Sum1p Repression von SPS18 unter Ölsäure-Mediumsbeding.

The one-year project described herein is concerned with underpinning the role of the yeast transcription factor Sum1p in regulating SPS18 via a resident mid-sporulation element MSE in its promoter. The identity of SPS18 is not yet clear, but it shares its promoter region with the oleic acid-inducible gene SPS19 encoding peroxisomal 2,4- dienoyl-CoA reductase, involved in fatty acid ß-oxidation. Although both genes are transcribed in sporulating diploid cells, SPS19 is highly expressed in haploid cells grown on fatty acids whereas SPS18 is not. Oleic acid- dependent transcription of SPS19 is due in part to an oleate response element ORE that binds Pip2p-Oaf1p representing the molecular sensor for fatty acids in the growth medium. In addition, the gene abides to regulation by a type 1 upstream activating sequence UAS1 that is targeted by the transcription factor Adr1p responsible for derepression of genes when glucose is scarce. Previous work using a set of lacZ-reporter genes with window deletions in the SPS18/SPS19 promoter identified an SPS18-repressing fragment that contained an MSE. Under vegetative conditions, certain MSEs are targeted by the Sum1p repressor in association with Hst1p. The present study proposes to address the issue of whether Sum1p in combination with Hst1p acts to shield SPS18 from oleic acid-dependent transcriptional up-regulation applied to the divergent SPS19 by acting as a repressor at the MSE. The envisioned studies should not only shed further light on the regulatory mechanism affecting the divergent SPS18/SPS19 promoter, but additionally might lead to the identification of novel circuits that maintain unidirectional transcriptional activation in the compact genome of yeast.

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 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 mid-sporulation 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 polymerase chain reaction demonstrated that in haploid sum1Δ, hst1Δ, or rfm1Δ cells, oleic acid-dependent expression of SPS18 was higher compared with the situation in wild-type cells, but in the sum1Δ 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.