Interallelic interaction and gene regulation in budding yeast

Abstract

InDrosophila, homologous chromosome pairing leads to "transvection," in which the enhancer of a gene can regulate the allelic transcription intrans.Interallelic interactions were also observed in vegetative diploid budding yeast, but their functional significance is unknown. Here, we show that aGAL1reporter can interact with its homologous allele and affect its expression. By ectopically inserting two allelic reporters, one driven by wild-typeGAL1promoter (WTGAL1pr) and the other by a mutant promoter with delayed response to galactose induction, we found that the two reporters physically associate, and the WTGAL1prtriggers synchronized firing of the defective promoter and accelerates its activation without affecting its steady-state expression level. This interaction and the transregulatory effect disappear when the same reporters are located at nonallelic sites. We further demonstrated that the activator Gal4 is essential for the interallelic interaction, and the transregulation requires fully activated WTGAL1prtranscription. The mechanism of this phenomenon was further discussed. Taken together, our data revealed the existence of interallelic gene regulation in yeast, which serves as a starting point for understanding long-distance gene regulation in this genetically tractable system.

Keywords: homologous pairing; interallelic interaction; interallelic regulation; single-cell gene expression; transvection.

Fig. 1.

Interallelic gene regulation measured by FACS. (A) Strain constructs. We constructed diploid strains containing WT GAL1pr driving gfp (GFP with frame-shift mutation) and mutant promoter (Δ1 or Δ2) driving WT GFP. The two reporters were integrated at allelic loci on ChrII. Yellow bar, Gal4 binding site; parentheses, deleted sequence; arrow, TSS; red bar, integration site; purple circle, centromere. Same notations apply to the figures below. (B) Histograms of Δ1pr-GFP FACS data at three time points of induction. Δ1pr-GFP/-, diploid cells with one ChrII containing Δ1pr-GFP and the other intact; Δ1pr-GFP/wt-gfp, diploid cells with one ChrII containing Δ1pr-GFP and the other containing wt-gfp at the allelic locus. To enhance the difference between the WT and mutant promoter, the stains were induced with 3% galactose + 2% raffinose + 0.1% glucose (for the 4 h and 6 h data). For the steady-state measurement, the strains were induced with 3% galactose + 2% raffinose (no glucose) for 15 h. (C) Fraction of activated cells containing Δ1pr-GFP or Δ2pr-GFP in the presence or absence of the allelic WT GAL1pr-gfp. (D) The steady-state level of GFP expression in these strains (normalized by that of WT GAL1pr-GFP) (D). Note that the induction rate was increased, but not the steady-state level. (E) The same as in C except that the Δ1pr-GFP and WT GAL1pr-gfp are at nonallelic location.

Fig. 2.

Interallelic gene regulation measured by time-lapse fluorescence microscopy. (A) Strain constructs. The diploid strains contain Δ1pr-YFP and WT GAL1pr-GFP at allelic (II/II′) or nonallelic (II/XVI) locations. (B) Time-lapse live-cell imaging of the allelic strain during induction with 3% galactose + 2% raffinose + 0.05% glucose. (C) GFP and YFP intensity (after cross-talk elimination) as a function of time in three single cells, each with a different color. The solid/stippled arrows point to the activation time in the GFP/YFP traces, respectively. (D) Histogram of the difference in GFP and YFP activation time (ΔT) in the allelic vs. the nonallelic strain. The activation of GFP and YFP are more synchronized in the allelic case. Total trace number: allelic, 82; nonallelic, 128. (E) Activated fraction as a function of time. The plot shows the cumulative distribution of the activation time derived from the single-cell traces in the allelic (Left) and the nonallelic (Right) strain. The GFP activation follows similar rate in these two strains, but YFP activation is faster in the allelic case. (F) Steady-state YFP intensity in Δ1pr-YFP(II)/-, Δ1pr-YFP(II)/wt-GFP(II), and Δ1pr-YFP(II)/wt-GFP(XVI) strains. The box plot shows the distribution of the steady-state level in single cells, which was quantified as the average YFP intensity >4 h after activation.

Fig. 3.

Inducible trans interaction between Δ1 and the WT GAL1 promoter. (A) Design of the 3C assay. Two flanking KanMx segments and XhoI cutting sites were inserted for PCR purpose. Cross-linked chromosomes were digested with XhoI, ligated, and subjected to PCR between primers P1 and P2. The two alleles were either inserted at allelic (II/II′) or nonallelic loci (II/V). (B and C) Representative gel showing the result of the 3C assay. The P1/P2 PCR product was only visible between alleles after galactose induction. The interaction between two cis regions on ChrXV was detected as a positive control. (D) The intensity of the P1/P2 3C signal as a function of induction time. The intensity was quantified by qPCR and normalized by the 3C signal of the positive control.

Fig. 4.

Interallelic interaction and regulation between varied alleles. (A) Interallelic interaction and regulation between Δ1pr-GFP and Δ4pr-gfp. In Δ4pr, the entire UAS from the WT GAL1pr was deleted. (Lower Left) Gel image of the 3C measurement between the two alleles. (Lower Right) Activated fraction of Δ1pr-GFP as a function of time with or without allelic Δ4pr-gfp. (B) The same as A except that the Δ4pr was replaced by ΔTATApr, where the TATA element of the WT GAL1pr was mutated (Methods).