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. 2019 May 13;8:e42499. doi: 10.7554/eLife.42499

Figure 4. Rgt1 expression, interaction partners, and competing domains regulate HAS1pr-TDA1pr pairing.

(A) RGT1 and SDD4 are upregulated in saturated cultures. RNA-seq expression levels for LEU3, SDD4, RGT1 in exponentially growing (Exp) and saturated (Sat) cultures. FPKM = fragments per kilobase per million read pairs. Bars indicate mean ± s.e.m. of biological triplicates. **p<0.01, ***p<0.001 (Student’s t-test). (B) Individual TF overexpression is insufficient for pairing. Shown are HAS1pr-TDA1pr homolog pairing frequencies with and without estradiol-induced overexpression of LEU3, SDD4, RGT1 in S. cerevisiae x S. uvarum hybrids during exponential growth, measured by 3C and normalized to contacts between HAS1pr-TDA1pr and LCB1 on S. cerevisiae chrXIII. Bars indicate mean ± s.d. of technical triplicates. (C) Effects of Rgt1 interaction partner deletions on ectopic HAS1pr-TDA1pr pairing in saturated conditions. Bar plot of median relative abundance in 3C library compared to the genomic library, with overlaid scatter plot of individual barcoded deletion strains. Retested controls are shown in same colors as in Figure 2; potential interaction partners are shown in shades of purple. (D) Regions of Rgt1 necessary for pairing in saturated culture conditions, with overlaid predicted disorder (black line, right y-axis) and secondary structure (pink and blue highlights). Each bar represents the ratio of the frequency of each 10 amino acid deletion in the 3C library compared to the genomic library. Error bars indicate the two biological replicates. (E) Domain deletions and phosphorylation site mutations tested in panel F. (F) Effects of Rgt1 domain deletions and phosphorylation site mutations on ectopic HAS1pr-TDA1pr pairing in saturated cultures, plotted as in panel C. Rgt1 indicates the strains with an ectopic wild-type (WT) or mutant copy of Rgt1 in addition to a deletion of the endogenous RGT1 locus.

Figure 4.

Figure 4—figure supplement 1. Overexpression of RGT1 is not sufficient for HAS1pr-TDA1pr pairing.

Figure 4—figure supplement 1.

(A) Estradiol-induced overexpression of TF genes. Bars show RNA levels of S. cerevisiae LEU3, SDD4, and RGT1 with (red) and without (gray) estradiol-induced overexpression in S. cerevisiae x S. uvarum hybrids, measured by reverse transcription qPCR (RT-qPCR) and normalized to S. cerevisiae ACT1 expression. Bars indicate mean ± s.e.m. of technical triplicates. *p<0.05, **p<0.01 (Student’s t-test). (B) RGT1 overexpression does not result in increased pairing. Frequency of contacts between HAS1pr-TDA1pr alleles as measured by 3C, normalized to the frequency of contacts between S. cerevisiae HAS1pr-TDA1pr and S. cerevisiae LCB1 in S. cerevisiae x S. uvarum hybrids (strain YMD3920) with (OE) or without (WT) a plasmid carrying a copy of epitope-tagged RGT1-6xHis-HA-3C-pA under a GAL1 promoter, grown in glucose (red), raffinose (blue), or galactose (green). Bars indicate mean ± s.d. of technical triplicates.
Figure 4—figure supplement 2. Regions of Rgt1 required for pairing correspond to regulatory domains.

Figure 4—figure supplement 2.

Regions of Rgt1 necessary for HAS1pr-TDA1pr pairing in saturated conditions, as in Figure 4D, shown as ratio of the frequency of each 10 amino acid deletion in the 3C library compared to the genomic library. Error bars indicate the two biological replicates. Highlighted regions indicate the previously mapped functional domains of Rgt1 (Polish et al., 2005).