Candida albicans Swi/Snf and Mediator Complexes Differentially Regulate Mrr1-Induced MDR1 Expression and Fluconazole Resistance

Zhongle Liu; Lawrence C Myers

doi:10.1128/AAC.01344-17

. 2017 Oct 24;61(11):e01344-17. doi: 10.1128/AAC.01344-17

Candida albicans Swi/Snf and Mediator Complexes Differentially Regulate Mrr1-Induced MDR1 Expression and Fluconazole Resistance

Zhongle Liu ^b, Lawrence C Myers ^a,^b,^✉

PMCID: PMC5655090 PMID: 28807921

ABSTRACT

Long-term azole treatment of patients with chronic Candida albicans infections can lead to drug resistance. Gain-of-function (GOF) mutations in the transcription factor Mrr1 and the consequent transcriptional activation of MDR1, a drug efflux coding gene, is a common pathway by which this human fungal pathogen acquires fluconazole resistance. This work elucidates the previously unknown downstream transcription mechanisms utilized by hyperactive Mrr1. We identified the Swi/Snf chromatin remodeling complex as a key coactivator for Mrr1, which is required to maintain basal and induced open chromatin, and Mrr1 occupancy, at the MDR1 promoter. Deletion of snf2, the catalytic subunit of Swi/Snf, largely abrogates the increases in MDR1 expression and fluconazole MIC observed in MRR1^GOF mutant strains. Mediator positively and negatively regulates key Mrr1 target promoters. Deletion of the Mediator tail module med3 subunit reduces, but does not eliminate, the increased MDR1 expression and fluconazole MIC conferred by MRR1^GOF mutations. Eliminating the kinase activity of the Mediator Ssn3 subunit suppresses the decreased MDR1 expression and fluconazole MIC of the snf2 null mutation in MRR1^GOF strains. Ssn3 deletion also suppresses MDR1 promoter histone displacement defects in snf2 null mutants. The combination of this work with studies on other hyperactive zinc cluster transcription factors that confer azole resistance in fungal pathogens reveals a complex picture where the induction of drug efflux pump expression requires the coordination of multiple coactivators. The observed variations in transcription factor and target promoter dependence of this process may make the search for azole sensitivity-restoring small molecules more complicated.

KEYWORDS: azole resistance, Candida albicans, Mediator, Mrr1, Swi/Snf complex

INTRODUCTION

Fungus-specific zinc cluster [Zn(II)Cys6] family transcription factors regulate drug resistance-related efflux pump genes in multiple fungal pathogens, including Candida glabrata Pdr1, Candida albicans Tac1 and Mrr1, and Candida dubliniensis Mrr1 (1 –3). Induction of drug efflux pump expression by hyperactive zinc cluster transcription factors is an important mechanism by which human fungal pathogens develop azole drug resistance (1 –3). Azole-resistant clinical isolates from multiple species have been found to have gain-of-function (GOF) mutations in these transcription factors (1 –3). These zinc cluster transcription factors driving azole resistance share a similar domain structure that includes an N-terminal DNA binding domain, a C-terminal transcriptional activation domain, and a middle region of ∼700 amino acids that regulates the activity of the transcription factor (4, 5). The mechanism used by hyperactive zinc cluster transcription factors to activate their targets, however, is not systemically understood. This gap in knowledge makes it uncertain whether the hyperactive zinc cluster transcription factors use a common mechanism to drive efflux pump expression in azole resistance. This question is of critical importance given the recent identification of small molecules that can inhibit interactions between C. glabrata Pdr1 and coactivator targets to reduce resistance (6).

Seminal studies in Saccharomyces cerevisiae and C. glabrata identified Mediator as a key coactivator for the transcriptional activation of drug efflux pump genes by the Pdr family of zinc cluster transcription factors (3, 7, 8). Mediator is a conserved eukaryotic multisubunit complex that can serve as a functional intermediary between transcription activators and the RNA polymerase II machinery (9, 10). Mediator has a modular structure consisting of a head, middle, and tail, as well as a variably associated Cdk8 kinase module (11, 12). The fungal tail module contains multiple targets for different transcription activation domains, including targets for S. cerevisiae and C. glabrata Pdr1 (6, 7, 9, 10). Disruption of an interaction between Pdr1 and the Mediator tail module subunit Med15 genetically or through the use of a small molecule increases azole sensitivity in C. glabrata (6, 7). In addition to serving as a coactivator, Mediator has also been shown to function as a corepressor. Although considerably less is known about Mediator's corepressor function, it appears to reside primarily in its Cdk8 kinase module (9, 10, 13).

Compared to S. cerevisiae and C. glabrata, the mechanisms used to induce drug efflux pump expression in C. albicans are less well understood. C. albicans is a major opportunistic fungal pathogen that usually exists as a benign human commensal but can cause life-threatening systemic infections in immunocompromised individuals (14 –16). The goal of this study was to determine the coactivator requirements for C. albicans fluconazole resistance-related transcription factor Mrr1 (for multidrug resistance regulator). MRR1^GOF mutations induce drug efflux gene expression and are commonly found in fluconazole-resistant C. albicans clinical isolates (1, 2, 17, 18). Mrr1 binding sites have been identified in a number of gene promoters (19), including the major facilitator transporter MDR1. Hyperactivation of Mrr1 by xenobiotics and the subsequent expression of MDR1 play a key role in detoxifying compounds such as benomyl (17, 19 –21). Expression of MDR1 is the major, but not the exclusive, fluconazole resistance pathway in strains with MRR1^GOF mutations (17, 19). The role of additional Mrr1 target genes, such as aldo-keto reductases IFD1 and IFD6 (17, 19), have not been individually tested. In addition to Mrr1, the MDR1 promoter is also regulated by Cap1, a transcription factor that regulates the oxidative stress response and is responsive to H₂O₂ treatment (19 –22). An earlier study of Mrr1-mediated transcription activation showed that although the SAGA (Spt-Ada-Gcn5-acetyltransferase) coactivator complex is recruited to the MDR1 promoter, its histone acetyltransferase activity is not required for MDR1 induction by benomyl or MRR1^GOF mutations (23, 24).

Here, we tested the dependence of hyperactive Mrr1 on Mediator complex and the Swi/Snf ATP-dependent chromatin remodeling complex, which has been found to be recruited by hyperactive S. cerevisiae Pdr1 to target promoters (25). The Swi/Snf complex disrupts DNA-histone contacts and facilitates the local sliding or eviction of nucleosomes to modify DNA accessibility, and it often serves as a coactivator at highly induced promoters (26 –28). Our results show that MDR1 expression and fluconazole resistance in MRR1^GOF mutant strains were reduced by loss of Swi/Snf and, to a lesser extent, Mediator tail module function. Measurement of Mrr1, Swi/Snf, Mediator, and histone occupancy at multiple Mrr1 target promoters has allowed us to dissect the contributions on Swi/Snf and Mediator to Mrr1 activation. It has also allowed us to identify a novel negative genetic interaction between the Cdk8 module of Mediator and chromatin remodeling at Mrr1 target genes.

RESULTS

Swi/Snf and Mediator govern fluconazole resistance in MRR1^GOF mutant strains.

To determine the importance of coactivators in facilitating the increased azole resistance observed in MRR1^GOF mutant strains, we investigated the role of the Swi/Snf and Mediator complexes. Deletion of snf2, the catalytic subunit of the Swi/Snf complex, is known to completely disable the ability of the Swi/Snf complex to remodel chromatin in S. cerevisiae (29, 30). A C. albicans snf2 deletion strain is viable (31), and we found that deletion of snf2 significantly sensitizes MRR1^GOF mutant strains to fluconazole (Table 1). The fluconazole MIC of three MRR1^GOF (Q350L, P683S, and N803D) mutant strains dropped from 6 to 8 μg/ml to 0.75 to 1.5 μg/ml in the snf2 null background (Table 1). These differences in MIC observed at 30°C were also observed at 37°C, at a similar level, for several selected strains (data not shown). There also appeared to be a small general increase in fluconazole sensitivity in the snf2 null background with wild-type MRR1 (Table 1). A major reduction in fluconazole MIC in an snf2 null background is not general to all C. albicans zinc cluster GOF mutants that have increased efflux pump expression. Deletion of snf2 in strains with TAC1^GOF mutations, which induce the expression of the Cdr1 and Cdr2 efflux pumps (32 –34), led to only minor decreases in fluconazole MIC (Table 1). In addition to chromatin-based coactivators such as Swi/Snf, the Mediator tail module plays a critical coactivator role at highly induced promoters (9, 10). Deletion of the MED3 (35, 36) subunit gene of Mediator specifically disrupts the assembly of the tail module of the complex, which is thought to be the primary target for activation domains (7, 10, 37, 38). The med3 deletion reduced the fluconazole MIC in MRR1^GOF mutants but to a much lesser degree than snf2 deletion (Table 1). As opposed to the differential effect of snf2 deletion on fluconazole MIC MRR1^GOF and TAC1^GOF mutant strains, the med3 null showed a similar fold reduction in fluconazole MIC for both zinc cluster transcription factors (Table 1). An antagonistic relationship between the tail module and the Cdk8 module of Mediator has previously been observed for the regulation of large subsets of genes in S. cerevisiae (39). An increased fluconazole MIC in MRR1^GOF mutant strains in an ssn3 null background showed that a similar relationship might exist at the MRR1^GOF regulon (Table 1). The increased fluconazole MIC in this background required hyperactive Mrr1 and is specific for Mrr1 versus Tac1, as there is no increase in fluconazole MIC in the MRR1 wild strain or TAC1^GOF mutants in an ssn3 null background (Table 1). To reveal the origin of the coactivator dependence of MRR1^GOF-driven azole resistance, we proceeded to analyze the promoters of the MRR1^GOF regulon in molecular detail.

TABLE 1.

Fluconazole MIC of MRR1^GOF and TAC1^GOF mutants in snf2, med3, mdr1, and ssn3 null backgrounds

MRR1 or TAC1 allele	Fluconazole MIC^a (μg/ml)
	WT^b		snf2Δ/Δ^c(MDR1)	ssn3Δ/Δ		snf2Δ/Δ ssn3Δ/Δ^c		med3Δ/Δ (MDR1)
	MDR1	mdr1Δ/Δ	snf2Δ/Δ^c(MDR1)	MDR1	mdr1Δ/Δ	MDR1	mdr1Δ/Δ	med3Δ/Δ (MDR1)
Mrr1^WT	0.75–1 (yLM417)	0.75–1 (yLM623)	0.38–0.75 (yLM427)	0.75–1 (yLM446)	0.75 (yLM637)	0.25–0.38 (yLM456)	0.25–0.5 (yLM651)	0.75 (yLM436)
Mrr1^Q350L	6–8 (yLM418)	1 (yLM624)	0.75–1 (yLM428)	12 (yLM447)	1 (yLM638)	24–32 (yLM457)	1.5 (yLM652)	3 (yLM437)
Mrr1^P683S	6 (yLM419)	—^d	0.75–1 (yLM429)	12 (yLM448)	—	—	—	2 (yLM438)
Mrr1^N803D	6–8 (yLM420)	1.5 (yLM625)	0.75–1.5 (yLM430)	24 (yLM449)	2 (yLM639)	48–64 (yLM459)	3 (yLM653)	3 (yLM439)
TAC1^WT	0.75 (yLM167)	—	0.5–0.75 (yLM472)	0.75–1 (yLM236)	—	—	—	0.5–0.75 (yLM232)
TAC1^T225A	16 (ACY67)	—	8–12 (yLM474)	12 (yLM237)	—	—	—	4–6 (yLM233)
TAC1^A736V	16–24 (ACY13)	—	16–24 (yLM476)	12–16 (yLM238)	—	—	—	6–8 (yLM234)
TAC1^G980E	16–24 (ACY71)	—	16–24 (yLM478)	12–16 (yLM239)	—	—	—	8–12 (yLM235)

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Fluconazole MICs were measured by Etest at 30°C on YPD plates. Plates were incubated for 36 h before readout if not otherwise specified. Intermediate values, between scale marks, are presented as intervals. The exact strain used for MIC measurement is in parentheses. Data in italics were generated by our work presented in detail in a companion article (48).

WT refers to a strain background with wild-type SNF2, SSN3, and MED3.

Strains with snf2 deletion were incubated for 48 h before MIC readout.

—, not determined.

Mrr1-mediated gene activation requires the Swi/Snf complex.

To determine whether the Swi/Snf complex serves as a coactivator for hyperactive Mrr1, we systematically investigated the expression and chromatin structure of MDR1 and two other target genes (IFD6 [C1_04140W_A] and IFD1 [C1_04010C_A]). Benomyl is a potent inducer of Mrr1 transcriptional activation (17, 19 –21). Consistent with the fluconazole resistance data (Table 1), reverse transcription-quantitative PCR (RT-qPCR) analyses showed that benomyl-induced activation of MRR1 target genes is severely impaired in the snf2 null background (Fig. 1A to C). Earlier studies have shown that there is positive feedback from Mrr1 activation to MRR1 expression (17, 19). The modest upregulation of MRR1 mRNA level upon benomyl treatment is unaffected in the snf2 null mutant (Fig. 1D). The decreased MDR1, IFD6, and IFD1 induction in the snf2 null mutant is not due to decreased expression of MRR1. Similar to benomyl hyperactivation, increased MDR1, IFD6, and IFD1 expression in MRR1^GOF (Q350L, P683S [not shown], and N803D) strains is dependent on SNF2 (Fig. 1E to G). MRR1^GOF expression was also unaffected by the deletion of snf2 (see Fig. S1A and B in the supplemental material). H₂O₂ induction of MDR1 expression (20, 21) was largely independent of Mrr1 yet was severely compromised in the snf2 null mutant (Fig. 1H). In addition to activated expression, uninduced MDR1 expression is also decreased in an snf2 null strain (Fig. 1H). Uninduced MDR1 expression, however, was not decreased by deletion of MRR1 in an snf2 deletion background (Fig. 1H) or at other Mrr1 target promoters (see the legend to Fig. 1). Therefore, the Swi/Snf dependence of the MDR1 promoter is independent of Mrr1 hyperactivation and is likely to be a general property of the promoter rather than a specific property determined by Mrr1.

FIG 1 — Induction of Mrr1 target genes is dependent on the Snf2 subunit of the Swi/Snf complex. (A to D) RT-qPCR analysis of benomyl (BEN) induction of *MDR1* (A), *IFD6* (B), *IFD1* (C), and *MRR1* (D) in *SNF2*^+/+ and *snf2*^Δ/Δ strains (yLM417 and yLM427, respectively) treated with 50 μg/ml benomyl for the indicated period of time. As with all RT-qPCR analyses in this work, *ACT1* abundance was used as an internal reference. The relative expression level of *MDR1*, *IFD6*, *IFD1*, and *MRR1* in the untreated *SNF2*^+/+ strain (yLM417) was 0.02, 0.92, 0.42, and 0.72, respectively. The expression of each gene, in the absence of benomyl, in the *SNF2*^+/+ strain (yLM417) was set to 1. (E to G) RT-qPCR analysis of *MDR1* (E), *IFD6* (F), and *IFD1* (G) in *SNF2*^+/+ (+) and *snf2*Δ/Δ (Δ) strains with GOF *MRR1* mutant *MRR1*^Q350L (yLM418 and yLM428, respectively) or *MRR1*^N803D (yLM420 and yLM430, respectively). The magnitude of gene activation in the *SNF2*^+/+ *MRR1*^WT strain (yLM417) induced by 15 min of exposure to 8 μg/ml or 40 μg/ml benomyl is included for comparison. The basal expression level of each gene measured in the untreated *SNF2*^+/+ *MRR1*^WT strain (yLM417) was set to 1. (H) RT-qPCR analysis of *MDR1* transcription activation induced by 0.005% H₂O₂ (∼1.5 mM) in *SNF2*^+/+ and *snf2*Δ/Δ strains in a wild-type *MRR1* (yLM417 and yLM427, respectively) or *mrr1* null (SCMRR1M4A and yLM426, respectively) background. The uninduced *MDR1* expression level in the *SNF2*^+/+ *MRR1*^WT strain (yLM417) was set to 1.

Snf2 and Mrr1 bind cooperatively to the MDR1 promoter.

DNA-bound transcription factors frequently interact with the Swi/Snf complex to facilitate activated transcription at specific promoters (26 –28). We performed chromatin immunoprecipitation (ChIP) assays to determine how Mrr1 and Swi/Snf influence each other's occupancy at Mrr1 target gene promoters. Genome-wide ChIP analysis using a C-terminally tagged Mrr1^GOF mutant previously revealed peaks of Mrr1 occupancy in the promoters of its target genes (19). Here, we tagged a series of Mrr1 variants for ChIP by placing a 6His3Flag tag at their N termini. Unlike a C-terminal hemagglutinin (HA) tag, which hyperactivates wild-type Mrr1 (19), the N-terminal tag generally did not interfere with the drug inducibility of wild-type Mrr1 or the hyperactivity of Mrr1^GOF mutants (Fig. S2). ChIP experiments showed a peak of Mrr1 occupancy at the MDR1 promoter under noninducing growth conditions that increased in response to benomyl stimuli (Fig. 2A and B). The peak of Mrr1 occupancy (Fig. 2A) spans a region of the MDR1 promoter that contains a previously identified benomyl response element (BRE) (21), which exhibited enriched Mrr1 binding (19). This peak of Mrr1 occupancy was dramatically reduced in an snf2 null strain (Fig. 2B). Akin to Mrr1, there is a peak of Snf2 (Swi/Snf complex) occupancy at the Mrr1 binding peak at the MDR1 promoter (Fig. 2C). This peak is independent of Mrr1 hyperactivation but is largely dependent on the presence of MRR1, as Snf2 occupancy decreases almost 10-fold in an mrr1 null strain (Fig. 2C). The mutual dependence of Mrr1 and Snf2 occupancy at the MDR1 promoter is consistent with their requirements for MDR1 transcriptional activation (17) (Fig. 1). The occupancy of Mrr1^GOF mutant proteins at the MDR1 promoter was also reduced in an snf2 null background (Fig. 2D). Compared to the MDR1 promoter, the relationship between Mrr1 and Snf2 occupancy appears to be more directional at the IFD6 promoter (Fig. 2E and F). Mrr1 occupancy at the IFD6 promoter was enriched near two adjacent consensus Mrr1 binding motifs (19) (Fig. 2E and F). Deletion of snf2 did not have a statistically significant effect on Mrr1 occupancy at the IFD6 promoter (Fig. 2F); however, Snf2 occupancy showed a strong decrease in the mrr1 null strain (Fig. 2G). This finding suggests the decreased IFD6 expression in the snf2 null strain (Fig. 1) originates from a step downstream of Mrr1 binding.

Swi/Snf affects nucleosome density at the promoters of Mrr1 target genes.

Swi/Snf dependence of Mrr1 target gene expression (Fig. 1) and the dependence of Swi/Snf occupancy on Mrr1 (Fig. 2) suggested that chromatin remodeling is involved in the activation of these genes. To test this hypothesis, we used ChIP to monitor the pattern of histone occupancy at the MDR1 promoter and its response to drug-induced gene activation. Under noninducing conditions there is a local minimum in histone H3 (Fig. 3A) and histone H4 (Fig. S3A) density in the MDR1 promoter at the same location that exhibited a peak of Mrr1 and Snf2 occupancy (Fig. 2). Analysis of the ChIP data by displacement index plots (40) showed that hyperactivation of Mrr1 by benomyl resulted in nucleosome depletion at the MDR1 promoter, as monitored by the loss of H3 (Fig. 3A and B) and H4 (Fig. S3A) occupancy. The increased histone displacement observed at earlier time points of induction (15 min) was partially reduced at a longer time point (90 min) (Fig. S4A and B), when MDR1 gene induction began to attenuate (Fig. S4C). Deletion of snf2 resulted in loss of the peak of histone displacement as well as a broad increase in histone occupancy at the MDR1 promoter in both the uninduced and induced state (Fig. 3A and B and Fig. S3B and C). Even in the absence of snf2, however, benomyl-treated cells did show a small increase in histone displacement (Fig. 3A and B). Hyperactivation of Mrr1 by GOF mutations also resulted in increased histone displacement at the MDR1 promoter versus the histone occupancy observed in the uninduced wild-type MRR1 background (Fig. 3C and D and Fig. S3C). The increase in histone displacement was, however, smaller than that observed with benomyl induction (Fig. 3C and D). As with benomyl activation, histone displacement in the MRR1^GOF mutant strains was strongly dependent on SNF2 (Fig. 3C and D and Fig. S3C). In addition to the MDR1 promoter, histone occupancy at the IFD6 promoter showed a similar dependence on Swi/Snf as the MDR1 promoter in both the absence and presence of benomyl (Fig. S5). Swi/Snf helps maintain low nucleosome occupancy around the Mrr1 binding regions in the uninduced state and facilitates Mrr1 binding at the MDR1 promoter. Additionally, Swi/Snf was critical for the enhanced histone displacement observed at these promoters upon Mrr1 hyperactivation.

FIG 3 — Swi/Snf maintains low nucleosome occupancy surrounding the Mrr1 binding site at the *MDR1* promoter. (A) Histone H3 ChIP assays were performed using cells from *SNF2*^+/+ and *snf2*Δ/Δ strains (yLM417 and yLM427, respectively) grown in the absence and presence of benomyl (8 or 40 μg/ml for 15 min) using an anti-H3 antibody. For each ChIP product, the recovery rate (percent input) of DNA fragments representing the +1 nucleosome of the *ADE2* gene was set to 1 to calculate the relative H3 occupancy (relative nucleosome [Nuc.] density) at the 6 regions (m-1 to m-5) tested within the *MDR1* locus. (B) Histone H3 displacement index plot of data presented in panel A. The displacement index was calculated by first regrouping the relative H3/nucleosome occupancy shown in panel A by the 6 DNA regions tested (m-1 to m-5). The data in each group were normalized by setting the H3 occupancy measured in the untreated *SNF2*^+/+ strain (yLM417) to 1. The negative logarithm (base 2) of each normalized data point (H3 displacement index) was calculated and plotted. (C) Histone H3 ChIP assays were performed using cells from *SNF2*^+/+ and *snf2*Δ/Δ strains with *MRR1*^Q350L (yLM418 and yLM428, respectively) or *MRR1*^N803D (yLM420 and yLM430, respectively) using an anti-H3 antibody. Histone H3 occupancy in an *SNF2*^+/+ *MRR*^WT strain (yLM417) in the absence and presence of benomyl (8 or 40 μg/ml for 15 min) was assessed in parallel for comparison. (D) Data shown in panel C were replotted in histone displacement index form.

Mediator positively and negatively regulates transcriptional activation by hyperactive Mrr1.

As might be expected from the reduction in MRR1^GOF-induced fluconazole resistance in the med3 null background (Table 1), Mediator is both present at and affects the expression of hyperactive Mrr1 target genes. Unlike Swi/Snf, a pronounced peak of Mediator occupancy (represented by head module subunit Med17) is observed at the MDR1 promoter only under conditions of Mrr1 hyperactivation (Fig. 4A). This peak of Mediator occupancy is located at the site of enriched Mrr1 occupancy (Fig. 2), dependent on Mrr1 (Fig. 4A), and contained the Cdk8 module (represented by the Ssn3 subunit) of Mediator (Fig. 4B). The decreased fluconazole MIC in the med3 null mutant correlates with a large decrease in Mediator occupancy at the induced MDR1 (Fig. 4C) and IFD6 (Fig. S6) promoters upon med3 deletion. The decreased fluconazole resistance and Mediator occupancy observed in the med3 null mutant was also accompanied by a decrease in the induction of MDR1 expression by hyperactive Mrr1. The impact of MED3 on Mrr1 target genes, however, was more complex than that of Swi/Snf.

FIG 4 — Med3 and Mrr1 dependence of Mediator occupancy at the *MDR1* promoter. (A) Med17 ChIP assays showing Mediator occupancy at the *MDR1* promoter in *MRR1*^+/+ and *mrr1*Δ/Δ strains (yLM470 and yLM462, respectively), with one copy of their *MED17* C termini tagged by 3×HA (WT/HA), in the absence and presence of benomyl treatment (50 μg/ml for 15 min). Cultures of an *MRR1*^+/+ *MED17* untagged wild-type strain (WT/WT; SC5314) were processed in parallel. The recovery rate (percent input) of DNA fragments containing m-1 amplicon in the ChIP product obtained from untreated SC5314 culture was set to 1 to calculate the relative recovery at other loci. (B) Ssn3 ChIP assays showing the Mediator Cdk8 module occupancy at the *MDR1* promoter in an *MRR1*^+/+ untagged strain (WT/WT) or a strain with both copies of *SSN3* C termini tagged by 3×HA (HA/HA; yLM481) in the absence and presence of benomyl treatment (50 μg/ml for 15 min). The recovery rate was calculated as described for panel A. (C) Med17 ChIP assays showing Mediator occupancy at the *MDR1* promoter in *MED3*^+/+ and *med3*Δ/Δ strains with one copy of the *MED17* C termini tagged by 3×HA (WT/HA; yLM482 and yLM483, respectively), as well as a *MED3*^+/+ strain with two copies of untagged native *MED17* (WT/WT; SC5314) treated with benomyl at the indicated concentration for 15 min. Recovery rate was calculated as described for panel A.

The initial induction of MDR1 and IFD6 expression by benomyl is strongly impaired in the med3 null strain, but this difference is reduced at the later time points (Fig. 5A and B). Benomyl induction of IFD1, which was strongly dependent on Swi/Snf (Fig. 1C), was not affected by med3 deletion at earlier time points and actually showed increased expression at the later time points of benomyl induction in the med3 null mutant (Fig. 5C). Similar to the snf2 null mutant, the observed med3-dependent changes in these Mrr1 target genes do not appear to be a result of changes in MRR1 expression in the med3 null strain (Fig. 5D). Expression of MDR1 and IFD6 in MRR1^GOF mutant strains is impaired in the med3 null background to a similar degree as that in the snf2 null background (Fig. 5E and F). IFD1 again showed a different pattern, as its expression in MRR1^GOF strains was largely unaffected by med3 deletion (Fig. 5G). The observed decreases in gene expression in the med3 null MRR1^GOF strains cannot be attributed to changes in expression of the MRR1^GOF mutants (Fig. 5H) or decreased Mrr1^GOF mutant occupancy at the MDR1 promoter (Fig. 5I). The higher fluconazole MIC in the med3 null MRR1^GOF strains compared to the snf2 null MRR1^GOF strains (Table 1) is somewhat surprising given the comparable effect of med3 and snf2 deletion on MDR1 expression in the MRR1^GOF mutant strains (Fig. 5E). Despite the similar effects of med3 and snf2 deletion on MDR1 expression in the MRR1^GOF mutant strains, there are mechanistic differences between the null mutants. The deletion of med3 resulted in a distinct increase in Mrr1^GOF mutant occupancy at the MDR1 promoter (Fig. 5I) compared to the large decrease observed in the snf2 null mutant (Fig. 2D). Unlike the snf2 null mutant (Fig. 3), deletion of med3 (Fig. 5J and K) does not impact nucleosome density at the MDR1 promoter under noninducing conditions. The med3 null strain also was still able to facilitate histone displacement at the MDR1 promoter after treatment with benomyl and even showed a stronger decrease in histone occupancy versus the wild type at low benomyl concentrations (Fig. 5J and K).

FIG 5 — Expression of Mrr1 target genes and Mrr1 and histone H3 occupancy at the *MDR1* promoter in *med3* null background. (A to D) RT-qPCR analysis of *MDR1* (A), *IFD6* (B), *IFD1* (C), and *MRR1* (D) expression in *MED3*^+/+ and *med3*Δ/Δ strains with wild-type *MRR1* (yLM417 and yLM436, respectively) treated with 50 μg/ml benomyl for the indicated periods of time. The expression level of each gene measured in the untreated culture of the *MED3*^+/+ strain (yLM417) was set to 1. The relative expression level of *MDR1*, *IFD6*, *IFD1*, and *MRR1* in the untreated *med3* null strain (yLM436) was 0.40, 0.42, 1.33, and 1.19, respectively. Parallel analysis of an *snf2Δ/Δ MRR1*^WT strain (yLM427) was performed for comparison. (E to G) RT-qPCR analyses of *MDR1* (E), *IFD6* (F), and *IFD1* (G) expression in *MED3*^+/+ strains with an *MRR1*^Q350L or *MRR1*^N803D allele (yLM418 and yLM420) and their respective *med3*Δ/Δ derivatives (yLM437 and yLM439) and *snf2*Δ/Δ derivatives (yLM428 and yLM430). Benomyl induction (50 μg/ml for 15 min) of each gene in an *MRR1*^WT *MED3*^+/+ strain (WT; yLM417) was included for comparison. The expression level of each gene of the untreated *MRR1*^WT *MED3*^+/+ strain (yLM417) was set to 1. (H) RT-qPCR analysis comparing expression of *MRR1*^GOF mutants in *SNF2/MED3* wild-type, *snf2* null, and *med3* null backgrounds using samples tested in panels E to G. *MRR1*^Q350L abundance (relative to *ACT1*) in the *SNF2*^+/+*MED3*^+/+ background (yLM418) was set to 1. (I) ChIP assays of Mrr1^WT and Mrr1^N803D occupancy at the *MDR1* promoter in strains expressing N-terminal 6His3Flag-tagged wild-type Mrr1 (*HF-WT*; yLM421) or GOF mutant Mrr1^N803D (*HF-N803D*; yLM424) and their *med3*Δ/Δ derivatives (yLM440 and yLM443, respectively), as well as a strain with untagged wild-type Mrr1 (WT; yLM417). The recovery rate (percent input) of DNA fragments containing m-3 amplicon in the ChIP product obtained from the untagged strain (yLM417) was set to 1 to calculate the relative recovery at other loci. (J) Histone H3 ChIP assays at the *MDR1* promoter in *MED3*^+/+ and *med3*Δ/Δ strains (yLM417 and yLM436, respectively) treated with benomyl at the indicated concentration for 15 min before fixation. (K) Data shown in panel J replotted into histone displacement index form. The *MDR1* promoter histone H3 occupancy measured from the untreated *MED3*^+/+ strain (yLM417) was set as the reference (with zero H3 displacement index).

Ssn3 negatively regulates the activation of Mrr1 target genes.

To determine whether the Cdk8 module exerted a repressive effect on Mrr1 target gene activation that might explain the increase in fluconazole MIC in MRR1^GOF mutants in the ssn3 null background, we analyzed the SSN3 dependence of Mrr1 target gene induction. The benomyl induction of MDR1 (Fig. 6A and B), IFD6 (Fig. 6C), and IFD1 (Fig. 6D) were all enhanced in the ssn3 null mutant strain, although the effect on MDR1 expression was pronounced only under partial benomyl induction conditions (Fig. 6A and B). An increase in MRR1 expression itself was also noted in the ssn3 deletion background in the presence of benomyl (Fig. 6E). Deletion of ssn3 in the MRR1^GOF mutant strains resulted in a minor increase in MDR1 (Fig. 6F) and an ∼2-fold increase in IFD1 (Fig. 6H) and MRR1 (Fig. 6I). Despite a substantial increase in benomyl induction of IFD6 expression in the ssn3 null strain (Fig. 6C), IFD6 expression was largely unaffected in strains with MRR1^GOF mutations in the ssn3 null versus SSN3 wild-type background (Fig. 6G). A broader investigation of Mrr1 target genes in MRR1^GOF mutant strains showed that most were repressed by Ssn3, activated by Snf2, and variably affected by the deletion of med3 (Fig. S7). The magnitude of the effect of ssn3 deletion on MDR1 expression does not seem to adequately explain the observed increase in fluconazole resistance (Table 1). Other genes, including ERG11, CDR1, and CDR2, whose overexpression can lead to fluconazole resistance (2, 18, 32), were also found to be largely unaffected in MRR1^GOF mutant strains in the ssn3 null background (Fig. S8).

FIG 6 — Derepression of Mrr1 target genes in the absence of Mediator kinase module subunit Ssn3. (A and B) RT-qPCR analysis of *MDR1* expression in *SSN3*^+/+ and *ssn3*Δ/Δ strains (yLM417 and yLM446, respectively) treated with 50 μg/ml benomyl for the indicated time (A) or in an independent experiment treated with 8 μg/ml or 40 μg/ml benomyl for 15 min (B). *MDR1* expression in the untreated *SSN3*^+/+ strain (yLM417) was set to 1. The relative basal expression of *MDR1* in the *ssn3* null background (yLM446) was 0.52 (A) and 0.51 (B). (C to E) RT-qPCR analysis of *IFD6* (C), *IFD1* (D), and *MRR1* (E) expression in cDNA samples analyzed in panel A. The expression level of each gene in the untreated *SSN3*^+/+ strain (yLM417) was set to 1. The relative expression levels of *IFD6*, *IFD1*, and *MRR1* in the untreated *ssn3* null mutant (yLM446) were 0.42, 0.52, and 0.92, respectively. (F to I) RT-qPCR analysis of *MDR1* (F), *IFD6* (G), *IFD1* (H), and *MRR1* (I) expression in strains with the *MRR1*^Q350L or *MRR1*^N803D allele (yLM418 and yLM420, respectively) and their *ssn3*Δ/Δ derivatives (yLM447 and yLM449, respectively). The expression of each gene measured in the untreated *SSN3*^+/+ *MRR1*^WT strain (yLM417) was set to 1. Analysis of this strain after 15 min of exposure to 8 μg/ml or 40 μg/ml benomyl was included for comparison. For panel I, the *MRR1* expression level in the untreated *SSN3*^+/+ *MRR1*^Q350L strain (yLM418) was set to 1.

Ssn3 negatively regulates Mrr1 occupancy and histone displacement at the MDR1 and IFD6 promoters.

The Swi/Snf dependence of gene expression from, and histone displacement at, the promoters of Mrr1 target genes (Fig. 1 and 3) prompted us to determine whether the impact of SSN3 on expression of Mrr1 target genes was related to chromatin structure. The ssn3 null strain showed increased histone displacement at the MDR1 promoter under benomyl-induced conditions but not under noninduced conditions (Fig. 7A and B). As with the increase in MDR1 benomyl induction in the ssn3 null strain (Fig. 6B), the increase in histone displacement was more pronounced at a lower benomyl concentration (Fig. 7A and B). Earlier we observed a positive correlation between histone displacement and Mrr1 occupancy at the MDR1 promoter (Fig. 3A and B and 2B). Concordant with this observation, the ssn3 null strain also showed increased Mrr1 occupancy, versus an SSN3 wild-type strain, at the MDR1 promoter under conditions of benomyl induction (Fig. 7C). Increased histone displacement (Fig. 7D and E) and Mrr1 occupancy (Fig. 7F) were also observed at the MDR1 promoter in the MRR1^GOF ssn3Δ/Δ strains, even though neither was associated with a major increase in MDR1 gene expression (Fig. 6F). At the activated IFD6 promoter, histone displacement (Fig. S9A to D) and Mrr1 occupancy (Fig. S9E and F) were similarly increased in the ssn3 null strain. Interestingly, Mrr1 occupancy at both the MDR1 and IFD6 promoters was decreased by GOF mutations, versus a strain with wild-type Mrr1, in an SSN3 wild-type background (Fig. 2D and 7F and Fig. S9F).

FIG 7 — Histone H3 and Mrr1 occupancy at the *MDR1* promoter in *ssn3* deletion strains. (A and B) Histone H3 ChIP assays at the *MDR1* promoter in *SSN3*^+/+ and *ssn3*Δ/Δ strains (yLM417 and yLM446, respectively) treated with 8 μg/ml or 40 μg/ml benomyl for 15 min. Histone H3 occupancy in the untreated *SSN3*^+/+ strain (yLM417) was set as the reference (with zero H3 displacement index) when data from panel A were transformed into the H3 displacement index plot shown in panel B. (C) Mrr1 ChIP assays at the *MDR1* promoter in *SSN3*^+/+ and *ssn3*Δ/Δ strains expressing N-terminal 6His3Flag-tagged wild-type Mrr1 (yLM421 and yLM450, respectively) treated with the indicated concentration of benomyl for 15 min. The recovery rate (percent input) of DNA fragments containing m-3 amplicon in a ChIP product obtained from an *SSN3*^+/+ strain expressing untagged wild-type Mrr1 (yLM417; not shown) was set to 1 to calculate the relative recovery at additional loci. (D and E) Histone H3 ChIP assays at the *MDR1* promoter in *SSN3*^+/+ strains with the *MRR1*^Q350L or *MRR1*^N803D allele (yLM418 and yLM420, respectively) and their ssn3Δ/Δ derivatives (yLM447 and yLM449, respectively). The *SSN3*^+/+ strain with wild-type *MRR1* (yLM417), in the absence and presence of benomyl treatment (8 μg/ml or 40 μg/ml for 15 min), was analyzed in parallel for comparison. Histone H3 occupancy in the untreated *SSN3*^+/+ *MRR1*^WT strain (yLM417) was set as the reference (with zero H3 displacement index) when the data from panel D were transformed into the H3 displacement index plot shown in panel E. (F) ChIP analysis of Mrr1^WT and Mrr1^N803D occupancy at the *MDR1* promoter in strains expressing N-terminal 6His3Flag-tagged wild-type Mrr1 (*HF-WT*; yLM421) or GOF mutant Mrr1^N803D (*HF-N803D*; yLM424) and their ssn3Δ/Δ derivatives (yLM450 and yLM453, respectively), as well as an *SSN3*^+/+ strain with untagged wild-type Mrr1 (WT; yLM417). The recovery rate (percent input) of DNA fragments containing m-3 amplicon in the ChIP product obtained from an untagged strain (yLM417) was set to 1 to calculate the relative recovery at additional loci.

SNF2-dependent changes in gene expression and nucleosome occupancy at Mrr1 target promoters and azole sensitivity are partially suppressed by ssn3 deletion.

The opposing effects of ssn3 and snf2 deletion on MRR1^GOF-driven fluconazole resistance, Mrr1 target gene expression, and nucleosome and Mrr1 occupancy prompted us to perform an epistasis analysis between SSN3 and SNF2. We generated an ssn3Δ/Δ snf2Δ/Δ double mutant and determined whether ssn3 deletion could suppress any of the Mrr1-related effects on azole resistance and gene expression observed in the snf2 deletion strain. Deletion of ssn3 in an snf2Δ/Δ background fully or partially restores the expression levels of benomyl-induced MRR1 target genes without dramatically increasing the expression of MRR1 (Fig. 8A to E). Restoration of MDR1 gene induction in the ssn3Δ/Δ snf2Δ/Δ strain was accompanied by an increase in histone displacement (Fig. 8F and G) and Mrr1 occupancy (Fig. 8H) versus the snf2Δ/Δ strain under benomyl induction conditions. Consistent with observations of the ssn3 null single mutant (Fig. 6 and 7), histone displacement (Fig. 8F and G) and Mrr1 occupancy (Fig. 8H) at the MDR1 promoter, as well as MDR1 basal expression (Fig. 8E), were largely unaffected under noninducing conditions in the ssn3Δ/Δ snf2Δ/Δ strain compared to the snf2Δ/Δ strain. The ssn3Δ/Δ snf2Δ/Δ strain showed a similar pattern of changes in histone displacement at the IFD6 promoter (Fig. S10). Akin to the effects on benomyl induction, the SNF2 dependence of MDR1 (Fig. 9A), IFD6 (Fig. 9B), and IFD1 (Fig. 9C) overexpression in hyperactive MRR1^GOF mutants was suppressed by deletion of ssn3 in the MRR1^GOF snf2Δ/Δ strains. For both MRR1^GOF mutants, the ssn3Δ/Δ snf2Δ/Δ strain showed slightly higher levels of MRR1 expression as well (Fig. 9D). Comparing the expression of additional Mrr1 target genes (19) revealed that snf2-dependent decreases in MRR1^GOF-activated transcription generally were suppressed by deletion of ssn3 (Fig. 9E). Concordant with the restored expression levels, the strong decrease in histone displacement observed at the MDR1 promoter in the MRR1^GOF mutant snf2Δ/Δ strains was suppressed by deletion of ssn3 (Fig. 10A and B). A similar phenomenon was observed at the IFD6 promoter in the ssn3Δ/Δ snf2Δ/Δ MRR1^GOF strain (Fig. S11). Interestingly, higher levels of Mrr1^GOF mutant occupancy were observed at the MDR1 promoter in the ssn3Δ/Δ snf2Δ/Δ background compared to either single deletion or the wild-type background (Fig. 10C). Lastly, we found that deletion of ssn3 in snf2Δ/Δ strains bearing MRR1^Q350L or MRR1^N803D mutants not only suppressed the effects of snf2 deletion on fluconazole MIC but actually increased the fluconazole MIC beyond that observed in the ssn3Δ/Δ, snf2Δ/Δ, or SSN3 SNF2 strains (Table 1). Similar to the ssn3 null strains, the expression levels of MDR1 in the MRR1^GOF ssn3Δ/Δ snf2Δ/Δ strains (Fig. 9A and E) do not appear to completely explain their fluconazole MICs. Deletion of mdr1 strongly decreased fluconazole MIC in the ssn3Δ/Δ and ssn3Δ/Δ snf2Δ/Δ MRR1^GOF strains (Table 1), suggesting that resistance was still conferred through a largely MDR1-dependent mechanism. The residual resistance seen in mdr1Δ/Δ ssn3Δ/Δ (snf2Δ/Δ) MRR1^GOF strains may result from derepression of another Mrr1 target gene(s) that makes smaller contributions to azole resistance. Deletion of oye32 or C7_03780C, two Mrr1 target genes that are derepressed in the mutant backgrounds (Fig. 9E), did not decrease fluconazole MICs in ssn3Δ/Δ (snf2Δ/Δ) MRR1^GOF strains (Table S1). Our understanding of the origin of MDR1-independent resistance mechanisms in these strains remains incomplete.

FIG 8 — Benomyl induced Mrr1 target gene expression, histone H3 occupancy, and Mrr1 occupancy in an *ssn3 snf2* double deletion background. (A to D) RT-qPCR analysis of benomyl induction of *MDR1* (A), *IFD6* (B), *IFD1* (C), and *MRR1* (D) expression in an *snf2*Δ/Δ *ssn3*Δ/Δ strain (yLM456) treated with 50 μg/ml benomyl for the indicated period of time. The strain was treated and analyzed in parallel with an *SNF2*^+/+ *SSN3*^+/+ strain (WT; yLM417), an *SNF2*^+/+ *ssn3*Δ/Δ strain (*ssn3*Δ/Δ; yLM446), and an *snf2Δ/Δ SSN3*^+/+ strain (*snf2*Δ/Δ; yLM427). The expression level of each gene measured in the untreated *SNF2*^+/+ *SSN3*^+/+ *MRR1*^WT strain (yLM417) was set to 1. The relative expression level of *MDR1*, *IFD6*, *IFD1*, and *MRR1* in the untreated *snf2*Δ/Δ *ssn3*Δ/Δ strain (yLM456) was 0.03, 0.86, 0.50, and 0.55, respectively. (E) *MDR1* expression in the strains described in panels A to D after 15 min of treatment with benomyl (8 μg/ml or 40 μg/ml). The *MDR1* expression level in the untreated *SNF2*^+/+ *SSN3*^+/+strain (yLM417) was set to 1. (F and G) Histone H3 ChIP assays at the *MDR1* promoter in the *snf2*Δ/Δ *ssn3*Δ/Δ strain (yLM456) treated with 8 μg/ml or 40 μg/ml benomyl for 15 min. Parallel analyses were performed in an *SNF2*^+/+ *SSN3*^+/+ strain (WT; yLM417), an *SNF2*^+/+ *ssn3*Δ/Δ strain (*ssn3*^Δ/Δ; yLM446), and an *snf2Δ/Δ SSN3*^+/+ strain (*snf2*^Δ/Δ; yLM427). Histone H3 occupancy in the untreated *SNF2*^+/+ *SSN3*^+/+ *MRR1*^WT strain (yLM417) was set as the reference (with zero H3 displacement index) when the data from panel F were transformed into the H3 displacement index plot shown in panel G. (H) ChIP assays of Mrr1 occupancy at the *MDR1* promoter in *SNF2*^+/+ *SSN3*^+/+, *SNF2*^+/+ *ssn3Δ/Δ snf2Δ/Δ SSN3*^+/+, and *snf2*Δ/Δ *ssn3*Δ/Δ strains expressing N-terminal 6His3Flag-tagged wild-type Mrr1 (yLM421, yLM450, yLM431, and yLM450, respectively) treated with 50 μg/m benomyl for 15 min. The recovery rate (percent input) of DNA fragments containing m-3 amplicon in a ChIP product obtained from the untreated *SNF2*^+/+*SSN3*^+/+ strain expressing untagged wild-type Mrr1 (yLM417; not shown) was set to 1 to calculate the relative recovery at additional loci.

FIG 9 — GOF mutant-induced Mrr1 target gene expression in an *ssn3 snf2* double deletion background. (A to D) RT-qPCR analysis of *MDR1* (A), *IFD6* (B), *IFD1* (C), and *MRR1* (D) expression in *snf2*Δ/Δ *ssn3*Δ/Δ double deletion strains expressing *MRR1*^Q350L or *MRR1*^N803D (yLM457 and yLM459, respectively). *SNF2*^+/+ *SSN3*^+/+ strains (yLM418 and yLM420, respectively), *SNF2*^+/+ *ssn3*Δ/Δ strains (yLM447 and yLM449, respectively), and *snf2Δ/Δ SSN3*^+/+ strains (yLM428 and yLM430, respectively) expressing the same *MRR1* mutants were analyzed in parallel. Benomyl induction (8 μg/ml or 40 μg/ml for 15 min) in the *SNF2*^+/+ *SSN3*^+/+ *MRR1*^WT strain (yLM417) was included for comparison, and the expression of each gene in this strain, in the absence of benomyl, was set to 1. (E) Clustered heat map comparing Mrr1 target gene expression, measured by RT-qPCR, among *SNF2*^+/+ *ssn3*Δ/Δ, *snf2Δ/Δ SSN3*^+/+, and *snf2*Δ/Δ *ssn3*Δ/Δ strains expressing *MRR1*^Q350L (yLM447, yLM428, and yLM457, respectively) or *MRR1*^N803D (yLM449, yLM430, and yLM459, respectively). The expression level of each tested gene in *SSN3*/*SNF2* wild-type strains expressing *MRR1*^Q350L (yLM418) or *MRR1*^N803D (yLM420) was individually set to 1 (not shown) to calculate the fold changes (in logarithmic form [base 2]) in the *snf2* and/or *ssn3* deletion strains. Data from three independent experiments (1 to 3) were clustered by MultiExperiment Viewer (MeV). Changes (log₂) from −3.5 to 3.5 are represented on a chromatic scale.

FIG 10 — GOF mutation induced histone H3 occupancy and Mrr1 occupancy in an *ssn3 snf2* double deletion background. (A and B) Histone H3 ChIP assays at the *MDR1* promoter in *SNF2*^+/+ *SSN3*^+/+ strains with *MRR1*^Q350L or *MRR1*^N803D (yLM418 and yLM420, respectively), their *ssn3*Δ/Δ derivatives (yLM447 and yLM449, respectively), *snf2*Δ/Δ derivatives (yLM428 and yLM430, respectively), and *snf2*Δ/Δ *ssn3*Δ/Δ derivatives (yLM457 and yLM459, respectively). The *MDR1* promoter histone H3 occupancy in the *SNF2*^+/+ *SSN3*^+/+ *MRR1*^WT (yLM417) strain in the absence and presence of benomyl treatment (8 μg/ml or 40 μg/ml for 15 min) was included for comparison. The *MDR1* promoter histone H3 occupancy in the untreated *SNF2*^+/+ *SSN3*^+/+ *MRR1*^WT sample was set as the reference (with zero H3 displacement index) when the data from panel A were transformed into the H3 displacement index plot shown in panel B. (C) Mrr1^N803D occupancy at the *MDR1* promoter in *SNF2*^+/+ *SSN3*^+/+, *SNF2*^+/+ *ssn3*Δ/Δ, *snf2*^Δ/Δ *SSN3*^+/+, and *snf2*Δ/Δ *ssn3*Δ/Δ strains expressing N-terminal 6His3Flag-tagged GOF mutant Mrr1^N803D (*HF-N803D*) (yLM424, yLM453, yLM433, and yLM460, respectively). The *SNF2*^+/+ *SSN3*^+/+ strain, expressing tagged wild-type Mrr1 (*HF-WT*; yLM421) in the absence and presence of benomyl (40 μg/ml for 15 min), was analyzed in parallel for comparison. The recovery rate (percent input) of DNA fragments containing m-3 amplicon in a ChIP product obtained from the *SNF2*^+/+ *SSN3*^+/+ *MRR1*^WT strain (yLM417; not shown) was set to 1 to calculate the relative recovery at additional loci.

Specific disruption of Ssn3 kinase activity is sufficient to potentiate MRR1 target gene induction.

The Cdk8 module plays distinct, yet overlapping, structural and enzymatic roles in gene regulation (13, 39, 41 –43). To determine whether the hyperactive Mrr1-associated azole resistance and gene expression properties associated with the ssn3 null background are phenocopied by specific abolition of the Ssn3 kinase activity, we analyzed these properties in an ssn3 kinase-dead mutant (ssn3^D325A [44]) strain. The kinase-dead mutant strain recapitulated the increases in both benomyl-induced histone displacement (Fig. 11A and B) and gene expression (Fig. 11C) observed for the MDR1 gene in the ssn3 null strain. Similar effects at the IFD6 promoter were observed in the kinase-dead mutant (ssn3^D325A) strain (Fig. S12). We also noted that hyperactive Mrr1 underwent a mobility shift on SDS-PAGE that is dependent on SSN3 (Fig. S13). This mobility shift is seen only when Mrr1 target genes are activated in an MRR1-dependent fashion (i.e., benomyl treatment or MRR1^GOF mutation [Fig. S13A to C] but not H₂O₂ induction [Fig. S13A and B]). The SSN3 dependence of the Mrr1 mobility shift (Fig. S13D and E), as well as our documentation of a phosphorylation-dependent Tac1 mobility shift (48) and the characterization of phosphorylation of other zinc cluster transcription factors (45, 46), suggest that the mobility shift is due to phosphorylation. The loss of Mediator recruitment to MRR1-induced promoters in the med3 null strain also correlates with a loss of the Mrr1 mobility shift (Fig. S13E and F), while snf2 deletion does not result in a loss of the hyperactivation-associated Mrr1 mobility shift (Fig. S13G and S1A). This finding implies that benomyl and GOF mutations change the conformation of Mrr1 to facilitate an interaction with Mediator, either on or off chromatin, and allow phosphorylation of Mrr1 by Ssn3. Although the functional significance of this Ssn3/Med3-dependent mobility shift is unclear, it could serve as a biomarker for Mrr1 hyperactivation.

FIG 11 — Benomyl-induced changes in histone H3 occupancy and gene expression at the *MDR1* promoter in an *ssn3* kinase-dead mutant. (A and B) Histone H3 ChIP assays at the *MDR1* promoter in an *ssn3* null strain (yLM265) and *ssn3* null strains expressing one copy of *SSN3*^WT (Δ/WT; yLM279) or *ssn3*^D325A (Δ/*D325A*; yLM276) treated with the indicated concentration of benomyl for 15 min. Data from panel A were transformed into the H3 displacement index form shown in panel B by setting the *MDR1* promoter nucleosome density profile measured in the untreated wild-type *SSN3* (yLM279) strain as the reference (with zero displacement index). (C) RT-qPCR analysis of benomyl induction of *MDR1* in an *ssn3* null strain and ssn3 null strains expressing one copy of *SSN3*^WT or *ssn3*^D325A saved from the experiment described in panels A and B, prior to fixation. The *MDR1* expression level in the untreated strain with wild-type *SSN3* (yLM279) was set to 1.

Effects of SSN3 on Mrr1 target gene induction occur mainly through an Mrr1-dependent mechanism.

Since the major effects of ssn3 deletion on Mrr1 target genes occur under conditions of activation (Fig. 6 and 7), we tested whether these effects were Mrr1 dependent. Specifically, benomyl-induced gene expression was analyzed by RT-qPCR in an ssn3Δ/Δ mrr1Δ/Δ strain. Consistent with observations in earlier work (17, 19), the response of the MDR1, IFD6, and IFD1 promoters to benomyl is severely compromised in an mrr1 null background (Fig. 12A to C). There is no increase in uninduced or benomyl-induced levels of IFD6 or IFD1 upon ssn3 deletion in an mrr1 null background (Fig. 12B and C) compared to the increase observed in an ssn3 null mutant in benomyl-activated wild-type MRR1 backgrounds (Fig. 6 and Fig. S12). Even though the MDR1 promoter lost most of its benomyl induction in the mrr1 null background, deletion of ssn3 in this background, similar to that of MRR1 wild-type strains (Fig. 6 and 11), led to a 4-fold increase in the benomyl induction of MDR1 (Fig. 12A). In no case, however, were the decreases in MDR1, IFD6, and IFD1 expression in the snf2 mrr1 double deletion background suppressed by the deletion of ssn3 (Fig. 12A). The MDR1 promoter in the ssn3Δ/Δ mrr1Δ/Δ strain also exhibited an increase in nucleosome displacement compared to the SSN3 mrr1Δ/Δ strain, while deletion of ssn3 in the mrr1Δ/Δ snf2Δ/Δ strain did not affect nucleosome displacement at MDR1 or IFD6 under any conditions (Fig. 12D to G).

FIG 12 — *MRR1* dependence of gene expression and histone displacement at the benomyl-activated *MDR1* and *IFD6* promoters in *ssn3* null mutants. (A to C) RT-qPCR analysis of *MDR1* (A), *IFD6* (B), and *IFD1* (C) expression in an *mrr1* deletion strain (SCMRR1M4A) and its *ssn3*Δ/Δ derivative (yLM445), *snf2*Δ/Δ derivative (yLM426), and *snf2*Δ/Δ *ssn3*Δ/Δ derivative (yLM455), together with an *SNF2*^+/+ *SSN3*^+/+ *MRR1*^WT strain (yLM417) treated with the indicated concentration of benomyl for 15 min. Expression of each gene measured in the untreated *SNF2*^+/+ *SSN3*^+/+ *MRR1*^WT strain (yLM417) was set to 1. (D and E) Histone H3 ChIP assays at the *MDR1* promoter in the strains tested in panels A to C in the absence and presence of 50 μg/ml benomyl for 15 min. The *MDR1* promoter histone H3 occupancy measured in the untreated *SNF2*^+/+ *SSN3*^+/+ *MRR1*^WT strain (yLM417) was set as the reference (with zero H3 displacement index) when the data from panel D were transformed into the displacement index plot shown in panel E. (F and G) Histone H3 ChIP assays at the *IFD6* promoter using the samples from panel D and testing them with *IFD6* promoter-specific primers. The *IFD6* promoter histone H3 occupancy measured in the untreated *SNF2*^+/+ *SSN3*^+/+ *MRR1*^WT strain (yLM417) was set as the reference (with zero H3 displacement index) when the data from panel F were transformed into the H3 displacement index plot shown in panel G.

DISCUSSION

C. albicans Mrr1 is a member of an important class of fungal zinc cluster transcription factors that regulate drug efflux pump expression and drug resistance in fungal pathogens (1 –3, 47). The Swi/Snf and Mediator-dependent mechanisms of hyperactive Mrr1 induced transcription, and their impact on fluconazole MIC, reported in this work, facilitates a careful comparison of the zinc cluster transcription factors in C. albicans and other fungal pathogens that will help guide future efforts to broadly address drug resistance in fungi.

The Swi/Snf complex is required for hyperactive Mrr1-induced gene activation and elevated fluconazole MIC in MRR1^GOF strains.

The impact of the Swi/Snf complex on fluconazole resistance in MRR1^GOF strains is among the most severe observed among the coactivators studied to date for C. albicans zinc cluster transcription factor GOF azole resistance mutants (23, 48). The deletion of snf2 reduced the fluconazole MIC in the MRR1^GOF strains to levels comparable to those of azole-sensitive MRR1^WT strains. The decreased fluconazole MIC is consistent with substantial decreases in induction of MDR1 upon hyperactivation of Mrr1 by exposure to benomyl or by either of two distinct GOF mutations. Although the impact of Swi/Snf on fluconazole resistance is observed mainly with hyperactive Mrr1, Swi/Snf is present and actively remodeling chromatin at the promoters of Mrr1 target genes in the uninduced state. This is reflected in a working model of the interplay of Mrr1, Swi/Snf, and Mediator based on the data reported here and is shown in Fig. 13. This model is not intended to be comprehensive or exclusive but provides a basic framework for interpreting the data. Our work supports the hypothesis that GOF mutations or xenobiotic treatment releases the Mrr1 activation domain from autoinhibition mediated by the identified inhibitory domain (5) and/or other effects mediated by the Mrr1 middle region (4). This potentiation of the Mrr1 activation domain apparently is not required to recruit Swi/Snf or to begin the process of chromatin remodeling (Fig. 13). The Mrr1 dependence of Swi/Snf occupancy at Mrr1 target promoters suggests that there is another interaction surface of Mrr1 that helps recruit Swi/Snf to initiate chromatin remodeling even under noninducing conditions. This event is necessary, but not sufficient, to induce high expression levels of the critical Mrr1 target genes. ChIP data showing a loss of Mrr1 occupancy in the snf2 null strain suggests that Swi/Snf can help prepare the chromatin at the MDR1 promoter for Mrr1 binding and, ultimately, activation (Fig. 13). Interestingly, this was not true at all Mrr1 target genes, as Mrr1 binding was not dependent on Swi/Snf at the IFD6 promoter. The finding that IFD6 expression was still dependent on Swi/Snf, despite the Swi/Snf-independent Mrr1 occupancy, supports a model where Swi/Snf also plays a role downstream of Mrr1 binding to facilitate the hyperactive Mrr1-induced gene expression. This model is further supported by the observation that hyperactivation of Mrr1 leads to additional Swi/Snf-dependent histone displacement at the target promoters beyond that observed in the noninduced state. It is not clear how Swi/Snf chromatin remodeling activity is potentiated during Mrr1 target gene induction. Since Swi/Snf recruitment is not increased by activation, it is likely that the histone eviction activity of Swi/Snf becomes more efficient at active Mrr1 target promoters. This could be directly driven by an unmasked Mrr1 activation domain (49) or occur through changes in histone modification or recruitment of histone chaperones during transcription activation (26 –28). A general role for Swi/Snf in converting inactive chromatin to active chromatin at the MDR1 promoter, independent of Mrr1 hyperactivation, was provided by data showing that the Mrr1-independent induction of MDR1 by H₂O₂ was also dependent on Swi/Snf. The complete dependence of MDR1-facilitated azole resistance on Swi/Snf stood in stark contrast to the three azole-resistant TAC1^GOF strains we tested. The TAC1^GOF strains, which overexpress the CDR1 drug efflux pump gene, exhibited little to no decrease in fluconazole MIC in an snf2 null background. The combination of this result with the observation that deletion of snf2 did not impact CDR1 expression in the TAC1^GOF strains (48) reveals that although the zinc cluster transcription factors that drive azole resistance in C. albicans share a domain structure, their coactivator dependence can vary widely. It is an open question whether the pattern of cofactor dependence we have observed originates from a difference in the sequence of the transcription factors, the structure of chromatin at the MDR1 and CDR1 promoters, or both. Although the azole resistance of the TAC1^GOF and the MRR1^GOF strains differed in their dependence on Swi/Snf, they both had a similar dependence on the Mediator tail module.

FIG 13 — Simplified model of the interplay between Mrr1, coactivators, chromatin remodeling, and *MDR1* transcription upon hyperactivation of Mrr1 by benomyl. Prior to adding benomyl, Mrr1 in its inactive form (Mrr1-I) cooperates with the Swi/Snf complex to promote nucleosome (red cylinders) displacement (represented by the arrows below the nucleosomes) from the *MDR1* promoter that permits Mrr1 binding. Upon addition of benomyl, Mrr1 is converted to its hyperactive form (Mrr1-A) and recruits Mediator to the *MDR1* promoter, and Mediator acts as a coactivator to facilitate high levels of *MDR1* transcription. Concurrent with the increased *MDR1* transcription, there is an increase (represented by thicker arrows) in net nucleosome displacement in the *MDR1* promoter compared to the noninduced state. We speculate that the increase in nucleosome displacement comes from a potentiation of Swi/Snf activity and the action of a second, currently unidentified chromatin remodeling complex (CRC?), whose recruitment is facilitated by Mediator and Mrr1-A. The observation that removal of the function of the Cdk8 module of Mediator (the *ssn3* null mutant) leads to increased net histone displacement in the induced state (independent of Swi/Snf activity) suggests that the Cdk8 module is negatively regulating this unidentified chromatin remodeling complex. Figure S14 in the supplemental material shows how this model accommodates the observations made in separate and combined loss of Swi/Snf and Cdk8 module function strains. Although not shown in this model, the data suggest that a second transcription factor, such as Cap1, plays a role weakly redundant to that of Mrr1 in the induction of *MDR1*.

The Mediator complex positively and negatively regulates Mrr1-induced gene activation and fluconazole resistance in MRR1^GOF strains.

Consistent with previous data showing direct interactions between activation domains and the Mediator tail module (7, 9, 10, 37, 38), hyperactivation of MRR1 facilitates Mediator recruitment to target gene promoters in a manner that is dependent on an intact Mediator tail module (Fig. 13). It is likely that there is a constitutively exposed portion of Mrr1 responsible for interaction with Swi/Snf, while there is another sequence that is only exposed upon Mrr1 hyperactivation that interacts with the Mediator tail module. The fluconazole resistance conferred by MRR1^GOF mutations was also decreased in the med3 null mutant but to a lesser extent than the loss of Swi/Snf function. The fold decrease in fluconazole MIC and MDR1 expression in the MRR1^GOF med3 null strains was similar to that of the fluconazole MIC and CDR1 expression changes observed in TAC1^GOF med3 null strains (48). The almost equivalent drop in MRR1^GOF-induced MDR1 expression in the med3 and snf2 null mutants was surprising given the differences in fluconazole MIC in these strains. Although the origin of this difference in MIC is currently unclear, the possibilities include increased expression of some another azole resistance gene(s) in the med3 null mutant or decreased expression of such a gene(s) in the snf2 null strain. Another difference in the med3 null mutant compared to the snf2 null mutant was that expression changes in hyperactive Mrr1-induced genes exhibit stronger promoter dependence.

The IFD1 promoter, when induced by either benomyl or MRR1^GOF, showed very little difference in a med3 null background versus a wild-type background compared to MDR1 and IFD6. The variable dependence of Mrr1 target promoters on MED3 may originate from a combination of positive and negative regulation by Mediator at these promoters. Occupancy of the Cdk8 kinase module, which we showed had a repressive effect on the induction of Mrr1 targets, decreases at Mrr1 target promoters upon deletion of med3. It is interesting that the promoter which exhibited the greatest derepression in the ssn3 null strain, IFD1, is also the promoter least affected by the deletion of med3. This relationship may be a general trend, as none of a larger set of MRR1^GOF target genes derepressed in the ssn3 null mutant showed a strong dependence on med3 for their expression (Fig. S7). At these promoters, any med3-dependent decrease in expression might be masked by the decreased recruitment of the repressive activity of the Cdk8 kinase module, resulting in unchanged expression levels or even net increases. The loss of the mobility shift of Mrr1, which most likely is due to multisite phosphorylation, may provide a clue to how Ssn3 could negatively regulate these genes. Identification and characterization of modified sites on Mrr1 will be required to properly assess the significance of this phenomena. Precisely how the kinase activity of Ssn3 exerts its repressive effect on the Mrr1-target promoters is unclear, but our epistasis analysis of SNF2 and SSN3 revealed a surprising connection to chromatin.

Swi/Snf and the Cdk8 kinase module combine to regulate chromatin remodeling during Mrr1-dependent transcription activation.

The opposing effects of snf2 and ssn3 loss of function on MDR1 expression and fluconazole MIC in MRR1^GOF strains prompted the question of whether these processes worked through intersecting or parallel pathways. Given the primary role of Swi/Snf in remodeling chromatin at Mrr1 target genes, we were surprised that ssn3 deletion suppressed the decrease in MDR1 expression and fluconazole MIC in MRR1^GOF in the snf2 null background. Intrigued by how loss of ssn3 function could overcome the high levels of histone occupancy at Mrr1 target gene promoters in the absence of Swi/Snf function, we probed chromatin structure in the ssn3Δ/Δ snf2Δ/Δ strain. We found that MDR1 (and IFD6) depression by ssn3 deletion in the snf2 null background was in fact accompanied by an Swi/Snf-independent increase in histone displacement. The effect of SSN3 on histone displacement at Mrr1 target promoters only occurs under inducing conditions in both the ssn3 null and ssn3 snf2 double deletion backgrounds. Since Mediator recruitment is also dependent on Mrr1 hyperactivation, this finding suggests recruitment of Ssn3, via its membership in the Mediator complex, is important for its negative regulation of histone displacement (Fig. 13). Most of the current models for Ssn3-facilitated transcription repression do not focus on a direct link between Ssn3 and nucleosome density during promoter induction (9, 10, 13). One possibility is that Ssn3 facilitates a process that assembles repressive chromatin. A second possibility is that Ssn3 negatively regulates a chromatin remodeling complex, such as RSC or ISWI (26 –28), that functions independently of Swi/Snf (Fig. 13). A pathway that involves histone modification could impact either of these processes. Recently, it has been shown that the S. cerevisiae Cdk8 module can exert a repressive impact on gene expression through a transcription factor-dependent effect on histone methylation (50).

Mrr1 directs the positive action of Swi/Snf and negative action of the Mediator Cdk8 module at Mrr1 target genes.

The regulation of MDR1 is complex and involves a number of other transcription factors in addition to Mrr1 (19, 51). We found that Mrr1 is important for both SNF2 and SSN3 to impact the expression of MDR1, IFD6, and IFD1 under inducing conditions (Fig. 13). Deletion of snf2 in an mrr1 null background does, however, result in further decreases in the expression of MDR1, IFD6, and IFD1 beyond those caused by an mrr1 deletion alone. This finding suggests that each of these promoters has some property that confers a low level of Swi/Snf dependence in the absence of Mrr1. This result is consistent with the ChIP results showing that there is residual Swi/Snf occupancy in an mrr1 null mutant despite the large decrease in Swi/Snf occupancy in this strain. The Mrr1-independent Swi/Snf occupancy may explain why the MDR1 and IFD6 promoters do not show increased histone occupancy in an mrr1 deletion background. The impact of SSN3 on MDR1, IFD6, and IFD1 gene expression appears to be promoter dependent. Deletion of ssn3, in the absence of Mrr1, has no impact on the expression of IFD6 and IFD1 or histone displacement at the IFD6 promoter. ssn3 deletion does, however, result in an ∼4-fold derepression of induced MDR1 expression in the absence of Mrr1, which is similar to the fold derepression observed in an MRR1 wild-type background. A similar residual impact of ssn3 deletion on histone displacement at the MDR1 promoter is observed in the absence of Mrr1. One of the additional transcription factors that regulates MDR1, such as Cap1, may be subject to negative regulation by SSN3. Interestingly, this secondary mechanism appears to require the Swi/Snf complex, as deletion of ssn3 does not result in increased histone displacement at the MDR1 promoter in the snf2Δ/Δ mrr1Δ/Δ strain (Fig. 12D and E). These results also support a model in which at least one other transcription factor at the MDR1 promoter mediates a weak Mrr1-independent response to benomyl. Previous studies have shown that Cap1 can cooperate with Mrr1 to induce the MDR1 promoter in response to benomyl (19, 22). It is possible that Cap1 senses the oxidative stress induced by benomyl (19, 22, 52) and weakly activates the MDR1 promoter in the absence of Mrr1. Benomyl induction of the IFD6 promoter, which is not facilitated by Cap1 (22), shows stringent dependence on Mrr1.

Impact of Mrr1 target genes other than MDR1 on fluconazole resistance.

The Mediator med3 and ssn3 null mutants, and particularly the ssn3 snf2 double mutant, all have observed fluconazole MICs in MRR1^GOF strains that were higher than expected based on their MDR1 expression levels. MDR1 still accounts for most of the fluconazole resistance in MRR1^GOF mutants in the ssn3 null and ssn3 snf2 double deletion backgrounds, similar to what is observed in an SSN3 SNF2 wild-type background (17, 19). Residual increases in MIC seen in mdr1Δ/Δ ssn3Δ/Δ (snf2Δ/Δ) MRR1^GOF strains support the idea that Mrr1 target genes contribute to resistance (17, 19). A broader assessment of derepressed Mrr1 target genes, particularly in the mdr1Δ/Δ ssn3Δ/Δ snf2Δ/Δ MRR1^GOF backgrounds, may help to identify secondary contributors to azole resistance.

Summary.

This work, combined with other studies, suggests that the coactivator requirements for each zinc cluster transcription factor involved in azole resistance are largely independent of the mechanism of hyperactivation (xenobiotic or type of GOF mutation) but can be quite different depending on the particular transcription factor and target gene in question. Moreover, the manipulation of MDR1 expression by disruption of one or more of these downstream mechanisms does not always have an absolutely predictive effect on the fluconazole MIC in that particular strain. A comparative knowledge of these different mechanisms is important, as therapeutic intervention downstream of zinc cluster transcription factor hyperactivation is considered to reduce drug resistance. Work in C. glabrata suggests small molecules that interfere with interactions between transcription factors, which drive efflux pump expression, and Mediator could be effective at restoring azole sensitivity to this pathogen with an intrinsically high resistance to fluconazole (6). If such a strategy were to be undertaken in C. albicans, the differential coactivator dependence of Mrr1^GOF and Tac1^GOF efflux pump gene activation we have identified here indicates that the small molecules have to be selected for the specific transcription factor/coactivator pair involved. In particular, the work in this paper indicates that strains which have become resistant via GOF mutations in Mrr1 would benefit from small molecules that inhibit Mrr1-Swi/Snf interactions or an inhibitor of C. albicans Swi/Snf itself. Interestingly, the human Swi/Snf complex has recently been identified as an attractive target for developing anticancer agents (53). Our results showing that hyperactivation of Mrr1 via xenobiotic (benomyl) exposure and via a GOF mutation work through the same mechanism suggest a second strategy for restoring azole sensitivity to a resistant strain. Work in C. glabrata has shown that xenobiotic activators of PDR1 work by binding directly to the transcription factor (7). Analogous to the targeting of the estrogen receptor by tamoxifen, screening for benomyl-like antagonists of Mrr1 could help identify small molecules that selectively stabilized the inactive form of the transcription factor, even in the presence of a GOF mutation.

MATERIALS AND METHODS

Details for construction of C. albicans strains and related DNA vectors are provided in the supplemental material. Strain information, including strain names, parental strain names, and genotypes, are listed in Table S2 in the supplemental material. Vectors for expressing N-terminal 6His3Flag-tagged MRR1 variants, SAT1 flipper plasmids for gene disruption, and primers for testing their correct integration are listed in Table S3. Deletion of mdr1 in C. albicans was performed by using a transient CRISPR-Cas9 system (54, 55), with modifications that are described in the supplemental material in detail. Table S4 lists PCR primers used in the study. C-terminal tagging of SNF2 and MED17 in SC5314 and SCMRR1M4A (mrr1Δ/Δ) (17) was performed using the previously described methods and constructs (36). Specifically, a MED17-3HA-SAT1 tagging cassette and an SNF2-3HA-SAT1 tagging cassette were amplified from the pFA-3HA-SAT1 template by ZL648/ZL649 and ZL794/ZL795, respectively. Correct integration was confirmed at the 5' junction by ZL650/LM21 and ZL623/LM21, respectively, and at the 3' junction by Kpp063/ZL651 and Kpp063/ZL594, respectively. The C-terminal tagging was also confirmed by immunoblotting with an anti-HA antibody (3F10; Roche). The same method was also used to tag MED17 in DSY2937-35 (33) and its med3Δ/Δ derivative (yLM232). All transformations were performed by electroporation and selected on either YPD plus Clonat plates (1% yeast extract, 2% peptone, 2% glucose, 2% agar, 0.1 mM uridine, 100 μg/ml Clonat) or Sc-Ura plates (6.7 g/liter Difco yeast nitrogen base without amino acids [BD], 2 g/liter drop-out mix synthetic without uracil [US Biological], 2% glucose, 2% agar).

Cell growth and drug treatment.

Cells were grown in liquid YPD medium (1% yeast extract, 2% peptone, 2% glucose, 0.1 mM uridine) at 30°C in this study. Benomyl treatment was performed by adding 10 mg/ml benomyl (Sigma) dimethyl sulfoxide (DMSO) stock to mid-log-phase cultures to the final concentration specified in the figure legends for each experiment. H₂O₂ treatment was performed by adding fresh 30% H₂O₂ (Fisher) to mid-log-phase cultures to a final concentration of 0.005%. Fluconazole treatment was performed by adding fresh 4 mg/ml fluconazole (Tokyo Chemical Industry) DMSO stock into mid-log-phase cultures to a final concentration of 50 μg/ml.

Fluconazole MIC measurement by Etest.

Overnight YPD cultures were diluted in a 0.85% NaCl solution to an optical density (OD) sufficient to form an even lawn when swabbed on a YPD plate (supplemented with 0.1 mM uridine). After placement of fluconazole Etest strips (MIC range, 0.016 to 256 μg/ml; bioMérieux), plates were incubated 36 to 48 h at 30°C before readout.

Immunoblotting.

Immunoblotting was used to compare 6His3Flag-Mrr1 protein levels and gel mobility between different strains or conditions. Approximately 10 OD₆₀₀ units of cells (10⁸ cells) were collected, briefly washed by cold water, and frozen in liquid nitrogen for whole-cell lysate preparation by following the method described in reference 32, except that the trichloroacetic acid (TCA)-precipitated pellets were resuspended in 150 μl loading buffer (40 mM Tris-HCl, pH 6.8, 8 M urea, 5% SDS, 0.1 M EDTA, 1% β-mercaptoethanol, and 0.1 mg/ml bromophenol blue) instead of 50 μl. Samples were resolved on 6% SDS-PAGE gel, transferred to a nitrocellulose membrane (120 mA; overnight), and probed by α-Flag (F7425; Sigma) antibody. The lower-molecular-weight region of the gel, containing no 6His3Flag-Mrr1 signal, was stained by Coomassie blue as a loading reference. The sensitive ECL substrate Clarity (Bio-Rad) was used for signal development.

RT-qPCR.

RNA samples were prepared from frozen cell pellets collected under the conditions specified in figure legends for each individual experiment and reverse transcribed as described previously (56). qPCR was performed using the relative standard curve method (StepOne; Life Technologies). ACT1 relative abundance measured by ZL712/ZL713 was used as an internal reference to compare MDR1 (ZL550/ZL551), IFD6 (ZL821/ZL822), IFD1 (ZL823/ZL824), and MRR1 (ZL694/ZL695) expression between different strains and conditions. qPCR primers for assessment of other Mrr1 target gene expression are listed in Table S4. Each RT-qPCR analysis was repeated at least twice, and the independent tests gave highly similar results. If not specified, the means from two sets of qPCR measurements on samples from one representative test are presented with the standard deviations from the two measurements, which was often too small to give visible error bars. A t test was applied when ambiguous changes were observed in independent replicates, and changes between the wild-type reference and mutants with a P value of >0.05 were judged not to be significant.

ChIP.

ChIP experiments were performed as described previously, with modifications (57). Fifty-milliliter cultures were fixed for 15 min by addition of formaldehyde to a final concentration of 1%. Cross-linking was stopped by adding 2.5 ml of 2.5 M glycine. After washing 3 times with 20 ml cold 1× phosphate-buffered saline (PBS), the cells were resuspended in 500 μl lysis buffer (50 mM HEPES-KOH, pH 7.5; 1% Triton X-100; 0.1% sodium deoxycholate; 1 mM EDTA; 150 mM NaCl) supplemented with protease inhibitor cocktail (Roche) and lysed by five 35-s bead-beatings (Biospec). Cell lysates were brought to 1 ml with lysis buffer and probe sonicated (Fisher) three times for 8 s at 30% amplitude after separation into two 1.5-ml tubes. Chromatin was further sheared by a Bio-Disruptor (high setting; 5 min four times for α-HA/Flag ChIP or 5 min five times for α-histone H3/H4 ChIP; 30 s on/30 s off). Two hundred microliters of sonicated cell lysate was incubated with 1.5 μl α-HA antibody (F7; Santa Cruz) or 1.5 μl α-Flag antibody (M2; Sigma) and 100 μl with 1 μl α-histone H3 antibody (ab1791; Abcam) or 1 μl α-histone H4 antibody (ab10158; Abcam) in low-adhesion tubes (USA Scientific) at 4°C overnight. Chromatin was captured by an additional 2-h incubation with 15 μl lysis buffer-equilibrated protein G Dynabeads (Life Technologies). Beads were washed twice in 500 μl wash buffer (50 mM HEPES-KOH, pH 7.5; 1% Triton X-100; 0.1% sodium deoxycholate; 1 mM EDTA; 500 mM NaCl) for 10 min, twice in 500 μl deoxycholate buffer (10 mM Tris-HCl, pH 8.0; 0.5% sodium deoxycholate; 1 mM EDTA; 0.5% NP-40; 0.25 M LiCl) for 10 min, and briefly in 500 μl TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) at room temperature. Precipitated chromatin was eluted by incubating the beads twice in 125 μl freshly made TES buffer (50 mM Tris-HCl, pH 8.0; 10 mM EDTA; 1.5% SDS) for 2 h at 65°C. The two eluates (∼250 μl in total volume) were pooled and the cross-links reversed at 65°C overnight. Forty microliters of each input cell lysate was reverse cross-linked in 200 μl TES buffer. After a brief RNase treatment (1 μl DNase-free RNase [Roche]; 30 min) at room temperature, samples were mixed with 1.5 ml PB buffer (Qiagen) and loaded onto a PCR purification column (Qiagen). The final ChIP products and input reference samples were eluted in 150 μl EB buffer (Qiagen).

Analysis of ChIP data.

The ChIP experiments that tested Snf2, Med17, Ssn3, and Mrr1 occupancy are presented in relative recovery of input format. Specifically, the abundance of DNA fragments containing a certain DNA region tested was compared between a ChIP product and the corresponding input reference sample by qPCR to calculate the absolute recovery rate. The absolute recovery rate for a specific ChIP product (specified in the figure legends) was set to 1 to calculate the relative recovery rate of each DNA region tested across ChIP products obtained in parallel.

Histone (H3 and H4) ChIP results are presented in two formats. In the relative nucleosome density format, the absolute recovery rate of each tested region in the MDR1 or IFD6 promoters for a given ChIP product was normalized using the absolute recovery of DNA fragments containing AZcp067/AZcp068 amplicon, which represents the nucleosome at position +1 of the C. albicans ADE2 gene (58). Histone H3 ChIP results were also presented in a histone displacement format similar to the method previously described (40). To convert the data to this format, the relative nucleosome density was first regrouped by the DNA region tested. Data in each group were then normalized by setting the value obtained from the non-benomyl-treated wild-type strain (yLM417 or SC5314, as specified in the figure legends) to 1. The negative base 2 logarithm of each normalized data point was plotted as the histone displacement index. A positive value for this index indicates a lower nucleosome density compared to the reference, and a negative value for this index indicates a higher nucleosome density compared to the reference.

Each ChIP analysis was repeated at least two times, and the independent tests gave consistent results. The means and standard deviations from two sets of qPCR measurements on samples from one representative experiment were presented, if not otherwise specified. A t test was applied when ambiguous changes were observed in independent replicates, and changes between the wild-type reference and mutants with a P value of >0.05 were judged not to be significant.

Supplementary Material

Supplemental material

supp_61_11_e01344-17__index.html^{(841B, html)}

ACKNOWLEDGMENTS

This study was supported by NIH 5R21AI113390 to L.C.M.

We thank Dominique Sanglard and Joachim Morschhäuser for providing strains. We also thank Gerry Fink for providing the C. albicans CRISPR system.

Footnotes

For a companion article on this topic, see https://doi.org/10.1128/AAC.01342-17.

Supplemental material for this article may be found at https://doi.org/10.1128/AAC.01344-17.

REFERENCES

1.Sanglard D, Coste A, Ferrari S. 2009. Antifungal drug resistance mechanisms in fungal pathogens from the perspective of transcriptional gene regulation. FEMS Yeast Res 9:1029–1050. doi: 10.1111/j.1567-1364.2009.00578.x. [DOI] [PubMed] [Google Scholar]
2.Morschhauser J. 2010. Regulation of multidrug resistance in pathogenic fungi. Fungal Genet Biol 47:94–106. doi: 10.1016/j.fgb.2009.08.002. [DOI] [PubMed] [Google Scholar]
3.Paul S, Moye-Rowley WS. 2014. Multidrug resistance in fungi: regulation of transporter-encoding gene expression. Front Physiol 5:143. doi: 10.3389/fphys.2014.00143. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.MacPherson S, Larochelle M, Turcotte B. 2006. A fungal family of transcriptional regulators: the zinc cluster proteins. Microbiol Mol Biol Rev 70:583–604. doi: 10.1128/MMBR.00015-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Schubert S, Popp C, Rogers PD, Morschhauser J. 2011. Functional dissection of a Candida albicans zinc cluster transcription factor, the multidrug resistance regulator Mrr1. Eukaryot Cell 10:1110–1121. doi: 10.1128/EC.05100-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Nishikawa JL, Boeszoermenyi A, Vale-Silva LA, Torelli R, Posteraro B, Sohn YJ, Ji F, Gelev V, Sanglard D, Sanguinetti M, Sadreyev RI, Mukherjee G, Bhyravabhotla J, Buhrlage SJ, Gray NS, Wagner G, Naar AM, Arthanari H. 2016. Inhibiting fungal multidrug resistance by disrupting an activator-Mediator interaction. Nature 530:485–489. doi: 10.1038/nature16963. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Thakur JK, Arthanari H, Yang F, Pan SJ, Fan X, Breger J, Frueh DP, Gulshan K, Li DK, Mylonakis E, Struhl K, Moye-Rowley WS, Cormack BP, Wagner G, Naar AM. 2008. A nuclear receptor-like pathway regulating multidrug resistance in fungi. Nature 452:604–609. doi: 10.1038/nature06836. [DOI] [PubMed] [Google Scholar]
8.Shahi P, Gulshan K, Naar AM, Moye-Rowley WS. 2010. Differential roles of transcriptional mediator subunits in regulation of multidrug resistance gene expression in Saccharomyces cerevisiae. Mol Biol Cell 21:2469–2482. doi: 10.1091/mbc.E09-10-0899. [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Poss ZC, Ebmeier CC, Taatjes DJ. 2013. The Mediator complex and transcription regulation. Crit Rev Biochem Mol Biol 48:575–608. doi: 10.3109/10409238.2013.840259. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Ansari SA, Morse RH. 2013. Mechanisms of Mediator complex action in transcriptional activation. Cell Mol Life Sci 70:2743–2756. doi: 10.1007/s00018-013-1265-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Tsai KL, Tomomori-Sato C, Sato S, Conaway RC, Conaway JW, Asturias FJ. 2014. Subunit architecture and functional modular rearrangements of the transcriptional mediator complex. Cell 157:1430–1444. doi: 10.1016/j.cell.2014.05.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Robinson PJ, Trnka MJ, Pellarin R, Greenberg CH, Bushnell DA, Davis R, Burlingame AL, Sali A, Kornberg RD. 2015. Molecular architecture of the yeast Mediator complex. eLife 4:e08719. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Nemet J, Jelicic B, Rubelj I, Sopta M. 2014. The two faces of Cdk8, a positive/negative regulator of transcription. Biochimie 97:22–27. doi: 10.1016/j.biochi.2013.10.004. [DOI] [PubMed] [Google Scholar]
14.Pfaller MA, Diekema DJ. 2010. Epidemiology of invasive mycoses in North America. Crit Rev Microbiol 36:1–53. doi: 10.3109/10408410903241444. [DOI] [PubMed] [Google Scholar]
15.Martins N, Ferreira IC, Barros L, Silva S, Henriques M. 2014. Candidiasis: predisposing factors, prevention, diagnosis and alternative treatment. Mycopathologia 177:223–240. doi: 10.1007/s11046-014-9749-1. [DOI] [PubMed] [Google Scholar]
16.Antinori S, Milazzo L, Sollima S, Galli M, Corbellino M. 2016. Candidemia and invasive candidiasis in adults: A narrative review. Eur J Intern Med 34:21–28. doi: 10.1016/j.ejim.2016.06.029. [DOI] [PubMed] [Google Scholar]
17.Morschhauser J, Barker KS, Liu TT, Bla BWJ, Homayouni R, Rogers PD. 2007. The transcription factor Mrr1p controls expression of the MDR1 efflux pump and mediates multidrug resistance in Candida albicans. PLoS Pathog 3:e164. doi: 10.1371/journal.ppat.0030164. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Dunkel N, Blass J, Rogers PD, Morschhauser J. 2008. Mutations in the multi-drug resistance regulator MRR1, followed by loss of heterozygosity, are the main cause of MDR1 overexpression in fluconazole-resistant Candida albicans strains. Mol Microbiol 69:827–840. doi: 10.1111/j.1365-2958.2008.06309.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Schubert S, Barker KS, Znaidi S, Schneider S, Dierolf F, Dunkel N, Aid M, Boucher G, Rogers PD, Raymond M, Morschhauser J. 2011. Regulation of efflux pump expression and drug resistance by the transcription factors Mrr1, Upc2, and Cap1 in Candida albicans. Antimicrob Agents Chemother 55:2212–2223. doi: 10.1128/AAC.01343-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Harry JB, Oliver BG, Song JL, Silver PM, Little JT, Choiniere J, White TC. 2005. Drug-induced regulation of the MDR1 promoter in Candida albicans. Antimicrob Agents Chemother 49:2785–2792. doi: 10.1128/AAC.49.7.2785-2792.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Rognon B, Kozovska Z, Coste AT, Pardini G, Sanglard D. 2006. Identification of promoter elements responsible for the regulation of MDR1 from Candida albicans, a major facilitator transporter involved in azole resistance. Microbiology 152:3701–3722. doi: 10.1099/mic.0.29277-0. [DOI] [PubMed] [Google Scholar]
22.Znaidi S, Barker KS, Weber S, Alarco AM, Liu TT, Boucher G, Rogers PD, Raymond M. 2009. Identification of the Candida albicans Cap1p regulon. Eukaryot Cell 8:806–820. doi: 10.1128/EC.00002-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Ramirez-Zavala B, Mogavero S, Scholler E, Sasse C, Rogers PD, Morschhauser J. 2014. SAGA/ADA complex subunit Ada2 is required for Cap1- but not Mrr1-mediated upregulation of the Candida albicans multidrug efflux pump MDR1. Antimicrob Agents Chemother 58:5102–5110. doi: 10.1128/AAC.03065-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Sellam A, Askew C, Epp E, Lavoie H, Whiteway M, Nantel A. 2009. Genome-wide mapping of the coactivator Ada2p yields insight into the functional roles of SAGA/ADA complex in Candida albicans. Mol Biol Cell 20:2389–2400. doi: 10.1091/mbc.E08-11-1093. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Gao C, Wang L, Milgrom E, Shen WC. 2004. On the mechanism of constitutive Pdr1 activator-mediated PDR5 transcription in Saccharomyces cerevisiae: evidence for enhanced recruitment of coactivators and altered nucleosome structures. J Biol Chem 279:42677–42686. doi: 10.1074/jbc.M406363200. [DOI] [PubMed] [Google Scholar]
26.Hargreaves DC, Crabtree GR. 2011. ATP-dependent chromatin remodeling: genetics, genomics and mechanisms. Cell Res 21:396–420. doi: 10.1038/cr.2011.32. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Rando OJ, Winston F. 2012. Chromatin and transcription in yeast. Genetics 190:351–387. doi: 10.1534/genetics.111.132266. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Becker PB, Workman JL. 2013. Nucleosome remodeling and epigenetics. Cold Spring Harb Perspect Biol 5:a017905. doi: 10.1101/cshperspect.a017905. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Cote J, Quinn J, Workman JL, Peterson CL. 1994. Stimulation of GAL4 derivative binding to nucleosomal DNA by the yeast SWI/SNF complex. Science 265:53–60. doi: 10.1126/science.8016655. [DOI] [PubMed] [Google Scholar]
30.Richmond E, Peterson CL. 1996. Functional analysis of the DNA-stimulated ATPase domain of yeast SWI2/SNF2. Nucleic Acids Res 24:3685–3692. doi: 10.1093/nar/24.19.3685. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Mao X, Cao F, Nie X, Liu H, Chen J. 2006. The Swi/Snf chromatin remodeling complex is essential for hyphal development in Candida albicans. FEBS Lett 580:2615–2622. doi: 10.1016/j.febslet.2006.04.009. [DOI] [PubMed] [Google Scholar]
32.Coste AT, Karababa M, Ischer F, Bille J, Sanglard D. 2004. TAC1, transcriptional activator of CDR genes, is a new transcription factor involved in the regulation of Candida albicans ABC transporters CDR1 and CDR2. Eukaryot Cell 3:1639–1652. doi: 10.1128/EC.3.6.1639-1652.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Coste A, Turner V, Ischer F, Morschhauser J, Forche A, Selmecki A, Berman J, Bille J, Sanglard D. 2006. A mutation in Tac1p, a transcription factor regulating CDR1 and CDR2, is coupled with loss of heterozygosity at chromosome 5 to mediate antifungal resistance in Candida albicans. Genetics 172:2139–2156. doi: 10.1534/genetics.105.054767. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Coste A, Selmecki A, Forche A, Diogo D, Bougnoux ME, d'Enfert C, Berman J, Sanglard D. 2007. Genotypic evolution of azole resistance mechanisms in sequential Candida albicans isolates. Eukaryot Cell 6:1889–1904. doi: 10.1128/EC.00151-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Myers LC, Gustafsson CM, Hayashibara KC, Brown PO, Kornberg RD. 1999. Mediator protein mutations that selectively abolish activated transcription. Proc Natl Acad Sci U S A 96:67–72. doi: 10.1073/pnas.96.1.67. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Zhang A, Petrov KO, Hyun ER, Liu Z, Gerber SA, Myers LC. 2012. The Tlo proteins are stoichiometric components of Candida albicans mediator anchored via the Med3 subunit. Eukaryot Cell 11:874–884. doi: 10.1128/EC.00095-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Brzovic PS, Heikaus CC, Kisselev L, Vernon R, Herbig E, Pacheco D, Warfield L, Littlefield P, Baker D, Klevit RE, Hahn S. 2011. The acidic transcription activator Gcn4 binds the mediator subunit Gal11/Med15 using a simple protein interface forming a fuzzy complex. Mol Cell 44:942–953. doi: 10.1016/j.molcel.2011.11.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Thakur JK, Arthanari H, Yang F, Chau KH, Wagner G, Naar AM. 2009. Mediator subunit Gal11p/MED15 is required for fatty acid-dependent gene activation by yeast transcription factor Oaf1p. J Biol Chem 284:4422–4428. doi: 10.1074/jbc.M808263200. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.van de Peppel J, Kettelarij N, van Bakel H, Kockelkorn TT, van Leenen D, Holstege FC. 2005. Mediator expression profiling epistasis reveals a signal transduction pathway with antagonistic submodules and highly specific downstream targets. Mol Cell 19:511–522. doi: 10.1016/j.molcel.2005.06.033. [DOI] [PubMed] [Google Scholar]
40.Erkina TY, Erkine AM. 2006. Displacement of histones at promoters of Saccharomyces cerevisiae heat shock genes is differentially associated with histone H3 acetylation. Mol Cell Biol 26:7587–7600. doi: 10.1128/MCB.00666-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Tsai KL, Sato S, Tomomori-Sato C, Conaway RC, Conaway JW, Asturias FJ. 2013. A conserved Mediator-CDK8 kinase module association regulates Mediator-RNA polymerase II interaction. Nat Struct Mol Biol 20:611–619. doi: 10.1038/nsmb.2549. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Elmlund H, Baraznenok V, Lindahl M, Samuelsen CO, Koeck PJ, Holmberg S, Hebert H, Gustafsson CM. 2006. The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II. Proc Natl Acad Sci U S A 103:15788–15793. doi: 10.1073/pnas.0607483103. [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Gonzalez D, Hamidi N, Del Sol R, Benschop JJ, Nancy T, Li C, Francis L, Tzouros M, Krijgsveld J, Holstege FC, Conlan RS. 2014. Suppression of Mediator is regulated by Cdk8-dependent Grr1 turnover of the Med3 coactivator. Proc Natl Acad Sci U S A 111:2500–2505. doi: 10.1073/pnas.1307525111. [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Chen C, Noble SM. 2012. Post-transcriptional regulation of the Sef1 transcription factor controls the virulence of Candida albicans in its mammalian host. PLoS Pathog 8:e1002956. doi: 10.1371/journal.ppat.1002956. [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Mamnun YM, Pandjaitan R, Mahe Y, Delahodde A, Kuchler K. 2002. The yeast zinc finger regulators Pdr1p and Pdr3p control pleiotropic drug resistance (PDR) as homo- and heterodimers in vivo. Mol Microbiol 46:1429–1440. doi: 10.1046/j.1365-2958.2002.03262.x. [DOI] [PubMed] [Google Scholar]
46.Leverentz MK, Reece RJ. 2006. Phosphorylation of Zn(II)2Cys6 proteins: a cause or effect of transcriptional activation? Biochem Soc Trans 34:794–797. doi: 10.1042/BST0340794. [DOI] [PubMed] [Google Scholar]
47.Morschhauser J. 2016. The development of fluconazole resistance in Candida albicans–an example of microevolution of a fungal pathogen. J Microbiol 54:192–201. doi: 10.1007/s12275-016-5628-4. [DOI] [PubMed] [Google Scholar]
48.Liu Z, Myers LC. 2017. Mediator tail module is required for Tac1-activated CDR1 expression and azole resistance in Candida albicans. Antimicrob Agents Chemother 61:e01342-17. doi: 10.1128/AAC.01342-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Gutierrez JL, Chandy M, Carrozza MJ, Workman JL. 2007. Activation domains drive nucleosome eviction by SWI/SNF. EMBO J 26:730–740. doi: 10.1038/sj.emboj.7601524. [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Law MJ, Finger MA. 2017. The Saccharomyces cerevisiae Cdk8 Mediator represses AQY1 transcription by inhibiting Set1p-dependent histone methylation. G3 (Bethesda) 7:1001–1010. doi: 10.1534/g3.117.039586. [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Mogavero S, Tavanti A, Senesi S, Rogers PD, Morschhauser J. 2011. Differential requirement of the transcription factor Mcm1 for activation of the Candida albicans multidrug efflux pump MDR1 by its regulators Mrr1 and Cap1. Antimicrob Agents Chemother 55:2061–2066. doi: 10.1128/AAC.01467-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Lucau-Danila A, Lelandais G, Kozovska Z, Tanty V, Delaveau T, Devaux F, Jacq C. 2005. Early expression of yeast genes affected by chemical stress. Mol Cell Biol 25:1860–1868. doi: 10.1128/MCB.25.5.1860-1868.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Zinzalla G. 2016. A new way forward in cancer drug discovery: inhibiting the SWI/SNF chromatin remodelling complex. Chembiochem 17:677–682. doi: 10.1002/cbic.201500565. [DOI] [PubMed] [Google Scholar]
54.Vyas VK, Barrasa MI, Fink GR. 2015. A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families. Sci Adv 1:e1500248. doi: 10.1126/sciadv.1500248. [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Min K, Ichikawa Y, Woolford CA, Mitchell AP. 2016. Candida albicans gene deletion with a transient CRISPR-Cas9 system. mSphere 1:e00130-16. doi: 10.1128/mSphere.00130-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Zhang A, Liu Z, Myers LC. 2013. Differential regulation of white-opaque switching by individual subunits of Candida albicans mediator. Eukaryot Cell 12:1293–1304. doi: 10.1128/EC.00137-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Zhu X, Wiren M, Sinha I, Rasmussen NN, Linder T, Holmberg S, Ekwall K, Gustafsson CM. 2006. Genome-wide occupancy profile of mediator and the Srb8-11 module reveals interactions with coding regions. Mol Cell 22:169–178. doi: 10.1016/j.molcel.2006.03.032. [DOI] [PubMed] [Google Scholar]
58.Lu Y, Su C, Mao X, Raniga PP, Liu H, Chen J. 2008. Efg1-mediated recruitment of NuA4 to promoters is required for hypha-specific Swi/Snf binding and activation in Candida albicans. Mol Biol Cell 19:4260–4272. doi: 10.1091/mbc.E08-02-0173. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[B1] 1.Sanglard D, Coste A, Ferrari S. 2009. Antifungal drug resistance mechanisms in fungal pathogens from the perspective of transcriptional gene regulation. FEMS Yeast Res 9:1029–1050. doi: 10.1111/j.1567-1364.2009.00578.x. [DOI] [PubMed] [Google Scholar]

[B2] 2.Morschhauser J. 2010. Regulation of multidrug resistance in pathogenic fungi. Fungal Genet Biol 47:94–106. doi: 10.1016/j.fgb.2009.08.002. [DOI] [PubMed] [Google Scholar]

[B3] 3.Paul S, Moye-Rowley WS. 2014. Multidrug resistance in fungi: regulation of transporter-encoding gene expression. Front Physiol 5:143. doi: 10.3389/fphys.2014.00143. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4.MacPherson S, Larochelle M, Turcotte B. 2006. A fungal family of transcriptional regulators: the zinc cluster proteins. Microbiol Mol Biol Rev 70:583–604. doi: 10.1128/MMBR.00015-06. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B5] 5.Schubert S, Popp C, Rogers PD, Morschhauser J. 2011. Functional dissection of a Candida albicans zinc cluster transcription factor, the multidrug resistance regulator Mrr1. Eukaryot Cell 10:1110–1121. doi: 10.1128/EC.05100-11. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6.Nishikawa JL, Boeszoermenyi A, Vale-Silva LA, Torelli R, Posteraro B, Sohn YJ, Ji F, Gelev V, Sanglard D, Sanguinetti M, Sadreyev RI, Mukherjee G, Bhyravabhotla J, Buhrlage SJ, Gray NS, Wagner G, Naar AM, Arthanari H. 2016. Inhibiting fungal multidrug resistance by disrupting an activator-Mediator interaction. Nature 530:485–489. doi: 10.1038/nature16963. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7.Thakur JK, Arthanari H, Yang F, Pan SJ, Fan X, Breger J, Frueh DP, Gulshan K, Li DK, Mylonakis E, Struhl K, Moye-Rowley WS, Cormack BP, Wagner G, Naar AM. 2008. A nuclear receptor-like pathway regulating multidrug resistance in fungi. Nature 452:604–609. doi: 10.1038/nature06836. [DOI] [PubMed] [Google Scholar]

[B8] 8.Shahi P, Gulshan K, Naar AM, Moye-Rowley WS. 2010. Differential roles of transcriptional mediator subunits in regulation of multidrug resistance gene expression in Saccharomyces cerevisiae. Mol Biol Cell 21:2469–2482. doi: 10.1091/mbc.E09-10-0899. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9.Poss ZC, Ebmeier CC, Taatjes DJ. 2013. The Mediator complex and transcription regulation. Crit Rev Biochem Mol Biol 48:575–608. doi: 10.3109/10409238.2013.840259. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10.Ansari SA, Morse RH. 2013. Mechanisms of Mediator complex action in transcriptional activation. Cell Mol Life Sci 70:2743–2756. doi: 10.1007/s00018-013-1265-9. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11.Tsai KL, Tomomori-Sato C, Sato S, Conaway RC, Conaway JW, Asturias FJ. 2014. Subunit architecture and functional modular rearrangements of the transcriptional mediator complex. Cell 157:1430–1444. doi: 10.1016/j.cell.2014.05.015. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12.Robinson PJ, Trnka MJ, Pellarin R, Greenberg CH, Bushnell DA, Davis R, Burlingame AL, Sali A, Kornberg RD. 2015. Molecular architecture of the yeast Mediator complex. eLife 4:e08719. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13.Nemet J, Jelicic B, Rubelj I, Sopta M. 2014. The two faces of Cdk8, a positive/negative regulator of transcription. Biochimie 97:22–27. doi: 10.1016/j.biochi.2013.10.004. [DOI] [PubMed] [Google Scholar]

[B14] 14.Pfaller MA, Diekema DJ. 2010. Epidemiology of invasive mycoses in North America. Crit Rev Microbiol 36:1–53. doi: 10.3109/10408410903241444. [DOI] [PubMed] [Google Scholar]

[B15] 15.Martins N, Ferreira IC, Barros L, Silva S, Henriques M. 2014. Candidiasis: predisposing factors, prevention, diagnosis and alternative treatment. Mycopathologia 177:223–240. doi: 10.1007/s11046-014-9749-1. [DOI] [PubMed] [Google Scholar]

[B16] 16.Antinori S, Milazzo L, Sollima S, Galli M, Corbellino M. 2016. Candidemia and invasive candidiasis in adults: A narrative review. Eur J Intern Med 34:21–28. doi: 10.1016/j.ejim.2016.06.029. [DOI] [PubMed] [Google Scholar]

[B17] 17.Morschhauser J, Barker KS, Liu TT, Bla BWJ, Homayouni R, Rogers PD. 2007. The transcription factor Mrr1p controls expression of the MDR1 efflux pump and mediates multidrug resistance in Candida albicans. PLoS Pathog 3:e164. doi: 10.1371/journal.ppat.0030164. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B18] 18.Dunkel N, Blass J, Rogers PD, Morschhauser J. 2008. Mutations in the multi-drug resistance regulator MRR1, followed by loss of heterozygosity, are the main cause of MDR1 overexpression in fluconazole-resistant Candida albicans strains. Mol Microbiol 69:827–840. doi: 10.1111/j.1365-2958.2008.06309.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B19] 19.Schubert S, Barker KS, Znaidi S, Schneider S, Dierolf F, Dunkel N, Aid M, Boucher G, Rogers PD, Raymond M, Morschhauser J. 2011. Regulation of efflux pump expression and drug resistance by the transcription factors Mrr1, Upc2, and Cap1 in Candida albicans. Antimicrob Agents Chemother 55:2212–2223. doi: 10.1128/AAC.01343-10. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B20] 20.Harry JB, Oliver BG, Song JL, Silver PM, Little JT, Choiniere J, White TC. 2005. Drug-induced regulation of the MDR1 promoter in Candida albicans. Antimicrob Agents Chemother 49:2785–2792. doi: 10.1128/AAC.49.7.2785-2792.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B21] 21.Rognon B, Kozovska Z, Coste AT, Pardini G, Sanglard D. 2006. Identification of promoter elements responsible for the regulation of MDR1 from Candida albicans, a major facilitator transporter involved in azole resistance. Microbiology 152:3701–3722. doi: 10.1099/mic.0.29277-0. [DOI] [PubMed] [Google Scholar]

[B22] 22.Znaidi S, Barker KS, Weber S, Alarco AM, Liu TT, Boucher G, Rogers PD, Raymond M. 2009. Identification of the Candida albicans Cap1p regulon. Eukaryot Cell 8:806–820. doi: 10.1128/EC.00002-09. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B23] 23.Ramirez-Zavala B, Mogavero S, Scholler E, Sasse C, Rogers PD, Morschhauser J. 2014. SAGA/ADA complex subunit Ada2 is required for Cap1- but not Mrr1-mediated upregulation of the Candida albicans multidrug efflux pump MDR1. Antimicrob Agents Chemother 58:5102–5110. doi: 10.1128/AAC.03065-14. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24.Sellam A, Askew C, Epp E, Lavoie H, Whiteway M, Nantel A. 2009. Genome-wide mapping of the coactivator Ada2p yields insight into the functional roles of SAGA/ADA complex in Candida albicans. Mol Biol Cell 20:2389–2400. doi: 10.1091/mbc.E08-11-1093. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B25] 25.Gao C, Wang L, Milgrom E, Shen WC. 2004. On the mechanism of constitutive Pdr1 activator-mediated PDR5 transcription in Saccharomyces cerevisiae: evidence for enhanced recruitment of coactivators and altered nucleosome structures. J Biol Chem 279:42677–42686. doi: 10.1074/jbc.M406363200. [DOI] [PubMed] [Google Scholar]

[B26] 26.Hargreaves DC, Crabtree GR. 2011. ATP-dependent chromatin remodeling: genetics, genomics and mechanisms. Cell Res 21:396–420. doi: 10.1038/cr.2011.32. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27.Rando OJ, Winston F. 2012. Chromatin and transcription in yeast. Genetics 190:351–387. doi: 10.1534/genetics.111.132266. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B28] 28.Becker PB, Workman JL. 2013. Nucleosome remodeling and epigenetics. Cold Spring Harb Perspect Biol 5:a017905. doi: 10.1101/cshperspect.a017905. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B29] 29.Cote J, Quinn J, Workman JL, Peterson CL. 1994. Stimulation of GAL4 derivative binding to nucleosomal DNA by the yeast SWI/SNF complex. Science 265:53–60. doi: 10.1126/science.8016655. [DOI] [PubMed] [Google Scholar]

[B30] 30.Richmond E, Peterson CL. 1996. Functional analysis of the DNA-stimulated ATPase domain of yeast SWI2/SNF2. Nucleic Acids Res 24:3685–3692. doi: 10.1093/nar/24.19.3685. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B31] 31.Mao X, Cao F, Nie X, Liu H, Chen J. 2006. The Swi/Snf chromatin remodeling complex is essential for hyphal development in Candida albicans. FEBS Lett 580:2615–2622. doi: 10.1016/j.febslet.2006.04.009. [DOI] [PubMed] [Google Scholar]

[B32] 32.Coste AT, Karababa M, Ischer F, Bille J, Sanglard D. 2004. TAC1, transcriptional activator of CDR genes, is a new transcription factor involved in the regulation of Candida albicans ABC transporters CDR1 and CDR2. Eukaryot Cell 3:1639–1652. doi: 10.1128/EC.3.6.1639-1652.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B33] 33.Coste A, Turner V, Ischer F, Morschhauser J, Forche A, Selmecki A, Berman J, Bille J, Sanglard D. 2006. A mutation in Tac1p, a transcription factor regulating CDR1 and CDR2, is coupled with loss of heterozygosity at chromosome 5 to mediate antifungal resistance in Candida albicans. Genetics 172:2139–2156. doi: 10.1534/genetics.105.054767. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B34] 34.Coste A, Selmecki A, Forche A, Diogo D, Bougnoux ME, d'Enfert C, Berman J, Sanglard D. 2007. Genotypic evolution of azole resistance mechanisms in sequential Candida albicans isolates. Eukaryot Cell 6:1889–1904. doi: 10.1128/EC.00151-07. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B35] 35.Myers LC, Gustafsson CM, Hayashibara KC, Brown PO, Kornberg RD. 1999. Mediator protein mutations that selectively abolish activated transcription. Proc Natl Acad Sci U S A 96:67–72. doi: 10.1073/pnas.96.1.67. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B36] 36.Zhang A, Petrov KO, Hyun ER, Liu Z, Gerber SA, Myers LC. 2012. The Tlo proteins are stoichiometric components of Candida albicans mediator anchored via the Med3 subunit. Eukaryot Cell 11:874–884. doi: 10.1128/EC.00095-12. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B37] 37.Brzovic PS, Heikaus CC, Kisselev L, Vernon R, Herbig E, Pacheco D, Warfield L, Littlefield P, Baker D, Klevit RE, Hahn S. 2011. The acidic transcription activator Gcn4 binds the mediator subunit Gal11/Med15 using a simple protein interface forming a fuzzy complex. Mol Cell 44:942–953. doi: 10.1016/j.molcel.2011.11.008. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B38] 38.Thakur JK, Arthanari H, Yang F, Chau KH, Wagner G, Naar AM. 2009. Mediator subunit Gal11p/MED15 is required for fatty acid-dependent gene activation by yeast transcription factor Oaf1p. J Biol Chem 284:4422–4428. doi: 10.1074/jbc.M808263200. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B39] 39.van de Peppel J, Kettelarij N, van Bakel H, Kockelkorn TT, van Leenen D, Holstege FC. 2005. Mediator expression profiling epistasis reveals a signal transduction pathway with antagonistic submodules and highly specific downstream targets. Mol Cell 19:511–522. doi: 10.1016/j.molcel.2005.06.033. [DOI] [PubMed] [Google Scholar]

[B40] 40.Erkina TY, Erkine AM. 2006. Displacement of histones at promoters of Saccharomyces cerevisiae heat shock genes is differentially associated with histone H3 acetylation. Mol Cell Biol 26:7587–7600. doi: 10.1128/MCB.00666-06. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B41] 41.Tsai KL, Sato S, Tomomori-Sato C, Conaway RC, Conaway JW, Asturias FJ. 2013. A conserved Mediator-CDK8 kinase module association regulates Mediator-RNA polymerase II interaction. Nat Struct Mol Biol 20:611–619. doi: 10.1038/nsmb.2549. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B42] 42.Elmlund H, Baraznenok V, Lindahl M, Samuelsen CO, Koeck PJ, Holmberg S, Hebert H, Gustafsson CM. 2006. The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II. Proc Natl Acad Sci U S A 103:15788–15793. doi: 10.1073/pnas.0607483103. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B43] 43.Gonzalez D, Hamidi N, Del Sol R, Benschop JJ, Nancy T, Li C, Francis L, Tzouros M, Krijgsveld J, Holstege FC, Conlan RS. 2014. Suppression of Mediator is regulated by Cdk8-dependent Grr1 turnover of the Med3 coactivator. Proc Natl Acad Sci U S A 111:2500–2505. doi: 10.1073/pnas.1307525111. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B44] 44.Chen C, Noble SM. 2012. Post-transcriptional regulation of the Sef1 transcription factor controls the virulence of Candida albicans in its mammalian host. PLoS Pathog 8:e1002956. doi: 10.1371/journal.ppat.1002956. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B45] 45.Mamnun YM, Pandjaitan R, Mahe Y, Delahodde A, Kuchler K. 2002. The yeast zinc finger regulators Pdr1p and Pdr3p control pleiotropic drug resistance (PDR) as homo- and heterodimers in vivo. Mol Microbiol 46:1429–1440. doi: 10.1046/j.1365-2958.2002.03262.x. [DOI] [PubMed] [Google Scholar]

[B46] 46.Leverentz MK, Reece RJ. 2006. Phosphorylation of Zn(II)2Cys6 proteins: a cause or effect of transcriptional activation? Biochem Soc Trans 34:794–797. doi: 10.1042/BST0340794. [DOI] [PubMed] [Google Scholar]

[B47] 47.Morschhauser J. 2016. The development of fluconazole resistance in Candida albicans–an example of microevolution of a fungal pathogen. J Microbiol 54:192–201. doi: 10.1007/s12275-016-5628-4. [DOI] [PubMed] [Google Scholar]

[B48] 48.Liu Z, Myers LC. 2017. Mediator tail module is required for Tac1-activated CDR1 expression and azole resistance in Candida albicans. Antimicrob Agents Chemother 61:e01342-17. doi: 10.1128/AAC.01342-17. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B49] 49.Gutierrez JL, Chandy M, Carrozza MJ, Workman JL. 2007. Activation domains drive nucleosome eviction by SWI/SNF. EMBO J 26:730–740. doi: 10.1038/sj.emboj.7601524. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B50] 50.Law MJ, Finger MA. 2017. The Saccharomyces cerevisiae Cdk8 Mediator represses AQY1 transcription by inhibiting Set1p-dependent histone methylation. G3 (Bethesda) 7:1001–1010. doi: 10.1534/g3.117.039586. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B51] 51.Mogavero S, Tavanti A, Senesi S, Rogers PD, Morschhauser J. 2011. Differential requirement of the transcription factor Mcm1 for activation of the Candida albicans multidrug efflux pump MDR1 by its regulators Mrr1 and Cap1. Antimicrob Agents Chemother 55:2061–2066. doi: 10.1128/AAC.01467-10. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B52] 52.Lucau-Danila A, Lelandais G, Kozovska Z, Tanty V, Delaveau T, Devaux F, Jacq C. 2005. Early expression of yeast genes affected by chemical stress. Mol Cell Biol 25:1860–1868. doi: 10.1128/MCB.25.5.1860-1868.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B53] 53.Zinzalla G. 2016. A new way forward in cancer drug discovery: inhibiting the SWI/SNF chromatin remodelling complex. Chembiochem 17:677–682. doi: 10.1002/cbic.201500565. [DOI] [PubMed] [Google Scholar]

[B54] 54.Vyas VK, Barrasa MI, Fink GR. 2015. A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families. Sci Adv 1:e1500248. doi: 10.1126/sciadv.1500248. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B55] 55.Min K, Ichikawa Y, Woolford CA, Mitchell AP. 2016. Candida albicans gene deletion with a transient CRISPR-Cas9 system. mSphere 1:e00130-16. doi: 10.1128/mSphere.00130-16. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B56] 56.Zhang A, Liu Z, Myers LC. 2013. Differential regulation of white-opaque switching by individual subunits of Candida albicans mediator. Eukaryot Cell 12:1293–1304. doi: 10.1128/EC.00137-13. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B57] 57.Zhu X, Wiren M, Sinha I, Rasmussen NN, Linder T, Holmberg S, Ekwall K, Gustafsson CM. 2006. Genome-wide occupancy profile of mediator and the Srb8-11 module reveals interactions with coding regions. Mol Cell 22:169–178. doi: 10.1016/j.molcel.2006.03.032. [DOI] [PubMed] [Google Scholar]

[B58] 58.Lu Y, Su C, Mao X, Raniga PP, Liu H, Chen J. 2008. Efg1-mediated recruitment of NuA4 to promoters is required for hypha-specific Swi/Snf binding and activation in Candida albicans. Mol Biol Cell 19:4260–4272. doi: 10.1091/mbc.E08-02-0173. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Candida albicans Swi/Snf and Mediator Complexes Differentially Regulate Mrr1-Induced MDR1 Expression and Fluconazole Resistance

Zhongle Liu

Lawrence C Myers

ABSTRACT

INTRODUCTION

RESULTS

Swi/Snf and Mediator govern fluconazole resistance in MRR1GOF mutant strains.

TABLE 1.

Mrr1-mediated gene activation requires the Swi/Snf complex.

FIG 1.

Snf2 and Mrr1 bind cooperatively to the MDR1 promoter.

FIG 2.

Swi/Snf affects nucleosome density at the promoters of Mrr1 target genes.

FIG 3.

Mediator positively and negatively regulates transcriptional activation by hyperactive Mrr1.

FIG 4.

FIG 5.

Ssn3 negatively regulates the activation of Mrr1 target genes.

FIG 6.

Ssn3 negatively regulates Mrr1 occupancy and histone displacement at the MDR1 and IFD6 promoters.

FIG 7.

SNF2-dependent changes in gene expression and nucleosome occupancy at Mrr1 target promoters and azole sensitivity are partially suppressed by ssn3 deletion.

FIG 8.

FIG 9.

FIG 10.

Specific disruption of Ssn3 kinase activity is sufficient to potentiate MRR1 target gene induction.

FIG 11.

Effects of SSN3 on Mrr1 target gene induction occur mainly through an Mrr1-dependent mechanism.

FIG 12.

DISCUSSION

The Swi/Snf complex is required for hyperactive Mrr1-induced gene activation and elevated fluconazole MIC in MRR1GOF strains.

FIG 13.

The Mediator complex positively and negatively regulates Mrr1-induced gene activation and fluconazole resistance in MRR1GOF strains.

Swi/Snf and the Cdk8 kinase module combine to regulate chromatin remodeling during Mrr1-dependent transcription activation.

Mrr1 directs the positive action of Swi/Snf and negative action of the Mediator Cdk8 module at Mrr1 target genes.

Impact of Mrr1 target genes other than MDR1 on fluconazole resistance.

Summary.

MATERIALS AND METHODS

Cell growth and drug treatment.

Fluconazole MIC measurement by Etest.

Immunoblotting.

RT-qPCR.

ChIP.

Analysis of ChIP data.

Supplementary Material

ACKNOWLEDGMENTS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Swi/Snf and Mediator govern fluconazole resistance in MRR1^GOF mutant strains.

The Swi/Snf complex is required for hyperactive Mrr1-induced gene activation and elevated fluconazole MIC in MRR1^GOF strains.

The Mediator complex positively and negatively regulates Mrr1-induced gene activation and fluconazole resistance in MRR1^GOF strains.