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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.


Genome-wide promoter association of Ncb2.(a) Charts showing the Ncb2 occupying and non-occupying gene promoters of C. albicans in an Ncb2 ChIP performed in Gu4 (AS) and Gu5 (AR) clinical isolates. (b) Ncb2 binding peaks localizes at the −50/−300 region encompassing the core promoter. Average binding profiles of Ncb2 for the promoter regions were determined relative to ATG start codon for both the isolates. Green line represents localization profile of Ncb2 in the AS isolate; whereas blue line represents localization profile of Ncb2 in AR isolate. Binding distance was calculated for the probe that has maximum normalized log ratio.
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f1: Genome-wide promoter association of Ncb2.(a) Charts showing the Ncb2 occupying and non-occupying gene promoters of C. albicans in an Ncb2 ChIP performed in Gu4 (AS) and Gu5 (AR) clinical isolates. (b) Ncb2 binding peaks localizes at the −50/−300 region encompassing the core promoter. Average binding profiles of Ncb2 for the promoter regions were determined relative to ATG start codon for both the isolates. Green line represents localization profile of Ncb2 in the AS isolate; whereas blue line represents localization profile of Ncb2 in AR isolate. Binding distance was calculated for the probe that has maximum normalized log ratio.

Mentions: Ncb2, a part of the heterodimeric NC2 complex that controls transcription of genes was earlier shown to be involved in the activated transcription of CDR1 gene in azole resistant (AR) clinical isolate of C. albicans4344. To understand the global impact of Ncb2 in acquisition of MDR in C. albicans, we performed a genome-wide recruitment analysis of the transcriptional regulator by employing ChIP-on-chip assay and compared it between azole sensitive (AS) and AR isolates. Three biological replicates were processed for each of the isolate to identify DNA regions that were occupied by Ncb2. By using polyclonal anti-Ncb2 antibody, the cross-linked protein-DNA complexes were pulled down43. Signal peaks detected in all the three experiments were chosen. Analysis of the genome-wide data revealed no major difference in terms of percentage occupancy of Ncb2 and varied between 25–30% in AR and AS isolates (Fig. 1a). For further analysis, by employing three different arbitrary cutoffs, all the promoters were ranked according to the P-values and fold enrichments (in comparison with input DNA); I) genes with P-values ≤ 0.055 and fold enrichments ≥1.5 (AS-1873 genes, AR-1550 genes), II) genes with P-values ≤ 0.055 and fold enrichments ≥2.0 (AS-1765 genes, AR-1546), III) genes with P-values ≤ 0.01 and fold enrichments ≥2.0 (AS-1119, AR-960). Furthermore, to compare the genomic binding of Ncb2 between the isolates, the average binding profile for the promoter (fragments: −1/−49, −50/−300, −301/−550, −551/−800, −801/−1250 and ≥1251, relative to the start codon) was computed which showed that Ncb2 mainly recruited at the core promoter regions434546 (Fig. 1b). Genome-wide occupancy data analysis also revealed that Ncb2 binds both inside and downstream of the genes (Supplementary Figs S1 and S2). The most common model for transcriptional activation is that the binding, just upstream of the transcription start site (TSS) helps in placing polymerase II at the beginning of the gene. Presence of TF binding sites inside or downstream of gene may act as regulatory sequence for the same gene or act as enhancer for the control of same gene or downstream gene (acting as a distal enhancer) in orientation and position independent manner474849. Notably, all binding sites may not be functional or represents a non productive binding site or could just be alternative TSS505152.


The global regulator Ncb2 escapes from the core promoter and impacts transcription in response to drug stress in Candida albicans
Genome-wide promoter association of Ncb2.(a) Charts showing the Ncb2 occupying and non-occupying gene promoters of C. albicans in an Ncb2 ChIP performed in Gu4 (AS) and Gu5 (AR) clinical isolates. (b) Ncb2 binding peaks localizes at the −50/−300 region encompassing the core promoter. Average binding profiles of Ncb2 for the promoter regions were determined relative to ATG start codon for both the isolates. Green line represents localization profile of Ncb2 in the AS isolate; whereas blue line represents localization profile of Ncb2 in AR isolate. Binding distance was calculated for the probe that has maximum normalized log ratio.
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Related In: Results  -  Collection

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f1: Genome-wide promoter association of Ncb2.(a) Charts showing the Ncb2 occupying and non-occupying gene promoters of C. albicans in an Ncb2 ChIP performed in Gu4 (AS) and Gu5 (AR) clinical isolates. (b) Ncb2 binding peaks localizes at the −50/−300 region encompassing the core promoter. Average binding profiles of Ncb2 for the promoter regions were determined relative to ATG start codon for both the isolates. Green line represents localization profile of Ncb2 in the AS isolate; whereas blue line represents localization profile of Ncb2 in AR isolate. Binding distance was calculated for the probe that has maximum normalized log ratio.
Mentions: Ncb2, a part of the heterodimeric NC2 complex that controls transcription of genes was earlier shown to be involved in the activated transcription of CDR1 gene in azole resistant (AR) clinical isolate of C. albicans4344. To understand the global impact of Ncb2 in acquisition of MDR in C. albicans, we performed a genome-wide recruitment analysis of the transcriptional regulator by employing ChIP-on-chip assay and compared it between azole sensitive (AS) and AR isolates. Three biological replicates were processed for each of the isolate to identify DNA regions that were occupied by Ncb2. By using polyclonal anti-Ncb2 antibody, the cross-linked protein-DNA complexes were pulled down43. Signal peaks detected in all the three experiments were chosen. Analysis of the genome-wide data revealed no major difference in terms of percentage occupancy of Ncb2 and varied between 25–30% in AR and AS isolates (Fig. 1a). For further analysis, by employing three different arbitrary cutoffs, all the promoters were ranked according to the P-values and fold enrichments (in comparison with input DNA); I) genes with P-values ≤ 0.055 and fold enrichments ≥1.5 (AS-1873 genes, AR-1550 genes), II) genes with P-values ≤ 0.055 and fold enrichments ≥2.0 (AS-1765 genes, AR-1546), III) genes with P-values ≤ 0.01 and fold enrichments ≥2.0 (AS-1119, AR-960). Furthermore, to compare the genomic binding of Ncb2 between the isolates, the average binding profile for the promoter (fragments: −1/−49, −50/−300, −301/−550, −551/−800, −801/−1250 and ≥1251, relative to the start codon) was computed which showed that Ncb2 mainly recruited at the core promoter regions434546 (Fig. 1b). Genome-wide occupancy data analysis also revealed that Ncb2 binds both inside and downstream of the genes (Supplementary Figs S1 and S2). The most common model for transcriptional activation is that the binding, just upstream of the transcription start site (TSS) helps in placing polymerase II at the beginning of the gene. Presence of TF binding sites inside or downstream of gene may act as regulatory sequence for the same gene or act as enhancer for the control of same gene or downstream gene (acting as a distal enhancer) in orientation and position independent manner474849. Notably, all binding sites may not be functional or represents a non productive binding site or could just be alternative TSS505152.

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.