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. 2007 Aug;27(15):5575-86.
doi: 10.1128/MCB.00459-07. Epub 2007 May 25.

Analysis of transcriptional activation at a distance in Saccharomyces cerevisiae

Krista C Dobi  1 , Fred Winston
Affiliations

Analysis of transcriptional activation at a distance in Saccharomyces cerevisiae

Krista C Dobi et al. Mol Cell Biol. 2007 Aug.

Abstract

Most fundamental aspects of transcription are conserved among eukaryotes. One striking difference between yeast Saccharomyces cerevisiae and metazoans, however, is the distance over which transcriptional activation occurs. In S. cerevisiae, upstream activation sequences (UASs) are generally located within a few hundred base pairs of a target gene, while in Drosophila and mammals, enhancers are often several kilobases away. To study the potential for long-distance activation in S. cerevisiae, we constructed and analyzed reporters in which the UAS-TATA distance varied. Our results show that UASs lose the ability to activate normal transcription as the UAS-TATA distance increases. Surprisingly, transcription does initiate, but proximally to the UAS, regardless of its location. To identify factors affecting long-distance activation, we screened for mutants allowing activation of a reporter when the UAS-TATA distance is 799 bp. These screens identified four loci, SIN4, SPT2, SPT10, and HTA1-HTB1, with sin4 mutations being the strongest. Our results strongly suggest that long-distance activation in S. cerevisiae is normally limited by Sin4 and other factors and that this constraint plays a role in ensuring UAS-core promoter specificity in the compact S. cerevisiae genome.

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Figures

FIG. 1.
FIG. 1.
The UAS-TATA distance affects HIS3 expression. (A) A schematic of the reporters used in this study. Four Gal4 binding sites from the GAL1 UAS were integrated at various distances 5′ of the HIS3 gene (see Materials and Methods). (B) Growth of strains with the HIS3 reporters integrated within the BPH1 ORF. UAS elements were positioned at the indicated distances 5′ of the HIS3 TATA. The strains shown are as follows: FY3 (the wild type [WT]), FY2549 (no UAS), FY2550 (UAS 280), FY2551 (UAS 380), FY2552 (UAS 493), FY2553 (UAS 574), FY2554 (UAS 690), and FY2555 (UAS 799). Patches of cells were grown on YPD, replica plated to the specified media, and grown for 2 days at 30°C. In this and all subsequent figures, patches for a particular growth medium were grown on the same plates, but have been arranged for ease of phenotype comparison between the different types of media. (C) Growth of strains with the HIS3 reporters integrated within the YBR281C ORF. The strains shown are as follows: FY3 (WT), FY2579 (no UAS), FY2557 (UAS 305), FY2558 (UAS 606), and FY2559 (UAS 806). Strains were grown and incubated as described for panel B. (D) Growth of reporter strains with LexA binding sites serving as the UAS. Strains with LexA sites at 123 (FY2581), 417 (FY2582), or 624 (FY2583) bp 5′ of HIS3 were transformed with LEU2-marked plasmids expressing LexA-Gcn4 or LexA-Gal4 fusion proteins or a vector-only control. Patches of cells were grown on SC-Leu, replica plated to the specified media, and grown for 3 days at 30°C. 3AT, 3-aminotriazole.
FIG. 2.
FIG. 2.
Phenotypic analyses of mutants permissive for long-distance activation. (A) Growth of mutant strains with the HIS3 UAS 799 reporter integrated into the BPH1 ORF. Cells were patched on YPD, replica plated to the specified media, and grown for 3 days at 30°C. The strains shown are as follows: FY2555 (wild type [WT] with reporter), FY2562 (sin4Δ), FY2570 (spt2Δ), FY2571 (spt10Δ), and FY2572 [(hta1-htb1)Δ]. (B) Growth of mutant strains containing the UAS 806 reporter integrated into the YBR281C ORF. The strains shown are as follows: FY2559 (WT with reporter), FY2560 (sin4Δ), and FY2580 (spt2Δ). Strains were grown and incubated as described for panel A. (C) Growth of strains containing a reporter with LexA binding sites positioned 642 bp 5′ of HIS3 in the BPH1 ORF. Strains were transformed with LEU2-marked plasmids expressing LexA-Gal4 or LexA-Gcn4 fusion protein or a vector-only control. The strains shown are as follows: FY2556 (WT with LexA reporter), FY2563 (sin4Δ), and FY2583 (spt2Δ). Strains were patched on SC-Leu, replica plated to the specified media, and grown for 3 days at 30°C. (D) Growth of wild-type, sin4Δ, and spt2Δ strains containing reporters with UAS elements positioned at the specified distances 5′ of the HIS3 TATA within BPH1 (right side of panel). The strains shown are as follows: FY2555 (WT UAS 799), FY2562 (sin4Δ UAS 799), FY2570 (spt2Δ UAS 799), FY2573 (WT UAS 1397), FY2574 (sin4Δ UAS 1397), FY2577 (spt2Δ UAS 1397), FY2575 (WT UAS 1995), FY2576 (sin4Δ UAS 1995), and FY2578 (spt2Δ UAS 1995). Strains were grown and incubated as described for panel A. (E) Growth of strains containing the BPH1 GAL UAS 799 reporter in Mediator mutants. The strains shown are as follows: FY3 (WT), FY2555 (WT with UAS 799 reporter), FY2562 (sin4Δ), FY2588 (gal11Δ), FY2589 (pgd1Δ), FY2590 (med2Δ), FY2591 (nut1Δ), FY2592 (med1Δ), FY2593 (srb10Δ), FY2602 (srb9Δ), srb2Δ (FY2594), FY2595 (srb5Δ), and FY2605 (soh1Δ). Strains were patched and grown for 4 days at 30°C.
FIG. 3.
FIG. 3.
Northern analysis of reporter strains. (A) Northern analysis of BPH1 GAL UAS reporter strains and mutants. A Northern blot was hybridized with a probe for HIS3 (top panel) and ACT1 (bottom panel). The strains shown are as follows: FY3 (lane 1), FY2549 (lane 2), FY2550 (lane 3), FY2551 (lane 4), FY2552 (lane 5), FY2553 (lane 6), FY2554 (lane 7), FY2555 (lane 8), FY2562 (lane 9), and FY2570 (lane 10). The numbers above lanes 3 to 10 indicate the UAS-TATA distance in base pairs. (B) Northern analysis of YBR281C GAL UAS reporter strains and mutants. The strains shown are as follows: FY3 (lane 1), FY2579 (lane 2), FY2557 (lane 3), FY2558 (lane 4), FY2559 (lane 5), FY2560 (lane 6), and FY2580 (lane 7). WT, wild type. Base pair distances are shown as in panel A.
FIG. 4.
FIG. 4.
5′ RACE analysis of reporter strains, with and without the ADH1 terminator. (A) Frequency of observed start sites among 5′ RACE clones (see Materials and Methods). The number of clones observed within each 50-bp region (represented by marks along the x axis) is plotted on the y axis, and the position along the BPH1 UAS 799 reporter gene is plotted along the x axis. Positions of the Gal4 binding sites and HIS3 promoter and coding regions are indicated. (B) Frequency of observed start sites among 5′ RACE clones of strains containing the terminator reporter (term.), the wild type (WT) (FY2585), and the sin4Δ mutant (FY2586). The number of clones observed is plotted on the y axis, and the position along the reporter is plotted on the x axis. Positions of the Gal4 binding sites, ADH1 terminator sequences, and HIS3 promoter and coding regions are indicated. A greater portion of the HIS3 coding region is shown in panel B to enable the depiction of start sites within the HIS3 ORF. Note that transcripts that terminate 5′ of the terminator will not be detected, as they will not anneal to the primer used.
FIG. 5.
FIG. 5.
Experiments with a reporter strain containing the ADH1 terminator. (A) Schematic of a BPH1 GAL UAS reporter with the ADH1 terminator (term.) sequences integrated between the UAS and HIS3. In this reporter strain, the UAS is 839 bp 5′ of the TATA. (B) Comparison of growth between strains containing the BPH1 GAL UAS 799 reporter and the BPH1 GAL UAS 839 reporter with the ADH1 terminator. The strains shown are as follows: FY2555 (wild-type [WT] UAS 799), FY2562 (sin4Δ UAS 799), FY2570 (spt2Δ UAS 799), FY2585 (WT term), FY2586 (sin4Δ term), and FY2587 (spt2Δ term). Strains were patched on YPD, replica plated to indicated media and grown for 3 days at 30°C. (C) Northern analysis comparing the BPH1 GAL UAS 799 reporter and the BPH1 GAL UAS 839 ADH1 terminator reporter. Blots were hybridized with a probe to HIS3 (top panel) and ACT1 (bottom panel). The strains shown are as follows: FY3 (lane 1), FY2555 (lane 2), FY2585 (lane 3), FY2562 (lane 4), FY2586 (lane 5), FY2570 (lane 6), and FY2587 (lane 7). −, absence of terminator; +, presence of terminator.
FIG. 6.
FIG. 6.
Molecular evidence for galactose-induced looping between the UAS and TATA. (A) Schematic of BPH1 GAL UAS 839 ADH1 terminator reporter (term.). Horizontal arrows denote the positions of same-strand oligonucleotide sequences that were used in 3C analysis (see Materials and Methods). Vertical arrows show the positions of some of the BfaI and MseI restriction enzyme cut sites along the reporter; there are 13 total sites between the TATA and Gal4 binding sites. (B) 3C analysis of looping between the UAS and TATA in the wild-type (WT) (FY2585) and the sin4Δ mutant (FY2586) strains (see Materials and Methods). Products of PCR using same-strand oligonucleotides correspond to the ligation of fragments containing the Gal4 binding sites and HIS3 TATA. Control PCR to normalize the amounts of DNA in the samples was performed using convergent oligonucleotides that amplify a region of chromosome V with no restriction enzyme cut sites. (C) Gal-induced looping between the UAS and TATA is dependent upon cross-linking and ligation. 3C analysis was performed as described in Materials and Methods, except without cross-linking (lanes 2 and 5) or addition of ligase (lanes 3 and 6). −, absence of formaldehyde and ligase; +, presence of formaldehyde and ligase.
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