Regulation of an intergenic transcript controls adjacent gene transcription in Saccharomyces cerevisiae - PubMed (original) (raw)

Regulation of an intergenic transcript controls adjacent gene transcription in Saccharomyces cerevisiae

Joseph A Martens et al. Genes Dev. 2005.

Abstract

Recent studies have revealed that transcription of noncoding, intergenic DNA is abundant among eukaryotes. However, the functions of this transcription are poorly understood. We have previously shown that in Saccharomyces cerevisiae, expression of an intergenic transcript, SRG1, represses the transcription of the adjacent gene, SER3, by transcription interference. We now show that SRG1 transcription is regulated by serine, thereby conferring regulation of SER3, a serine biosynthetic gene. This regulation requires Cha4, a serine-dependent activator that binds to the SRG1 promoter and is required for SRG1 induction in the presence of serine. Furthermore, two coactivator complexes, SAGA and Swi/Snf, are also directly required for activation of SRG1 and transcription interference of SER3. Taken together, our results elucidate a physiological role for intergenic transcription in the regulation of SER3. Moreover, our results demonstrate a mechanism by which intergenic transcription allows activators to act indirectly as repressors.

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Figures

Figure 1.

Figure 1.

Effect of serine on SER3 and SRG1 expression. (A) A schematic of SRG1 and SER3, showing TATA and putative UAS sequences conserved between S. cerevisiae and four related yeast strains. TATA elements are represented by black boxes, the putative SRG1 UAS elements by light-gray boxes 5′ of the SRG1 TATA (labeled 1–4), and the putative SER3 UAS elements by dark-gray boxes 5′ of SER3 TATA. The horizontal black bars mark the SRG1 and SER3 UAS regions that were amplified by PCR in ChIP experiments described later. The arrows indicate the orientation and positions of the SRG1 and SER3 transcripts. (B) Northern analysis of SER3 and SRG1 was performed on wild-type (FY2472) and srg1-1 (FY2471; contains a mutation of the SRG1 TATA) strains after a shift from SD + ser to SD medium. SNR190 served as a loading control. Total RNA was isolated at 15-min intervals. These data are representative of three independent experiments. (C) Northern analysis was performed on the strains described in B after the opposite shift, from SD to SD + ser. These data are representative of three independent experiments.

Figure 2.

Figure 2.

Identification of a serine response element in the SRG1 promoter. (A) Northern analysis of SER3 and SRG1 on a wild-type strain (FY2460) and on a series of srg1 promoter mutants. The srg1-20 mutant (FY2467) has the entire SRG1 UAS region deleted (Fig. 1A, regions 1–4). The srg1-21 (FY2464), srg1-22 (FY2465), srg1-23 (FY2466), and srg1-24 (FY2461) mutants are deletions of SRG1 UAS sequences 1, 2, 3, and 4, respectively (Fig. 1A). Total RNA was isolated from cells grown in SD + ser medium and from cells that had then been shifted to SD medium for 25 min. SNR190 served as a loading control. These data are representative of three independent experiments. (B) Northern analysis of SER3 and SRG1 was performed on wild-type (FY2460), _cha4_Δ (FY2459), srg1-25 (FY2463), and srg1-26 (FY2462) strains that were grown in SD + ser and SD medium as described in A. The srg1-25 mutant is a deletion of a putative Cha4-binding site in the SRG1 promoter and the srg1-26 mutant has a triple-point mutation within this putative Cha4-binding site. These data are representative of three independent experiments.

Figure 3.

Figure 3.

ChIP analysis of Cha4 association with the SRG1 promoter. (A) ChIP analysis of Cha4 was performed on wild-type (FY2470) and srg1-25 (FY2468) strains expressing Cha4-Flag and on an untagged control strain (FY1350). Cha4-Flag was immunoprecipitated with anti-Flag antibody from cells grown in SD + ser medium (+serine) and from cells that had been shifted from SD + ser to SD medium for 25 min (–serine). A representative set of PCR reactions that amplify the SRG1 UAS and SER3 UAS regions (see diagram in Fig. 1A) from twofold dilutions of chromatin is shown. The control primer set amplifies a region of chromosome V that lacks open reading frames (Komarnitsky et al. 2000). (B) Quantitation of ChIP analysis. The amount of SRG1 UAS or SER3 UAS that was amplified from immunoprecipitated DNA is expressed as a percentage of the amount of input DNA. Each bar represents the average and standard error from three independent experiments.

Figure 4.

Figure 4.

Repression of SER3 is dependent on the Spt3 and Spt8 subunits of SAGA. (A) Northern analysis of SER3. Total RNA was isolated from wild-type (FY3), _snf2_Δ (FY1360), _spt3_Δ (FY294), _spt8_Δ (FY50), _spt7_Δ (FY963), _spt20_Δ (FY1098), _ada1_Δ (FY1560), _gcn5_Δ (FY1600), _ada2_Δ (FY1548), _ada3_Δ (FY1596), _ubp8_Δ (FY2473), _sgf29_Δ (FY2474), and _sgf73_Δ (FY2475) strains that were grown in YPD. SNR190 served as a loading control. These data are representative of three independent experiments. (B) Northern analysis of SER3 and SRG1. Total RNA was isolated from wild-type (FY4), two _snf2_Δ (FY2150 and FY2151), and two _spt3_Δ (FY930 and FY2142) strains that were grown in SD + ser medium. RNA levels were averaged for at least three independent experiments. SER3 mRNA levels are 31.4 ± 2.7 and 27.5 ± 2.3 in _snf2_Δ and _spt3_Δ strains, respectively, as compared with wild type. SRG1 RNA levels are 0.59 ± 0.06 and 0.37 ± 0.07 in _snf2_Δ and _spt3_Δ strains, respectively, as compared with wild type.

Figure 5.

Figure 5.

ChIP analysis of Snf2 and Ada1 association to the SRG1 promoter. (A) ChIP analysis of Snf2 was performed on wild-type (FY2470), _cha4_Δ (FY2469), and srg1-25 (FY2468) strains expressing Snf2-myc and an untagged control strain (FY1350). Snf2-myc was immunoprecipitated with anti-myc A14 antibody from chromatin isolated from cells that were grown in SD + ser medium (+serine) and from cells that were shifted from SD + ser to SD medium for 25 min (–serine). The amounts of SRG1 UAS and SER3 UAS that were amplified from immunoprecipitated DNA are expressed as percentages of the amounts of input DNA. Each bar represents the average and standard error from three independent experiments. (B) ChIP analysis of Ada1. Ada1 was immunoprecipitated with anti-Ada1 antibody from the same chromatin that is described in A with the exception of the untagged control strain, which was replaced by an _ada1_Δ control strain (FY1560). Each bar represents the average amount and standard error of three independent experiments.

Figure 6.

Figure 6.

Effect of _snf2_Δ and _spt3_Δ on transcription interference by SRG1.(A) A schematic of gal1::SRG1p. The SRG1 UAS (gray boxes) and TATA (left-most black box) sequences were integrated into the GAL1 promoter, 5′ of the four Gal4-binding sites (white boxes). The arrow indicates the srg1-GAL1 RNA that is transcribed as a result of the SRG1 promoter insertion. The gal1::srg1-1p allele contains a similar insertion of the srg1-1 TATA mutant promoter. Under the conditions of the experiment, with cells grown on glucose, there is no transcription from the normal GAL1 initiation site; however, Gal4 is still bound under these growth conditions (Dudley et al. 1999b; Ren et al. 2000). (B) Northern analysis was performed on gal1::SRG1p (FY2476), gal1::srg1-1p (FY2477), _snf2_Δ gal1::SRG1p (FY2478), and _spt3_Δ gal1::SRG1p (FY2479) strains that were grown in YPD medium. Transcription from the SRG1 promoter was detected using a probe to the GAL1 UAS (SRG1-GAL1). SNR190 RNA was measured as a loading control. (C) ChIP analysis of Gal4 was performed on the same strains described in B. The amount of GAL1 UAS that was PCR-amplified from immunoprecipitated DNA was calculated relative to the amount amplified from input DNA. Each bar represents the average and standard error from three experiments.

Figure 7.

Figure 7.

A model for the coordinated regulation of serine biosynthesis and catabolism by Cha4. (Top) In the presence of high serine levels, Cha4 indirectly represses the serine biosynthetic gene SER3 via activation of SRG1 and directly activates the serine catabolic gene CHA1. (Bottome) Under serine-starvation conditions, when Cha4 is no longer able to recruit SAGA and Swi/Snf, the expression states of SER3 and CHA1 are reversed. In this model, the expression of SER3 also requires putative activators (Act.) that bind to the previously identified SER3 UAS (Martens et al. 2004). Thus, Cha4 can act as both an activator and as a repressor in response to serine.

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