The Candida glabrata Upc2A transcription factor is a global regulator of antifungal drug resistance pathways

The G898D mutant form of Upc2A confers elevated fluconazole resistance in C. glabrata.

“This mutant Upc2A drove elevated fluconazole resistance and behaved like the hyperactive S. cerevisiae factor.”

• Also stated in: § A gain-of-function form of Upc2A confers elevated fluconazole resistance · plain

  “Introduction of the G898D mutation into UPC2A led to the resulting factor exhibiting elevated fluconazole resistance when compared to either the tagged or untagged version of the wild-type gene.”

Loss of UPC2A causes a dramatic increase in fluconazole susceptibility.

“Loss of UPC2A caused a dramatic increase in fluconazole susceptibility.”

ERG11 is an example of a locus controlled by Upc2A but not Pdr1.

“The ERG11 gene is an example of a locus controlled by Upc2A but not Pdr1.”

The large group of Upc2A-bound genes includes loci not typically considered as target genes, such as components of the translational machinery.

“This large group of Upc2A-bound genes include loci not typically considered as target genes, such as components of the translational machinery”

The third largest class of promoters (85) were only bound by Upc2A under induced conditions (wild-type + fluconazole and G898D Upc2A +/- fluconazole).

“The third largest class of promoters (85) were only bound by Upc2A under conditions we consider induced (wild-type + fluconazole and G898D Upc2A +/- fluconazole).”

There were 565 promoters that could only be bound by Upc2A in the presence of fluconazole.

“Interestingly, there were 565 of these promoters that could only be bound by Upc2A in the presence of fluconazole while others were bound in multiple different conditions.”

The top 4 GO terms enriched in genes bound by both Upc2A and Pdr1 were associated with transmembrane transport or integral membrane components.

“The top 4 GO terms enriched in genes bound by both these Zn2Cys6-containing transcription factors were associated with transmembrane transport or integral membrane components (S2 Table).”

The largest number of Upc2A-bound genes was seen for the wild-type factor in the presence of fluconazole (>1000 promoters bound).

“The largest number of bound genes was seen for the wild-type factor in the presence of fluconazole (>1000 promoters bound).”

Over a two-hour fluconazole challenge, no differences in the levels of wild-type and G898D Upc2A are seen.

“Over the time course of fluconazole challenge (two hours), no differences in the levels of these two forms of Upc2A were seen.”

GO term enrichment analysis indicated that very different genes were associated with the three patterns of Upc2A DNA-binding.

“GO term enrichment analysis of these three different classes of promoters indicated that very different genes were associated with these patterns of Upc2A DNA-binding.”

Alignment of C. glabrata Upc2A and ScUpc2 indicated that G898 is the analogous position in Upc2A.

“Alignment of C. glabrata Upc2A and ScUpc2 indicated that G898 was the analogous position in Upc2A.”

Binding of wild-type Upc2A increases in the presence of fluconazole compared to absence of drug.

“Binding of wild-type Upc2A is represented by the read depth and can be seen to increase in the presence of fluconazole compared to in the absence of the drug.”

The class of genes bound under all 4 conditions was most predominantly enriched for proteins associated with the plasma membrane.

“The class of genes bound under all 4 different conditions was most predominantly enriched for proteins associated with the plasma membrane (S1B Table)”

Fluconazole elevates PDR1 and CDR1 by 4- or 7-fold, respectively, when wild-type UPC2A is present.

“The presence of fluconazole elevated PDR1 and CDR1 by 4- or 7-fold, respectively when the wild-type UPC2A gene was present.”

The ScUpc2 SREs are most closely fit to the right-hand element of the Upc2A SRE predicted by MEME-ChIP.

“These ScUpc2 SREs are most closely fit to the right-hand element of the Upc2A SRE predicted by MEME-ChIP.”

Transcription of the FKS1 gene, encoding a β-glucan synthase protein, is responsive to Upc2A.

“Transcription of the FKS1 gene, encoding a β-glucan synthase protein was also found to be responsive to Upc2A”

The inducible class of Upc2A-bound promoters showed the strongest enrichment for genes involved in fatty acid biosynthesis.

“the inducible class of Upc2A-bound promoters showed the strongest enrichment for genes involved in fatty acid biosynthesis (S1C Table).”

Comparison of genes bound by Upc2A with those previously found associated with Pdr1 indicated a high degree of overlap between these two target gene suites.

“Comparison of the genes bound by Upc2A with those we previously found to be associated with Pdr1 indicated a high degree of overlap between these two target gene suites.”

• Also stated in: § Overlap between Upc2A- and Pdr1-controlled genes by ChIP-seq · plain

  “Strikingly, more than 50% of Pdr1 target promoters were also associated with Upc2A binding (Fig 2C).”

upc2AΔ strains are hypersensitive to caspofungin.

“consistent with upc2AΔ strains being hypersensitive to caspofungin”

The next largest group of bound promoters (309) were bound under all 4 different conditions.

“The next largest group of bound promoters (309) were found to be bound under all 4 different conditions.”

UPC2A transcription is elevated approximately two-fold by fluconazole, irrespective of the UPC2A allele tested.

“UPC2A transcription was elevated approximately two-fold by fluconazole, irrespective of the UPC2A allele tested.”

More than 50% of Pdr1 target promoters were also associated with Upc2A binding.

“Strikingly, more than 50% of Pdr1 target promoters were also associated with Upc2A binding (Fig 2C).”

The levels of wild-type and G898D forms of Upc2A are not detectably different by western blotting with anti-Upc2A antiserum.

“The levels of the wild-type and G898D forms of Upc2A were not detectably different as shown by blotting with the anti-Upc2A polyclonal antiserum.”

Introduction of the G898D UPC2A allele modestly reduces fluconazole induction of PDR1 and CDR1 to 2-fold and 4-fold, respectively.

“Introduction of the G898D UPC2A allele modestly reduced fluconazole induction for these two genes to 2-fold for PDR1 and 4-fold for CDR1.”

Binding of G898D Upc2A is constant irrespective of the presence of drug.

“Binding of G898D Upc2A was constant, irrespective of the presence of the drug.”

The largest class of Upc2A-bound genes was enriched for components of the translation apparatus.

“The largest class of genes was enriched for components of the translation apparatus (S1A Table).”

None of the three FKS genes were altered in the presence of the gain-of-function form of UPC2A.

“None of these genes were altered in the presence of this gain-of-function form of UPC2A while both ERG1 and AUS1 were elevated (Fig 6B), confirming the functionality of this hypermorphic form of Upc2A.”

GO terms enriched in the 53 direct target genes included ergosterol biosynthesis, plasma membrane and cell wall biogenesis, membrane transport, sterol uptake and RNA polymerase II core promoter-binding factors.

“GO terms enriched in this set of genes included ergosterol biosynthesis, plasma membrane and cell wall biogenesis, membrane transport, sterol uptake and RNA polymerase II core promoter-binding factors.”

ERG1 contains an everted SRE repeat.

“Note that ERG1 contains an everted SRE repeat indicated by the arrows.”

Expression of Pdr1 was not affected when the CDR1 SRE was removed.

“Expression of Pdr1 was not affected when the CDR1 SRE was removed but fluconazole induction of Pdr1 was reduced when the SRE was removed from the PDR1 promoter.”

The only GO term enriched in the 64 genes that require Upc2A for FLC induction yet are not bound by Upc2A represents loci involved in ergosterol biosynthesis.

“The only GO term enriched in the 64 genes that require the presence of Upc2A for FLC induction (yet are not bound by Upc2A) represent loci involved in ergosterol biosynthesis.”

The genes in this class of enriched genes include UPC2B and homologues of S. cerevisiae NRG2 and ADR1.

“The genes in this class of enriched genes include UPC2B and homologues of S. cerevisiae NRG2 and ADR1.”

Two ERG genes were exceptions to the general trends of ERG gene regulation by fluconazole and Upc2A.

“There were two ERG genes that were exceptions to these general trends of regulation.”

ERG10, ERG13, HMG1 and ERG20 were not influenced by fluconazole challenge in wild-type cells.

“ERG10, ERG13, HMG1 and ERG20, which all encode early steps in ergosterol biosynthesis, were not influenced by fluconazole challenge in wild-type cells although expression of these genes was strongly depressed under these same conditions in the absence of UPC2A.”

The mutant form of the ERG1 SRE blocked formation of both complexes.

“The mutant form of this SRE blocked formation of both complexes.”

QDR2 and RTA1 showed higher levels of fluconazole induction in the upc2AΔ strain.

“SNQ2 and PDH1 were reduced to approximately 50% of their normal drug-induced levels in the upc2AΔ strain while QDR2 and RTA1 showed higher levels of fluconazole induction in the same strain.”

The ChIP-seq data predicted a potential SRE upstream of the FKS1 and FKS2 genes.

“The ChIP-seq data predicted a potential SRE upstream of the FKS1 and FKS2 genes (S1 and S2 Tables).”

Five of these nine ergosterol biosynthesis genes also contained SREs.

“Five of these 9 genes also contained SREs.”

The majority of ERG genes showed at least two-fold induction by fluconazole in wild-type cells when they corresponded to steps later in the ergosterol biosynthetic pathway.

“The majority of ERG genes showed at least two-fold induction by fluconazole in wild-type cells as long as these genes corresponded to steps later in the ergosterol biosynthetic pathway.”

ERG1 and AUS1 were elevated in the presence of the gain-of-function form of UPC2A.

“None of these genes were altered in the presence of this gain-of-function form of UPC2A while both ERG1 and AUS1 were elevated (Fig 6B), confirming the functionality of this hypermorphic form of Upc2A.”

Expression of Erg11 was unaffected in these backgrounds.

“Expression of Erg11 was unaffected in these backgrounds as these alterations were restricted to the CDR1 and PDR1 promoters.”

PDR1 was similarly induced irrespective of the UPC2A background.

“CDR1 fluconazole induction was reduced upon loss of Upc2A but PDR1 was similarly induced irrespective of the UPC2A background (Fig 3B).”

The final and largest class of genes found to be bound by Upc2A were less than two-fold induced by fluconazole.

“The final and largest class of genes found to be bound by Upc2A were less than two-fold induced by fluconazole.”

Upc2A DNA-binding to CDR1 is regulated in a manner similar to that seen for ERG11.

“Upc2A DNA-binding to CDR1 was regulated in a manner similar to that seen for ERG11.”

These Upc2A-dependent but indirect targets include ERG4, ERG8, ERG9, ERG24, ERG26 and ERG27.

“These Upc2A-dependent but indirect targets include ERG4, ERG8, ERG9, ERG24, ERG26 and ERG27.”

SNQ2 and PDH1 were reduced to approximately 50% of their normal drug-induced levels in the upc2AΔ strain.

“SNQ2 and PDH1 were reduced to approximately 50% of their normal drug-induced levels in the upc2AΔ strain while QDR2 and RTA1 showed higher levels of fluconazole induction in the same strain.”

The presence of the GOF form of UPC2A caused relatively small changes in gene transcription.

“The presence of the GOF form of UPC2A caused relatively small changes in gene transcription.”

Introduction of the mSRE into ERG1, CDR1 or PDR1 promoters led to a reduction in the level of fluconazole-induced β-galactosidase activity produced by each respective fusion gene.

“Introduction of the mSRE into ERG1, CDR1 or PDR1 promoters led to a reduction in the level of fluconazole-induced β-galactosidase activity produced by each respective fusion gene (Fig 5A).”

Both epitope-tagged UPC2A alleles behaved like the wild-type strain.

“Loss of UPC2A caused hypersensitivity to all these agents (Fig 6A) while both epitope-tagged alleles behaved like the wild-type strain.”

ERG1 was similarly drug induced in both wild-type and upc2AΔ cells.

“ERG1, one of the genes showing the best induction by fluconazole in wild-type cells and most prominent peak of Upc2A ChIP-seq density (See S3 Table), was similarly drug induced in both wild-type and upc2AΔ cells.”

There are 117 genes that are only FLC-inducible in the presence of Upc2A.

“However, there are 117 genes that are only FLC-inducible in the presence of Upc2A.”

The bound regions for Pdr1 and Upc2A extensively overlap in the upstream region of CDR1.

“The bound regions for Pdr1 and Upc2A extensively overlap in the upstream region of CDR1.”

The reduction in fluconazole resistance caused by mSRE-containing CDR1 was modestly enhanced by the simultaneous loss of the SRE from the PDR1 gene.

“This was modestly enhanced by the simultaneous loss of the SRE from the PDR1 gene.”

ERG8 was not seen to contain a detectable ChIP-seq peak for Upc2A binding.

“Strikingly, ERG8 was not seen to contain a detectable ChIP-seq peak for Upc2A binding.”

Only 11 genes were observed to be elevated at least 1.4-fold in the presence of G898D UPC2A.

“There were only 11 genes observed to be elevated at least 1.4-fold.”

Nine of the eleven genes elevated by G898D UPC2A encoded products involved in ergosterol biosynthesis.

“A striking feature shared by these genes was that nine of eleven encoded products that were involved in the biosynthesis of ergosterol.”

Pdr1-regulated genes that were also associated with SREs exhibited less dependence on the presence of Upc2A for fluconazole induction than most ERG genes.

“Pdr1-regulated genes that were also associated with SREs exhibited less dependence on the presence of Upc2A for fluconazole induction than seen for most ERG genes.”

ERG8 was similarly fluconazole-induced in both wild-type and upc2AΔ cells, irrespective of the presence of Upc2A.

“ERG8, encoding an enzyme that functions early in the ergosterol pathway, was also similarly fluconazole-induced in both gene backgrounds, irrespective of the presence of Upc2A.”

PDR16 and RSB1 were repressed by the presence of fluconazole in wild-type cells.

“Two other Pdr1 target genes (PDR16, RSB1) were repressed by the presence of fluconazole in wild-type cells and their expression lowered further when upc2AΔ cells were treated with fluconazole.”

CDR1 fluconazole induction was reduced upon loss of Upc2A.

“CDR1 fluconazole induction was reduced upon loss of Upc2A but PDR1 was similarly induced irrespective of the UPC2A background (Fig 3B).”

The presence of the G898D Upc2A protein led to enhanced expression of multiple genes involved in ergosterol biosynthesis.

“As we have seen for ERG11 (Fig 1B and 1C), the presence of the G898D Upc2A protein led to enhanced expression of multiple genes involved in ergosterol biosynthesis.”

The CDR1 SRE exhibited two DNase I hypersensitive sites linked to Upc2A binding.

“The CDR1 SRE exhibited two DNase I hypersensitive sites linked to Upc2A binding while the PDR1 SRE showed a clear protected region but no associated hypersensitive site.”

GO term analysis of this class indicated that the top enriched categories were involved in translation and the ribosome (60 total).

“GO term analysis of this class of genes indicated that the top enriched categories were involved in translation and the ribosome (60 total).”

The simplest class of fluconazole-induced genes are represented by the 53 direct target genes.

“The simplest class of fluconazole-induced genes are represented by the 53 direct target genes.”

The major reduction in fluconazole resistance was caused by the presence of the mSRE-containing form of the CDR1 gene.

“The major reduction in fluconazole resistance was caused by the presence of the mSRE-containing form of the CDR1 gene.”

PDR16 and RSB1 expression was lowered further when upc2AΔ cells were treated with fluconazole.

“Two other Pdr1 target genes (PDR16, RSB1) were repressed by the presence of fluconazole in wild-type cells and their expression lowered further when upc2AΔ cells were treated with fluconazole.”

Genes encoding enzymes that function later in ergosterol biosynthesis, such as ERG11, were induced by fluconazole at least two-fold in wild-type cells.

“Genes encoding enzymes that function later in ergosterol biosynthesis (like ERG11) were induced by fluconazole at least two-fold in wild-type cells but depressed by at least two-fold in a upc2AΔ background.”

The next highest categories were plasma membrane or amino acid transmembrane transport and represented 74 genes.

“The next highest categories were plasma membrane or amino acid transmembrane transport and represented 74 genes.”

Expression of ERG10, ERG13, HMG1 and ERG20 was strongly depressed under fluconazole conditions in the absence of UPC2A.

“ERG10, ERG13, HMG1 and ERG20, which all encode early steps in ergosterol biosynthesis, were not influenced by fluconazole challenge in wild-type cells although expression of these genes was strongly depressed under these same conditions in the absence of UPC2A.”

The wild-type ERG1 probe produced two different species of protein:DNA complex.

“The wild-type ERG1 probe produced two different species of protein:DNA complex, possibly corresponding to either one or two binding sites being occupied with Upc2A.”

Genes encoding enzymes that function later in ergosterol biosynthesis were depressed by at least two-fold in a upc2AΔ background.

“Genes encoding enzymes that function later in ergosterol biosynthesis (like ERG11) were induced by fluconazole at least two-fold in wild-type cells but depressed by at least two-fold in a upc2AΔ background.”

Fluconazole induction of Pdr1 was reduced when the SRE was removed from the PDR1 promoter.

“Expression of Pdr1 was not affected when the CDR1 SRE was removed but fluconazole induction of Pdr1 was reduced when the SRE was removed from the PDR1 promoter.”

Loss of UPC2A reduced basal expression of FKS1 by 50%.

“Loss of UPC2A reduced basal expression of FKS1 by 50% and had a modest effect on FKS2 (30% reduction) (Fig 6C).”

This region shows strong sequence conservation with the 893-898 region of C. glabrata Upc2A.

“This region shows strong sequence conservation with the 893–898 region of C. glabrata Upc2A.”

Caspofungin induction of FKS2 was unaffected by the absence of Upc2A.

“The addition of caspofungin strongly induced FKS2 expression as expected [35] with this induction unaffected by the absence of Upc2A.”

A binding site for Upc2A lies upstream of PDR1.

“Here we establish that a binding site for Upc2A lies upstream of PDR1 and is required for normal activation of PDR1 expression (Fig 5) as well as many of the other genes controlled directly by Pdr1.”

Loss of the SRE from the FKS1 promoter caused a significant reduction in FKS1-dependent expression of lacZ in the absence of caspofungin.

“Loss of the SRE from the FKS1 promoter caused a significant reduction in FKS1-dependent expression of lacZ in the absence of caspofungin (Fig 6E).”

The G898D UPC2A allele has a surprisingly limited effect on gene expression, triggering significant transcriptional changes almost exclusively in genes associated with ergosterol biosynthesis.

“The G898D UPC2A allele has a surprisingly limited effect on gene expression as this allele was seen to trigger significant transcriptional changes almost exclusively in genes associated with ergosterol biosynthesis (Table 2).”

The addition of caspofungin strongly induced FKS2 expression.

“The addition of caspofungin strongly induced FKS2 expression as expected [35] with this induction unaffected by the absence of Upc2A.”

• cites [35] SK Katiyar, 2012, Fks1 and Fks2 are functionally redundant but differentially regulated in Candida glabrata: implications for echinocandin resistance, Antimicrob Agents Chemother sourceNot checked yet

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Loss of UPC2A had a modest effect on FKS2, reducing it by 30%.

“Loss of UPC2A reduced basal expression of FKS1 by 50% and had a modest effect on FKS2 (30% reduction) (Fig 6C).”

A large increase in ChIP-seq peaks is seen for Upc2A when azole-treated cells are compared to untreated cells.

“First, a large increase in ChIP-seq peaks is seen for Upc2A when azole-treated cells are compared to untreated cells.”

The Upc2A binding site upstream of PDR1 is required for normal activation of PDR1 expression.

“Here we establish that a binding site for Upc2A lies upstream of PDR1 and is required for normal activation of PDR1 expression (Fig 5) as well as many of the other genes controlled directly by Pdr1.”

C. glabrata has high intrinsic resistance to fluconazole.

“First, this pathogen has a high intrinsic resistance to fluconazole [6].”

• cites [6] MC Arendrup, 2017, Multidrug-Resistant Candida: Epidemiology, Molecular Mechanisms, and Treatment, J Infect Dis sourceNot checked yet

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Candida glabrata commonly acquires resistance to azole drugs like fluconazole via mutations in a gene encoding a transcription factor called PDR1.

“Second, high level resistant isolates easily arise that contain gain-of-function (GOF) mutations in a transcription factor-encoding gene called PDR1 [7–9].”

• cites [7] JP Vermitsky, 2004, Azole resistance in Candida glabrata: coordinate upregulation of multidrug transporters and evidence for a Pdr1-like transcription factor, Antimicrob Agents Chemother sourceNot checked yet

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• cites [8] HF Tsai, 2006, Candida glabrata PDR1, a transcriptional regulator of a pleiotropic drug resistance network, mediates azole resistance in clinical isolates and petite mutants, Antimicrob Agents Chemother sourceNot checked yet

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• cites [9] SG Whaley, 2016, Azole Resistance in Candida glabrata, Current infectious disease reports sourceNot checked yet

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Our previous work indicated overlap between Upc2A and Pdr1 target genes.

“Since our previous work indicated overlap between Upc2A and Pdr1 target genes [26]”

• cites [26] BG Vu, 2018, Construction and Use of a Recyclable Marker To Examine the Role of Major Facilitator Superfamily Protein Members in Candida glabrata Drug Resistance Phenotypes, mSphereNot checked yet

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Loss of UPC2A is sufficient to strongly reduce fluconazole resistance of a GOF PDR1 mutant allele in C. glabrata.

“First, loss of UPC2A (C. glabrata homologue of C. albicans UPC2) was sufficient to strongly reduce fluconazole resistance of a GOF PDR1 mutant allele [17].”

• cites [17] SG Whaley, 2014, UPC2A is required for high-level azole antifungal resistance in Candida glabrata, Antimicrob Agents Chemother sourceNot checked yet

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The increased activation of ERG11 in the presence of G898D UPC2A is due to increased function of Upc2A rather than a change in its expression compared to wild-type.

“These data argue that the increased activation seen for ERG11 in the presence of the G898D UPC2A gene was due to increased function of Upc2A rather than a change in its expression compared to the wild-type factor.”

Genetic means of reducing flux through the ergosterol pathway lead to induction of the Pdr pathway, including PDR1 and CDR1, in a Upc2A-dependent manner.

“Second, genetic means of reducing the flux through the ergosterol pathway led to induction of the Pdr pathway, including PDR1 and CDR1, in a Upc2A-dependent manner [18].”

• cites [18] BG Vu, 2019, Evidence that Ergosterol Biosynthesis Modulates Activity of the Pdr1 Transcription Factor in Candida glabrata., MBioNot checked yet

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The overlap between Upc2A and Pdr1 transcriptional circuits extends well beyond PDR1 and CDR1.

“these new data indicate that the overlap between these two transcriptional circuits extends well beyond the initial two genes.”

Mutant PDR1 transcription factors exhibit enhanced target gene expression compared to the wild-type allele.

“These mutant transcription factors exhibit enhanced target gene expression when compared to the wild-type allele.”

There are no gain-of-function forms currently known for UPC2A.

“there are no gain-of-function (GOF) forms currently known for UPC2A.”

• cites [22] H Yang, 2015, Structural mechanism of ergosterol regulation by fungal sterol transcription factor Upc2, Nat Commun sourceNot checked yet

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• cites [23] CJ Heilmann, 2010, An A643T mutation in the transcription factor Upc2p causes constitutive ERG11 upregulation and increased fluconazole resistance in Candida albicans, Antimicrob Agents Chemother sourceNot checked yet

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• cites [24] SJ Hoot, 2011, An A643V amino acid substitution in Upc2p contributes to azole resistance in well-characterized clinical isolates of Candida albicans, Antimicrob Agents Chemother sourceNot checked yet

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Upc2A-mediated transcriptional activation extends far beyond its well-appreciated control of ERG gene expression.

“Our data provide a new view of the global importance of Upc2A-mediated transcriptional activation as extending far beyond its well-appreciated control of ERG gene expression.”

G898D UPC2A behaves as a hyperactive transcriptional activator in C. glabrata, as previously seen for G888D Upc2 in S. cerevisiae.

“This is consistent with G898D UPC2A behaving as a hyperactive transcriptional activator in C. glabrata as has previously been seen for G888D Upc2 in S. cerevisiae [25].”

• cites [25] BS Davies, 2005, Dual activators of the sterol biosynthetic pathway of Saccharomyces cerevisiae: similar activation/regulatory domains but different response mechanisms, Mol Cell Biol sourceNot checked yet

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The upc2-1 mutation is a change of glycine to aspartate at position G888D in the carboxy-terminus of ScUpc2.

“The relevant mutation (upc2-1) [19] is a change of a glycine to an aspartate residue located at position (G888D) in the carboxy-terminus of ScUpc2.”

• cites [19] JH Crowley, 1998, A mutation in a purported regulatory gene affects control of sterol uptake in Saccharomyces cerevisiae, J Bacteriol sourceNot checked yet

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Upc2A is a master regulator of lipid and membrane protein biosynthesis.

“These studies provide a new perspective of Upc2A as a master regulator of lipid and membrane protein biosynthesis.”

Fluconazole resistance in C. glabrata likely involves coordination of the Pdr1- and Upc2A-dependent transcriptional circuits.

“Together, these data indicated that fluconazole resistance in C. glabrata was likely to involve coordination of the Pdr1- and Upc2A-dependent transcriptional circuits.”

These genes illustrate a more complicated mode of regulation by Upc2A as they can be fluconazole-regulated in the presence or absence of this factor.

“These genes illustrate a more complicated mode of regulation by Upc2A as they can be fluconazole-regulated in the presence or absence of this factor.”

The SREs indicated in Fig 4A are likely required for Upc2A binding to each promoter.

“These data argue that the SREs indicated in Fig 4A are likely required for Upc2A binding to each promoter.”

The G898D Upc2A protein is a functional hypermorphic form of Upc2A.

“None of these genes were altered in the presence of this gain-of-function form of UPC2A while both ERG1 and AUS1 were elevated (Fig 6B), confirming the functionality of this hypermorphic form of Upc2A.”

Each SRE identified in ERG1, CDR1 and PDR1 is required for normal drug-induced promoter activation.

“While some degree of fluconazole inducibility was retained in each mSRE-containing promoter, these data indicate that each SRE identified above is required for normal drug induced promoter activation.”

• Also stated in: § Phenotypes caused by loss of SRE function · plain

  “Normal expression of lacZ gene fusions requires the presence of intact SREs in Upc2A target promoters.”

The vast number of fluconazole-induced genes do not depend on the presence of Upc2A.

“These data argue that the vast number of fluconazole-induced genes do not depend on the presence of Upc2A.”

The coordinate up-regulation of ergosterol biosynthesis genes is likely responsible for the observed increase in fluconazole resistance caused by the G898D UPC2A allele.

“This coordinate up-regulation is likely responsible for the observed increase in fluconazole resistance caused by this allele.”

The large number of Upc2A target genes illustrates the breadth of processes transcriptionally influenced by this factor.

“The large number of Upc2A target genes illustrates the breadth of processes that are transcriptionally influenced by this factor.”

Upc2A appears to coordinate a broad group of genes including a large number of plasma membrane-localized proteins.

“Upc2A appears to be coordinating a broad group of genes including a large number of plasma membrane-localized proteins (See S1 and S3 Tables).”

Comparing the target gene sets defined by Upc2A and Pdr1 suggests a hierarchical relationship between these two transcription factors.

“Comparing the members of the target gene sets defined by Upc2A and Pdr1 suggests a hierarchical relationship between these two transcription factors.”

Upc2A transcriptionally regulates FKS1 expression.

“Upc2A transcriptionally regulates FKS1 expression.”

Upc2A may provide a link between the genetically separable pathways of azole and echinocandin resistance.

“These two antifungal drugs have been considered to be defined by genetically separable pathways but here we provide evidence that Upc2A may provide a link between them.”

C. glabrata Upc2A accumulates in the nucleus upon ergosterol limitation.

“Based on detailed structural and subcellular localization studies on S. cerevisiae Upc2 [22], we propose that C. glabrata Upc2A accumulates in the nucleus upon ergosterol limitation.”

• cites [22] H Yang, 2015, Structural mechanism of ergosterol regulation by fungal sterol transcription factor Upc2, Nat Commun sourceNot checked yet

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• AssumptionRests on an assumption: Fluconazole treatment causes ergosterol limitation in C. glabrata.

Little was known about the full range of target genes of Upc2A-like factors.

“Detailed analyses demonstrated the crucial role of these Upc2A-like factors in regulation of ERG gene biosynthesis [30,37,38] but little was known about the full range of their target genes.”

• cites [36] PJ Espenshade, 2007, Regulation of sterol synthesis in eukaryotes, Annu Rev Genet sourceNot checked yet

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Loss of UPC2A leads to a profound increase in fluconazole susceptibility, even in the presence of a GOF form of PDR1.

“Loss of UPC2A leads to a profound increase in fluconazole susceptibility, even in the presence of a GOF form of PDR1 [17].”

• cites [17] SG Whaley, 2014, UPC2A is required for high-level azole antifungal resistance in Candida glabrata, Antimicrob Agents Chemother sourceNot checked yet

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Upc2A appears to be a central determinant of the composition of the plasma membrane compartment in C. glabrata.

“Coupled with its control of ergosterol in this membrane, Upc2A appears to be a central determinant of the composition of this membrane compartment in C. glabrata.”

This provides an illustration of the overlapping modes of regulation controlling gene expression in response to ergosterol limitation.

“This provides an illustration of the overlapping modes of regulation controlling gene expression in response to ergosterol limitation.”

Our data illuminate the complexity and interrelationship of expression of genes involved in ergosterol biosynthesis with plasma membrane proteins and even the cell wall.

“Finally, our data also illuminate the complexity and interrelationship of expression of genes involved in ergosterol biosynthesis with plasma membrane proteins and even the cell wall.”

The Upc2A binding site upstream of PDR1 is required for many of the other genes controlled directly by Pdr1.

“Here we establish that a binding site for Upc2A lies upstream of PDR1 and is required for normal activation of PDR1 expression (Fig 5) as well as many of the other genes controlled directly by Pdr1.”

Construction and analysis of G898D Upc2A demonstrated that there is no particular prohibition on hyperactive alleles of UPC2A existing in this pathogenic yeast.

“Construction and analysis of G898D Upc2A demonstrated that there is no particular prohibition on hyperactive alleles of UPC2A existing in this pathogenic yeast.”

The interaction between Upc2A and the FKS1 gene provides a connection between azole resistance and echinocandin resistance.

“The finding of the interaction between Upc2A and the FKS1 gene provides an interesting connection between azole resistance, well-known to be impacted by Upc2A [40], and echinocandin resistance.”

• cites [40] M Nagi, 2011, Transcription factors CgUPC2A and CgUPC2B regulate ergosterol biosynthetic genes in Candida glabrata, Genes Cells sourceNot checked yet

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The ERG genes are especially sensitive to the level of activity of Upc2A.

“While the full range of Upc2A target genes illustrate the global importance of this transcription factor, the ERG genes are especially sensitive to the level of activity of this regulator.”