation observed for the replicate experiments.

Mentions: The comparative analysis of the genome-wide binding profiles of Ncb2 in the AR and AS isolates also identified genes whose promoters were exclusively enriched in the AS isolate (Supplementary Table S6). GO slim analysis of the genes exclusively enriched in AS isolate showed that these are involved in transport, filamentous growth, response to drug, pathogenesis, and response to stress etc. (Supplementary Table S7). Four genes were chosen with different binding strength of Ncb2 to establish a relation between exclusive occupancy in AS isolate and gene transcription. The validation of the relation between Ncb2 exclusive occupancy in AS isolate and expression of this group of genes confirmed that the Ncb2 has a dual role both as a positive and negative regulator of gene expression (Fig. 4a,b; Supplementary Fig. S7). Interestingly, ChIP-coupled PCR from the ChIPed DNA obtained from AR and AS isolate of these genes confirmed that Ncb2 only occupies the genes of AS isolate (Fig. 4c,d; Supplementary Fig. S8).


The global regulator Ncb2 escapes from the core promoter and impacts transcription in response to drug stress in Candida albicans
Ncb2 acts as a positive as well as negative regulator of the gene expression of AS exclusive target genes.(a) To establish a relation between Ncb2 AS-specific enrichment and gene transcription, we employed semi-quantitative RT-PCR analysis for selected genes. Expression data shows that it acts to control gene expression positively and negatively in the AS isolate. CDR1 expression level was used as a control for genes induced in AR strain. ACT1 expression level was used for the normalization of the data. RT-PCR was performed from RNA isolated from three different preparations. Cropped gels were used for presentation. Complete gels are shown in Supplementary Fig. S7. (b) Bar diagram was used to find out the differences in the gene expression of genes exclusively occupied by Ncb2 in the AS isolate. Bars represent the standard deviation observed for the replicate experiments. (c) ChIP analysis of the promoter regions of selected genes of Ncb2 exclusive occupancy in AS isolate. Control and test represents experiment performed on cross-linked chromatin with pre-immune and anti-Ncb2 antibody. Precipitation of ADH1 and ACT1 promoter regions were used as positive and negative control for Ncb2 binding. For presentation purpose cropped gels are shown. For full gel see Supplementary Fig. S8. (d) Bar diagram showing the exclusive occupancy of Ncb2 in AS isolate as compared to AR isolate. Bars represent the standard deviation observed for the replicate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5382705&req=5

f4: Ncb2 acts as a positive as well as negative regulator of the gene expression of AS exclusive target genes.(a) To establish a relation between Ncb2 AS-specific enrichment and gene transcription, we employed semi-quantitative RT-PCR analysis for selected genes. Expression data shows that it acts to control gene expression positively and negatively in the AS isolate. CDR1 expression level was used as a control for genes induced in AR strain. ACT1 expression level was used for the normalization of the data. RT-PCR was performed from RNA isolated from three different preparations. Cropped gels were used for presentation. Complete gels are shown in Supplementary Fig. S7. (b) Bar diagram was used to find out the differences in the gene expression of genes exclusively occupied by Ncb2 in the AS isolate. Bars represent the standard deviation observed for the replicate experiments. (c) ChIP analysis of the promoter regions of selected genes of Ncb2 exclusive occupancy in AS isolate. Control and test represents experiment performed on cross-linked chromatin with pre-immune and anti-Ncb2 antibody. Precipitation of ADH1 and ACT1 promoter regions were used as positive and negative control for Ncb2 binding. For presentation purpose cropped gels are shown. For full gel see Supplementary Fig. S8. (d) Bar diagram showing the exclusive occupancy of Ncb2 in AS isolate as compared to AR isolate. Bars represent the standard deviation observed for the replicate experiments.
Mentions: The comparative analysis of the genome-wide binding profiles of Ncb2 in the AR and AS isolates also identified genes whose promoters were exclusively enriched in the AS isolate (Supplementary Table S6). GO slim analysis of the genes exclusively enriched in AS isolate showed that these are involved in transport, filamentous growth, response to drug, pathogenesis, and response to stress etc. (Supplementary Table S7). Four genes were chosen with different binding strength of Ncb2 to establish a relation between exclusive occupancy in AS isolate and gene transcription. The validation of the relation between Ncb2 exclusive occupancy in AS isolate and expression of this group of genes confirmed that the Ncb2 has a dual role both as a positive and negative regulator of gene expression (Fig. 4a,b; Supplementary Fig. S7). Interestingly, ChIP-coupled PCR from the ChIPed DNA obtained from AR and AS isolate of these genes confirmed that Ncb2 only occupies the genes of AS isolate (Fig. 4c,d; Supplementary Fig. S8).

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