Candida albicans Zn Cluster Transcription Factors Tac1 and Znc1 Are Activated by Farnesol To Upregulate a Transcriptional Program Including the Multidrug Efflux Pump CDR1

TAC1 dependence of CDR2, RTA3, and CDR1 induction by FOH and 1-DD. RT-qPCR analysis of CDR2 (A), RTA3 (B), and CDR1 (C) mRNA expression after treatment with FNZ (25 μM), FOH (50 μM), and 1-DD (50 μM) in a wild-type (TAC1^WT; yLM167 or yLM660) and tac1 deletion strain (tac1Δ/Δ; yLM166 or yLM663). The expression level of each gene, in the absence of treatment (0 min) in the wild-type background, was set to 1.

Znc1 and Tac1 contribute individually and in tandem to the regulation of specific FOH- and 1-DD-induced promoters. (A and B) RT-qPCR analysis of RTA3 (A) and CDR1 (B) mRNA expression in tac1Δ/Δ and znc1Δ/Δ strains treated with FNZ, FOH, or 1-DD. A wild-type strain (yLM660) and mutants carrying a tac1 deletion (yLM663), znc1 deletion (yLM661), or double deletion (yLM664) were treated with FNZ (25 μM), FOH (50 μM), or 1-DD (50 μM). CDR1 and RTA3 basal expression in the untreated wild-type strain (yLM660) was individually set to 1. (C) Immunoblot analysis of Cdr1 protein levels in tac1Δ/Δ, znc1Δ/Δ, and double deletion strains treated with FNZ or FOH. Wild-type (yLM665), tac1Δ/Δ (yLM666), znc1Δ/Δ (yLM667), and tac1Δ/Δ znc1Δ/Δ (yLM668) strains expressing C-terminally 3×HA-tagged Cdr1 were treated with FNZ (25 μM) or FOH (50 μM). Cell lysates were resolved on 6% SDS-PAGE gels and probed with an anti-HA antibody. Blot images, acquired at two exposure times, are presented, and Coomassie blue staining (CBS) images are presented as a loading control. (D to E) RT-qPCR analysis of CDR1 (D) and CDR2 (E) mRNA expression in tac1Δ/Δ and znc1Δ/Δ strains, and complementation controls, treated with FOH. A tac1Δ/Δ znc1Δ/Δ strain was complemented by ZNC1 (yLM676), TAC1 (yLM677), or both (yLM675) and treated with FOH (50 μM). Parallel experiments were also conducted in wild-type (yLM660), tac1 deletion (yLM663), parental tac1 znc1 double deletion (yLM664), and mock complementation (yLM678) strains for comparison. A plus sign labels a native or restored gene locus, while Δ and V (vector) mark an unrestored gene disruption and a mock complementation by introducing an empty vector. (F and G) RT-qPCR analysis of orf19.320 (F) and IFD1 (G) mRNA expression in the wild-type ZNC1 or znc1 deletion background (yLM660 and yLM661, respectively) treated with FNZ (25 μM), FOH (50 μM), and 1-DD (50 μM) for the indicated period of time. orf19.320 and IFD1 basal expression in the wild-type strain was individually set to 1. (H) qPCR analysis of orf19.320 expression in the complementation strains using cDNA samples tested in panels D and E. Noninduced expression levels in the wild-type strain (yLM660) were set to 1.

Direct hyperactivation of Tac1 and Znc1 by FOH and 1-DD. C. albicans one-hybrid assay assessing Tac1- and Znc1-dependent reporter activation under multiple inducing conditions. A C. albicans one-hybrid strain expressing the LexA DNA binding domain (yLM567) or the fusion of the LexA DBD with Tac1, Znc1, or Hal9 fragments (yLM568, yLM680 and yLM681, respectively) were treated with FNZ (25 μM), FOH (50 μM), and 1-DD (50 μM) for 1 h and 3 h before measurement of LacZ activity. Untreated (none) and methanol-treated (not shown) cultures showed the same level of basal activity. Statistical significance for reporter activation was determined by comparing the basal and induced LacZ activity in t tests (**, P < 0.01; ***, P < 0.001).

Tac1 and Znc1 occupancy at FOH target promoters. (A to E) ChIP analysis of Tac1 and Znc1 occupancy at CDR1 (A), RTA3 (B), CDR2 (C), orf19.320 (D), and IFD1 (E) upon treatment with FOH. A strain carrying two copies of N-terminally 6His3Flag-tagged TAC1 and two copies of C-terminally 3×HA-tagged ZNC1 (Znc1-3HA/HF-Tac1; yLM686) and a strain with native TAC1 and ZNC1 (untagged; yLM660) were treated with FOH (50 μM) or vehicle (Veh.; methanol) for 15 min before fixation. Each sample was immunoprecipitated by an anti-Flag antibody and an anti-HA antibody in separate reactions. Promoter regions tested for Tac1 and Znc1 binding and their relative positions to the known Tac1 cis elements at the CDR1, RTA3, and CDR2 promoters (DRE; blue boxes) or the CGG triplet motifs found at the orf19.320 and IFD1 promoters (PZMs; red boxes) are schematically shown in each panel. Percent recovery of input (Input%) at the CDR1 promoter 1-up region in the anti-Flag/anti-HA ChIP products obtained from the methanol-treated untagged strain was set to 1 to normalize Tac1/Znc1 binding across conditions and promoter regions. Hence, the strength of ChIP signals (y axis value) can be compared across panels. (F) Alignment of the CDR1, CDR2, and RTA3 DREs and the CGG triplet motifs (PZMs) at the orf19.320 and IFD1 promoters. The 13 nucleotide positions are numbered in order. Individual nucleotide substitutions that have been found to be deleterious in the CDR2 DRE (26) are highlighted in red. (G) ChIP analysis of Znc1 occupancy at target promoters in a tac1 deletion background. Wild-type (yLM684) and tac1 deletion (yLM685) strains expressing two copies of C-terminally 3×HA-tagged ZNC1 (HA/HA) were treated with 50 μM FOH for the indicated period of time before fixation for an anti-HA ChIP assay. Percent recovery of input (Input%) at the 1-up region in a wild-type strain with native Tac1 (+/+) and Znc1 (WT/WT) was set to 1 to normalize recoveries across conditions and promoters.

Mediator requirement for, and recruitment by, Tac1- and Znc1-dependent induction of CDR1 by farnesol. (A) RT-qPCR analysis of CDR1 mRNA expression in a wild-type strain (yLM167) and a med3 null strain (yLM232) after treatment with 50 μM FOH. FNZ (25 μM) induction (30) was performed as a reference. (B) Anti-HA ChIP analysis of Mediator occupancy at the CDR1 promoter in wild-type (yLM695), tac1Δ/Δ (yLM696), znc1Δ/Δ (yLM697), and tac1Δ/Δ znc1Δ/Δ (yLM698) strains expressing C-terminally 3×HA-tagged Med17 treated with 50 μM FOH. Percent recovery of input (Input%) at the 1-up region in a ChIP product obtained from a strain with native MED17 (WT/WT; yLM660) was set to 1.

Influence of Mrr2 on FOH and 1-DD target gene expression. (A) RT-qPCR analysis of CDR1 mRNA expression in a wild-type strain (yLM660) and tac1Δ/Δ znc1Δ/Δ strains (yLM701 to yLM704) generated from an otherwise wild-type, mrr2Δ/Δ, stb5Δ/Δ, or cta4Δ/Δ background and treated with FOH (50 μM) and 1-DD (50 μM). CDR1 expression, in the absence of treatment, in yLM660 was set to 1. (B) RT-qPCR analysis of CDR1 mRNA expression in wild-type (yLM660) and mrr2 null (yLM662) strains treated with FNZ (25 μM), FOH (50 μM), or 1-DD (50 μM). CDR1 expression level, in the absence of treatment, in the wild-type strain was set to 1. (C) Anti-HA immunoblot analysis of C-terminally 3×HA-tagged CDR1 protein expression in MRR2 wild-type strains (yLM665 and yLM705) and deletion mutants (yLM707 and yLM706) in the presence and absence of TAC1 and ZNC1 and treatment with FNZ (25 μM) or FOH (50 μM). Blot images acquired at different exposures are presented with a Coomassie blue stain (CBS) as a loading control.

Combinatorial control of gene expression by Tac1, Znc1, and Mrr2. A summary of the effect of TAC1, ZNC1, and MRR2 on gene expression induced by FNZ (25 μM), FOH (50 μM), and 1-DD (50 μM) is shown. The means of the relative gene expression after 15 and 30 min of exposure to the inducer were used to compare promoter activation in different strain backgrounds after being normalized to the induced expression level in the wild-type strain as percentages. Effects of transcription factor deletion on gene activation are visualized by a black-yellow chromatic scale. Transcription factor(s) dependence for each promoter was illustrated by the colors and symbols as coded in the legend box.

Feedback modulation of FOH induction by Cdr1. (A) RT-qPCR analysis of CDR1, CDR2, orf19.320, and orf19.344 mRNA expression in a wild-type strain (yLM660) and mutant strains carrying a deletion in cdr1 (yLM708), cdr2 (yLM709), or both (yLM710) after treatment with FOH (20 μM). Expression of each gene in the wild-type strain, in the absence of treatment, was set to 1. Symbol legends are in the box on the right. (B) RT-qPCR analysis of CDR2, RTA3, orf19.320, and PDR16 mRNA expression in wild-type (yLM660) and cdr1 null (yLM708) strains treated with 50 μM farnesyl acetate (FAc) or 50 μM FOH. Expression of each gene in the wild-type strain, in the absence of treatment, was set to 1. Symbol legends are in the box on the right. (C) RT-qPCR analysis of orf19.344 mRNA expression in TAC1/ZNC1 wild-type (WT; yLM660 and yLM708) and tac1Δ/Δ znc1Δ/Δ (yLM664 and yLM711) strains, derived from a wild-type or cdr1Δ/Δ background, after treatment with increasing concentrations of FOH. Expression of orf19.344 in the wild-type strain (yLM660), in the absence of treatment, was set to 1. (D) RT-qPCR analysis of orf19.320 mRNA expression in strains carrying a wild-type (TAC1^WT; yLM167) or a GOF mutant TAC1 allele (TAC1^A736V; yLM169) treated with 16 μM FOH. Expression of orf19.320 in the TAC1^WT strain, in the absence of treatment, was set to 1.

Dependence of C. albicans and C. dubliniensis farnesol sensitivity on CDR1 expression. (A) Colony formation analysis comparing cell viability between wild-type and cdr1 null strains after 1 to 6 h of FOH or vehicle (methanol) exposure. Wild-type (yLM660), cdr1 null (yLM708), cdr1 cdr2 double deletion (yLM710), and tac1 znc1 mrr2 triple deletion (yLM702) strains were each diluted from overnight YPD cultures and treated with 200 μM FOH in YPD medium. Aliquots, after an appropriate dilution (if needed), were spread on YPD plates at the indicated time points. Fold change in CFU was calculated by setting the CFU before treatment (not shown) to 1 and is presented in a logarithmic form (base 2). (B) Representative plate scans showing a high FOH concentration in agar medium reduces colony growth of a tac1Δ/Δ znc1Δ/Δ mrr2Δ/Δ strain. Dilutions of a wild-type strain (yLM660) and a tac1 znc1 mrr2 triple deletion mutant (yLM702) from overnight cultures were plated on YPD agar supplemented with 200 μM FOH or the same volume of vehicle (methanol). Plates were imaged after incubation for the indicated amounts of time at 30°C. (C) Colony formation analysis comparing cell viability between three C. dubliniensis strains (CD36, Wü284, and CD57) after 1 to 6 h of FOH or vehicle (methanol) exposure. Wild-type (yLM660) and cdr1 cdr2 double deletion (yLM710) C. albicans strains were tested in parallel as a control. The strains were each diluted from overnight YPD cultures and treated with 200 μM FOH in YPD medium. Aliquots, after an appropriate dilution (if needed), were spread on YPD plates at the indicated time points. Fold change in CFU was calculated by setting the CFU before treatment (not shown) to 1 and is presented in a logarithmic form (base 2).