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Transcriptional repressor Gal80 recruits corepressor complex Cyc8-Tup1 to structural genes of the Saccharomyces cerevisiae GAL regulon.

ABSTRACT: Under non-inducing conditions (absence of galactose), yeast structural genes of the GAL regulon are repressed by Gal80, preventing interaction of Gal4 bound to UAS_GAL promoter motifs with general factors of the transcriptional machinery. In this work, we show that Gal80 is also able to interact with histone deacetylase-recruiting corepressor proteins Cyc8 and Tup1, indicating an additional mechanism of gene repression. This is supported by our demonstration that a lexA-Gal80 fusion efficiently mediates repression of a reporter gene with an upstream lexA operator sequence. Corepressor interaction and in vivo gene repression could be mapped to a Gal80 minimal domain of 65 amino acids (aa 81-145). Site-directed mutagenesis of selected residues within this domain showed that a cluster of aromatic-hydrophobic amino acids (YLFV, aa 118-121) is important, although not solely responsible, for gene repression. Using chromatin immunoprecipitation, Cyc8 and Tup1 were shown to be present at the GAL1 promoter in a wild-type strain but not in a gal80 mutant strain under non-inducing (derepressing) growth conditions. Expression of a GAL1-lacZ fusion was elevated in a tup1 mutant (but not in a cyc8 mutant) grown in derepressing medium, indicating that Tup1 may be mainly responsible for this second mechanism of Gal80-dependent gene repression.

SUBMITTER: Lettow J

PROVIDER: S-EPMC8801411 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Saccharomyces cerevisiae normally cannot assimilate mannitol, a promising brown macroalgal carbon source for bioethanol production. To date, the molecular mechanisms underlying this inability remain unknown. Here, we found that cells acquiring mannitol-assimilating ability appeared from wild-type S. cerevisiae strain during prolonged culture in mannitol medium. Our microarray analysis revealed that genes for putative mannitol dehydrogenase and hexose transporters were up-regulated in cells acquiring mannitol-assimilating ability. Take account of our other results including complementation analysis and cell growth data, we demonstrated that this acquisition of mannitol-assimilating ability was due to the spontaneous mutation in the gene encoding Tup1 or Cyc8. Tup1-Cyc8 is the general corepressor complex involved in the repression of many kinds of genes. Thus, it is suggested that the inability of wild-type S. cerevisiae to assimilate mannitol can be attributed to the transcriptional repression of a set of genes involved in mannitol utilization by Tup1-Cyc8 corepressor. In other words, Tup1-Cyc8 is a key regulator of mannitol metabolism in S. cerevisiae. We also showed that S. cerevisiae strain which carries mutant allele of TUP1 or CYC8 produced ethanol from mannitol efficiently. Especially, strain carrying mutant allele of CYC8 showed high tolerance to salt, which is superior to other ethanologenic microorganisms. This characteristic is highly beneficial to produce bioethanol from marine biomass. Taken together, Tup1-Cyc8 can be an ideal target to develop a yeast-algal bioethanol production system. To figure out how Mtl+ strains (cells acquiring ability to grow in mannitol medium) had acquired the ability to assimilate mannitol, we performed genome-wide analysis by using Nimblegen microarrays. Yeast Saccharomyces cerevisiae cells (wild-type BY4742 strain and two Mtl+ strains, MK3619 and MK3683) were grown at 30Â°C to the logarithmic phase in SC or SM media. Total RNA was purified and the 4 RNA samples (BY4742 cells in SC as control, MK3619 cells in SM, MK3683 cells in both SC and SM) were analyzed with Nimblegen microarrays.

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Transcriptional repressor Gal80 recruits corepressor complex Cyc8-Tup1 to structural genes of the Saccharomyces cerevisiae GAL regulon.

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