Synergism of the Two Myb Domains of Tay1 Protein Results in High Affinity Binding to Telomeres

Double-stranded regions of the telomeres are recognized by proteins containing Myb-like domains conferring specificity toward telomeric repeats. Although biochemical and structural studies revealed basic molecular principles involved in DNA binding, relatively little is known about evolutionary pathways leading to various types of Myb domain-containing proteins in divergent species of eukaryotes. Recently we identified a novel type of telomere-binding protein YlTay1p from the yeast Yarrowia lipolytica containing two Myb domains (Myb1, Myb2) very similar to the Myb domain of mammalian TRF1 and TRF2. In this study we prepared mutant versions of YlTay1p lacking Myb1, Myb2, or both Myb domains and found that YlTay1p carrying either Myb domain exhibits preferential affinity to both Y. lipolytica (GGGTTAGTCA)n and human (TTAGGG)n telomeric sequences. Quantitative measurements of the protein binding to telomeric DNA revealed that the presence of both Myb domains is required for a high affinity of YlTay1p to either telomeric repeat. Additionally, we performed detailed thermodynamic analysis of the YlTay1p interaction with its cognate telomeric DNA, which is to our knowledge the first energetic description of a full-length telomeric-protein binding to DNA. Interestingly, when compared with human TRF1 and TRF2 proteins, YlTay1p exhibited higher affinity not only for Y. lipolytica telomeres but also for human telomeric sequences. The duplication of the Myb domain region in YlTay1p thus produces a synergistic effect on its affinity toward the cognate telomeric sequence, alleviating the need for homodimerization observed in TRF-like proteins possessing a single Myb domain

Functional information from clinically-derived drug resistant forms of the Candida glabrata Pdr1 transcription factor.

Azole drugs are the most frequently used antifungal agents. The pathogenic yeast Candida glabrata acquires resistance to azole drugs via single amino acid substitution mutations eliciting a gain-of-function (GOF) hyperactive phenotype in the Pdr1 transcription factor. These GOF mutants constitutively drive high transcription of target genes such as the ATP-binding cassette transporter-encoding CDR1 locus. Previous characterization of Pdr1 has demonstrated that this factor is negatively controlled by the action of a central regulatory domain (CRD) of ~700 amino acids, in which GOF mutations are often found. Our earlier experiments demonstrated that a Pdr1 derivative in which the CRD was deleted gave rise to a transcriptional regulator that could not be maintained as the sole copy of PDR1 in the cell owing to its toxically high activity. Using a set of GOF PDR1 alleles from azole-resistant clinical isolates, we have analyzed the mechanisms acting to repress Pdr1 transcriptional activity. Our data support the view that Pdr1-dependent transactivation is mediated by a complex network of transcriptional coactivators interacting with the extreme C-terminal part of Pdr1. These coactivators include but are not limited to the Mediator component Med15A. Activity of this C-terminal domain is controlled by the CRD and requires multiple regions across the C-terminus for normal function. We also provide genetic evidence for an element within the transactivation domain that mediates the interaction of Pdr1 with coactivators on one hand while restricting Pdr1 activity on the other hand. These data indicate that GOF mutations in PDR1 block nonidentical negative inputs that would otherwise restrain Pdr1 transcriptional activation. The strong C-terminal transactivation domain of Pdr1 uses multiple different protein regions to recruit coactivators

Jjj1 Is a Negative Regulator of Pdr1-Mediated Fluconazole Resistance in Candida glabrata

ABSTRACT The high prevalence of fluconazole resistance among clinical isolates of Candida glabrata has greatly hampered the utility of fluconazole for the treatment of invasive candidiasis. Fluconazole resistance in this yeast is almost exclusively due to activating mutations in the transcription factor Pdr1, which result in upregulation of the ABC transporter genes CDR1, PDH1, and SNQ2 and therefore increased fluconazole efflux. However, the regulation of Pdr1 is poorly understood. In order to identify genes that interact with the Pdr1 transcriptional pathway and influence the susceptibility of C. glabrata to fluconazole, we screened a collection of deletion mutants for those exhibiting increased resistance to fluconazole. Deletion of the gene coding for a protein homologous to the Saccharomyces cerevisiae J protein Jjj1 resulted in decreased fluconazole susceptibility. We used the SAT1 flipper method to generate independent deletion mutants for JJJ1 in an SDD clinical isolate. Expression of both CDR1 and PDR1 was increased in the absence of JJJ1. In the absence of CDR1 or PDR1, deletion of JJJ1 has only a modest effect on fluconazole susceptibility. Transcriptional profiling using transcriptome sequencing (RNA-seq) revealed upregulation of genes of the Pdr1 regulon in the absence of JJJ1. Jjj1 appears to be a negative regulator of fluconazole resistance in C. glabrata and acts primarily through upregulation of the ABC transporter gene CDR1 via activation of the Pdr1 transcriptional pathway. IMPORTANCE Candida glabrata is the second most common species of Candida recovered from patients with invasive candidiasis. The increasing number of infections due to C. glabrata, combined with its high rates of resistance to the commonly used, well-tolerated azole class of antifungal agents, has limited the use of this antifungal class. This has led to the preferential use of echinocandins as empirical treatment for serious Candida infections. The primary mechanism of resistance found in clinical isolates is the presence of an activating mutation in the gene encoding the transcription factor Pdr1 that results in upregulation of one or more of the efflux pumps Cdr1, Pdh1, and Snq2. By developing a better understanding of this mechanism of resistance to the azoles, it will be possible to develop strategies for reclaiming the utility of the azole antifungals against this important fungal pathogen