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 UASGAL 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.

Corepressors SsnF and RcoA Regulate Development and Aflatoxin B1 Biosynthesis in Aspergillus flavus NRRL 3357

“…In fact, RcoA and SsnF frequently modulate downstream gene expressions independently of CCR regulation. Cyc8 in yeast regulates GAL1 without Mig1 recruitment, and deletion of cyc8 does not elevate GAL1 expression [ 15 , 23 ]. In addition, Cyc8-Tup1 could directly interact with histones H3 and H4, influencing their acetylation [ 24 ].…”

Functional characterization and comparative analysis of gene repression-mediating domains interacting with yeast pleiotropic corepressors Sin3, Cyc8 and Tup1

“…Yeast strains, media and growth conditions Assays for gene repression in vivo were performed with S. cerevisiae strain RTS-lexA, containing an integrated CYC1-lacZ reporter gene with 4 lexA operator sites in its upstream region (MATα leu2 his3 trp1 ura3::lexA Op -CYC1-lacZ ::URA3; Lettow et al 2022). Transformants with effector plasmids encoding LexArepressor fusions (based on pRT-lexA: 2µm LEU2 MET25 PR -HA 3 -lexA DBD -NLS) were cultivated under double-selective conditions in synthetic complete media (SCD-Ura-Leu, 2% glucose).…”

“…These expression cassettes were also used to construct translational lexA-repressor fusions by insertion into the multi-cloning site of plasmid pRT-lexA (episomal plasmid containing lexA DBD fused with a nuclear localization sequence and activated by the MET25 promoter). Plasmids used to study CTI6, FKH1, GAL80 and OPI1 have been described (Aref and Schüller 2020; Aref et al 2021;Lettow et al 2022;Jäschke et al, 2011). Epitopetagged corepressor HA 3 -Sin3 (full-length) was synthesized in yeast or in E. coli, using expression plasmids pCW117 (Wagner et al 2001) or pSW11 (Grigat et al 2012) while bacterial expression plasmid pJL34 encodes a truncated Sin3 comprising PAH1 and PAH2.…”

Functional characterization and comparative analysis of gene repression-mediating domains interacting with yeast pleiotropic corepressors Sin3, Cyc8 and Tup1

“…Assays for gene repression in vivo were performed with S. cerevisiae strain RTS-lexA, containing an integrated CYC1-lacZ reporter gene with 4 lexA operator sites in its upstream region ( MAT α leu2 his3 trp1 ura3::lexA Op -CYC1-lacZ::URA3 ; Lettow et al 2022 ). Transformants with effector plasmids encoding LexA-repressor fusions (based on pRT-lexA: 2 µm LEU2 MET25 PR -HA 3 -lexA DBD -NLS) were cultivated under double-selective conditions in synthetic complete media (SCD-Ura-Leu, 2% glucose).…”

“…These expression cassettes were also used to construct translational lexA-repressor fusions by insertion into the multi-cloning site of plasmid pRT-lexA (episomal plasmid containing lexA DBD fused with a nuclear localization sequence and activated by the MET25 promoter). Plasmids used to study CTI6 , FKH1 , GAL80 and OPI1 have been described (Aref and Schüller 2020 ; Aref et al 2021 ; Lettow et al 2022 ; Jäschke et al 2011 ). Epitope-tagged corepressor HA 3 -Sin3 (full-length) was synthesized in yeast or in E. coli , using expression plasmids pCW117 (Wagner et al 2001 ) or pSW11 (Grigat et al 2012 ) while bacterial expression plasmid pJL34 encodes a truncated Sin3 comprising PAH1 and PAH2.…”