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. 2023 Jul 27;14:4528. doi: 10.1038/s41467-023-40209-w

Fig. 5. Cwt1 and Zcf11 comprise a transcription factor complex required for AA-induced AOX2 transactivation.

Fig. 5

a Strains were tested for sensitivity to AA or Inz-5 in SC-galactose. b Relative AOX2 transcript levels were measured following AA addition (10 μM) in YPGal. Basal AOX2 expression (time 0) in the WT background was set to 1. c C. albicans expressing Cwt1-3XHA with native Zcf11 or Zcf11-6XHis-3XFlag (Zcf11-HF) were grown in YPGal with/without AA treatment. Anti-HA and anti-Flag immunoprecipitation (IP) products were analyzed by immunoblotting. d Localization of Cwt1-GFP and Zcf11-GFP were monitored by fluorescence microscopy. Scale bar: 5 μm. e Binding profile of Cwt1-3XHA, Zcf11-3XHA, or Rtg1-3XHA at the AOX2 promoter before and after AA treatment (10 μM; 15 min; YPGal) were analyzed by anti-HA ChIP using the primer set in Fig. 4f. Background signal determined using an untagged WT strain without AA treatment. f Cwt1-3XHA binding at the CARbox was compared between WT and zcf11-deletion strains. AA treatment and background signal assessment were performed as in e. g WT or zcf11-deletion strains expressing Cwt1-3XHA or Zcf11-3XHA were treated with AA (10 μM; 15 min) in YPD or YPGal. Blots were probed using anti-HA antibody. CBS: Coomassie Blue staining of the blot showing comparable loading. h AOX2 promoters with mutation(s) in the CGG motif 2 or (and) motif 3 (see Supplementary Fig. 5f) in a strain expressing Cwt1-3XHA. Top left: AA-induced AOX2 expression from the WT and mutant promoters. Top right: Cwt1-3XHA binding at CARbox upon AA treatment in YPGal (10 μM; 15 min) by α-HA ChIP. Bottom: DNA sequence flanking CGG motifs 2 and 3. i, j AA-induced CARbox binding of Rtg1/Rtg3 (or Cwt1/Zcf11) as measured by ChIP was compared between WT and mutant strains after AA treatment (10 μM; 15 min; YPGal). p values: two-tailed t test (n = 3). k Mutation of the GTCA motifs abolish AA-induced Cwt1 binding at CARbox, as assessed by ChIP. b, e, h (left) present mean (SD) of technical triplicates from an experiment representative of biological duplicates. Data for the corresponding biological replicates are in source data file. f, h (right), i, j, k present mean (SD) of biological triplicates.