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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.


Ncb2 occupies different positions at promoter regions of ADAEC and ECE1 in AS and AR isolates.(a) CRIP assay showing the differential recruitment of Ncb2 at ADAEC promoter region in AS and AR isolates. The result demonstrate that in AS isolate Ncb2 recruited at the upstream of core promoter region whereas its recruitment was found at the core promoter near ATG codon in AR isolate. On the right side of the figure a bar diagram depicting the difference in enrichment profiles is shown. (b) Ncb2 occupy different positions at promoter regions of ECE1 in AS and AR isolates. CRIP assay showing the differential recruitment profiles of Ncb2 at gene promoter of ECE1. Its occupancy was found at upstream region of core promoter in AS isolates whereas it occupies at the core promoter region in AR isolates. On the right side of the figure a bar diagram representing the observed differences of Ncb2 occupancy in AS and AR isolates is shown. For demonstration cropped gel bands are shown. Complete gels are shown in Supplementary Fig. S12. (c) Ncb2 preferentially recruited at A-rich sequence motif of DNA. Logo of the Ncb2 DNA binding motif discovered by Rsat using 500 promoter regions that have highest enrichment profiles in ChIP-on-chip data.
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f6: Ncb2 occupies different positions at promoter regions of ADAEC and ECE1 in AS and AR isolates.(a) CRIP assay showing the differential recruitment of Ncb2 at ADAEC promoter region in AS and AR isolates. The result demonstrate that in AS isolate Ncb2 recruited at the upstream of core promoter region whereas its recruitment was found at the core promoter near ATG codon in AR isolate. On the right side of the figure a bar diagram depicting the difference in enrichment profiles is shown. (b) Ncb2 occupy different positions at promoter regions of ECE1 in AS and AR isolates. CRIP assay showing the differential recruitment profiles of Ncb2 at gene promoter of ECE1. Its occupancy was found at upstream region of core promoter in AS isolates whereas it occupies at the core promoter region in AR isolates. On the right side of the figure a bar diagram representing the observed differences of Ncb2 occupancy in AS and AR isolates is shown. For demonstration cropped gel bands are shown. Complete gels are shown in Supplementary Fig. S12. (c) Ncb2 preferentially recruited at A-rich sequence motif of DNA. Logo of the Ncb2 DNA binding motif discovered by Rsat using 500 promoter regions that have highest enrichment profiles in ChIP-on-chip data.

Mentions: Further, shift in the positional occupancy of Ncb2 was confirmed by using chromatin restriction digestion coupled immunoprecipitation (CRIP) assay. For this, genes that displayed shift in occupancy of Ncb2 and showed regulated gene expression in AS and AR isolates were chosen. CRIP assay was performed with genes such as PDX1, DFI1, ADAEC and ECE1 in AS and AR isolates. Their promoter DNA was separated into ATG codon containing core promoter and upstream of the core promoter fragments by using HinFI, EcoRV, XhoI and DpnI site as depicted in Fig. 5b. The fragmented chromatin was precipitated by using anti-Ncb2 antibody followed by PCR using two primer pairs for each gene (Fig. 5b). Interestingly, it was observed that in the AS isolate Ncb2 occupancy was detected at upstream of the core promoter regions while there was no occupancy at the core promoter near ATG codon (Figs 5c,d and 6a,b; Supplementary Figs S11 and S12). Of note, a totally reverse scenario was observed in the AR isolate where Ncb2 was exclusively recruited at the core promoter regions (Figs 5c,d and 6a,b; Supplementary Figs S11 and S12).


The global regulator Ncb2 escapes from the core promoter and impacts transcription in response to drug stress in Candida albicans
Ncb2 occupies different positions at promoter regions of ADAEC and ECE1 in AS and AR isolates.(a) CRIP assay showing the differential recruitment of Ncb2 at ADAEC promoter region in AS and AR isolates. The result demonstrate that in AS isolate Ncb2 recruited at the upstream of core promoter region whereas its recruitment was found at the core promoter near ATG codon in AR isolate. On the right side of the figure a bar diagram depicting the difference in enrichment profiles is shown. (b) Ncb2 occupy different positions at promoter regions of ECE1 in AS and AR isolates. CRIP assay showing the differential recruitment profiles of Ncb2 at gene promoter of ECE1. Its occupancy was found at upstream region of core promoter in AS isolates whereas it occupies at the core promoter region in AR isolates. On the right side of the figure a bar diagram representing the observed differences of Ncb2 occupancy in AS and AR isolates is shown. For demonstration cropped gel bands are shown. Complete gels are shown in Supplementary Fig. S12. (c) Ncb2 preferentially recruited at A-rich sequence motif of DNA. Logo of the Ncb2 DNA binding motif discovered by Rsat using 500 promoter regions that have highest enrichment profiles in ChIP-on-chip data.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5382705&req=5

f6: Ncb2 occupies different positions at promoter regions of ADAEC and ECE1 in AS and AR isolates.(a) CRIP assay showing the differential recruitment of Ncb2 at ADAEC promoter region in AS and AR isolates. The result demonstrate that in AS isolate Ncb2 recruited at the upstream of core promoter region whereas its recruitment was found at the core promoter near ATG codon in AR isolate. On the right side of the figure a bar diagram depicting the difference in enrichment profiles is shown. (b) Ncb2 occupy different positions at promoter regions of ECE1 in AS and AR isolates. CRIP assay showing the differential recruitment profiles of Ncb2 at gene promoter of ECE1. Its occupancy was found at upstream region of core promoter in AS isolates whereas it occupies at the core promoter region in AR isolates. On the right side of the figure a bar diagram representing the observed differences of Ncb2 occupancy in AS and AR isolates is shown. For demonstration cropped gel bands are shown. Complete gels are shown in Supplementary Fig. S12. (c) Ncb2 preferentially recruited at A-rich sequence motif of DNA. Logo of the Ncb2 DNA binding motif discovered by Rsat using 500 promoter regions that have highest enrichment profiles in ChIP-on-chip data.
Mentions: Further, shift in the positional occupancy of Ncb2 was confirmed by using chromatin restriction digestion coupled immunoprecipitation (CRIP) assay. For this, genes that displayed shift in occupancy of Ncb2 and showed regulated gene expression in AS and AR isolates were chosen. CRIP assay was performed with genes such as PDX1, DFI1, ADAEC and ECE1 in AS and AR isolates. Their promoter DNA was separated into ATG codon containing core promoter and upstream of the core promoter fragments by using HinFI, EcoRV, XhoI and DpnI site as depicted in Fig. 5b. The fragmented chromatin was precipitated by using anti-Ncb2 antibody followed by PCR using two primer pairs for each gene (Fig. 5b). Interestingly, it was observed that in the AS isolate Ncb2 occupancy was detected at upstream of the core promoter regions while there was no occupancy at the core promoter near ATG codon (Figs 5c,d and 6a,b; Supplementary Figs S11 and S12). Of note, a totally reverse scenario was observed in the AR isolate where Ncb2 was exclusively recruited at the core promoter regions (Figs 5c,d and 6a,b; Supplementary Figs S11 and S12).

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.