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.
Highlights
Transcriptional repression in eukaryotes can be executed by several molecular mechanisms such as prevention of nuclear import of an activator, inhibition of its DNA binding, or deactivation of transcriptional activation domains
Gal80 was able to interact with Cyc8 and Tup1 but not with Sin3. Since both corepressors were synthesized in E. coli, we conclude that Gal80 can directly interact with Cyc8 and Tup1 without contribution of additional yeast proteins
There is no complete agreement on all regulatory aspects of GAL gene control in S. cerevisiae, Gal80 is generally considered as an antagonist of Gal4, needed to shield its activation domain unless galactose induction becomes effective
Summary
Transcriptional repression in eukaryotes can be executed by several molecular mechanisms such as prevention of nuclear import of an activator, inhibition of its DNA binding, or deactivation of transcriptional activation domains. Generating a chromatin structure being refractory against access of transcription factors is of general importance for eukaryotic gene repression (Courey and Jia 2001). This is achieved by gene-specific repressor proteins utilizing a limited number of corepressors which recruit. Neither Cyc nor Tup is able to bind DNA, but depends on interaction with sequencespecific DNA-binding proteins (Mig, Rox, α2, and Crt, among others) to trigger gene repression. Structural repetitions within Cyc (10 tetratricopeptide repeats, TPR, found at the N-terminus; Tzamarias and Struhl 1995) and Tup (7 WD40 repeats at the C-terminus; Zhang et al 2002) are
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