Overlapping coactivator function is required for transcriptional activation by theCandida glabrataPdr1 transcription factor

Abstract

AbstractAzole resistance in the pathogenic yeastCandida glabratais a serious clinical complication and increasing in frequency. The majority of resistant organisms have been found to contain a substitution mutation in the Zn2Cys6 zinc cluster-containing transcription factor Pdr1. These mutations typically lead to this factor driving high, constitutive expression of target genes like the ATP-binding cassette transporter-encoding geneCDR1. Overexpression of Cdr1 is required for the observed elevated fluconazole resistance exhibited by strains containing one of these hyperactivePDR1alleles. While the identity of hyperactivePDR1alleles has been extensively documented, the mechanisms underlying how these gain-of-function (GOF) forms of Pdr1 lead to elevated target gene transcription are not well understood. We have used a tandem affinity purification (TAP)-tagged form of Pdr1 to identify coactivator proteins that biochemically purify with the wild-type and two different GOF forms of Pdr1. Three coactivator proteins were found to associate with Pdr1: the SWI/SNF complex Snf2 chromatin remodeling protein and two different components of the SAGA complex, Spt7 and Ngg1. We found that deletion mutants lacking eitherSNF2orSPT7exhibited growth defects, even in the absence of fluconazole challenge. To overcome these issues, we employed a conditional degradation system to acutely deplete these coactivators and determined that loss of either coactivator complex, SWI/SNF or SAGA, caused defects in Pdr1-dependent transcription. A double degron strain that could be depleted for both SWI/SNF and SAGA exhibited a profound defect inPDR1autoregulation, revealing that these complexes work together to ensure high level Pdr1-dependent gene transcription.