Avoiding unscheduled transcription in shared promoters: Saccharomyces cerevisiae Sum1p represses the divergent gene pair SPS18-SPS19 through a midsporulation element (MSE)

Abstract

The sporulation-specific gene SPS18 shares a common promoter region with the oleic acid-inducible gene SPS19. Both genes are transcribed in sporulating diploid cells, albeit unevenly in favour of SPS18, whereas in haploid cells grown on fatty acids only SPS19 is highly activated. Here, SPS19 oleate-response element (ORE) conferred activation on a basal CYC1-lacZ reporter gene equally in both orientations, but promoter analysis using SPS18-lacZ reporter constructs with deletions identified a repressing fragment containing a midsporulation element (MSE) that could be involved in imposing directionality towards SPS19 in oleic acid-induced cells. In sporulating diploids, MSEs recruit the Ndt80p transcription factor for activation, whereas under vegetative conditions, certain MSEs are targeted by the Sum1p repressor in association with Hst1p and Rfm1p. Quantitative real-time PCR demonstrated that in haploid sum1Delta, hst1Delta, or rfm1Delta cells, oleic acid-dependent expression of SPS18 was higher compared with the situation in wild-type cells, but in the sum1Delta mutant, this effect was diminished in the absence of Oaf1p or Pip2p. We conclude that SPS18 MSE is a functional element repressing the expression of both SPS18 and SPS19, and is a component of a stricture mechanism shielding SPS18 from the dramatic increase in ORE-dependent transcription of SPS19 in oleic acid-grown cells.

Fig. 1

Scheme of the SPS18–SPS19 intergenic region incorporated into the reporter genes used. A 1.4-kb XbaI–SphI fragment including the shared SPS18–SPS19 promoter region and a portion of the reading frames of both genes was used as template for site-directed mutagenesis. The distance between the two ATG translational start codons is 300 bp. The terminal 3′ G in the depicted sequence occurs 79 nucleotides upstream of the SPS18 ATG site, whereas the terminal 5′ C is 130 bp upstream of the SPS19 ATG triplet. The sequences representing UAS1_SPS19, SPS19 ORE, UAS^SPS19, and SPS18 MSE, are indicated as boxes below the sequence. Boxed regions above the sequence represent mutations introduced into the promoter that was incorporated within the various reporter genes used. An XhoI restriction site (CTCGAG) was substituted for the boxed DNA sequences designated M1 and M3, whereas regions designated M2, M4, and M5 were deleted. TATA-box sequences TATAAA or TATAAG occur 61 and 103 nucleotides 5′ of the SPS18 and SPS19 ATG start codons, respectively.

Fig. 2

Scheme of the promoter arrangement in the strains reported in Table 5. In the WT strain yAG561 expressing the WT reporter gene (a), Sum1p acting at the MSE is proposed to block oleic acid-induced transcriptional activation () due to Pip2p-Oaf1p binding to the ORE, so that no decrease in reporter-gene expression levels was etected when Pip2p-Oaf1p was absent in the mutant strain yAG1310 (b). However, when the MSE was mutated within the M3 reporter construct (c) and Sum1p could not act on its cognate element, Pip2p-Oaf1p could induce transcription of SPS18 (→) in the WT strain yAG565 beyond the levels attained by the pip2Δoaf1Δ mutant yAG1312 (d). Thick and thin arrows indicate high or low levels of transcription, respectively. UAS refers to an overlapping Adr1p-binding element.

Fig. 3

Scheme of the promoter arrangement in the strains reported in Table 6. In the WT strain yAG1230 expressing the WT SPS18–lacZ reporter construct (a), Sum1p could act at the MSE to block oleic acid-induced transcriptional activation () due to Pip2p-Oaf1p binding to the ORE, but in yAG1193 cells devoid of Sum1p (b); this latter activation proceeded unhindered (→), resulting in higher levels of reporter-gene activity. The thick arrows reflect higher levels of transcription compared with those depicted by the thin arrows. The Adr1p-binding element overlapping the ORE is referred to as UAS.