The Tup1-Ssn6 complex has been well characterized as a Saccharomyces cerevisiae general transcriptional repressor with functionally conserved homologues in metazoans. These homologues are essential for cell differentiation and many other developmental processes. The mechanism of repression of all of these proteins remains poorly understood. Srb10 (a cyclin-dependent kinase associated with the Mediator complex) and Hda1 (a class I histone deacetylase) have each been implicated in Tup1-mediated repression. We present a statistically based genome-wide analysis that reveals that Hda1 partially represses roughly 30% of Tup1-repressed genes, whereas Srb10 kinase activity contributes to the repression of ϳ15% of Tup1-repressed genes. These effects only partially overlap, suggesting that different Tup1-repression mechanisms predominate at different promoters. We also demonstrate a distinction between histone deacetylation and transcriptional repression. In an HDA1 deletion, many Tup1-repressed genes are hyperacetylated at lysine 18 of histone H3, yet are not derepressed, indicating deacetylation alone is not sufficient to repress most Tup1-controlled genes. In a strain lacking both Srb10 and Hda1 functions, more than half of the Tup1-repressed genes are still repressed, suggesting that Tup1-mediated repression occurs by multiple, partially overlapping mechanisms, at least one of which is unknown. INTRODUCTIONThe Tup1-Ssn6 complex is a general transcriptional repressor in Saccharomyces cerevisisae that controls a diverse set of genes generally characterized as being important for adaptation to nonstandard growth. Homologues of Tup1 have been identified in several other organisms (for example, unc-37 in Caenorhabditis elegans, Groucho in Drosophila, and TLE proteins in humans), and their repression functions are essential for embryonic development, cell differentiation, neurogenesis, and other developmental processes (Pflugrad et al., 1997;Fisher and Caudy, 1998;Levanon et al., 1998;Grbavec et al., 1999). Consequently, a better understanding of the mechanism of Tup1-mediated repression in yeast should illuminate this same process and its wide-ranging downstream consequences in other organisms. The Tup1-Ssn6 complex does not itself bind DNA but is recruited to target promoters through an association with sequence-specific DNA binding proteins; however, the crucial question of how transcriptional repression is established once this event occurs has not been clearly answered.Two models for Tup1-mediated repression are supported by a number of earlier observations. One proposes that Tup1 produces a transcriptionally repressed chromatin state by recruiting histone deacetylases (HDACs). Hda1, a class I HDAC, has emerged as the most likely deacetylase to be acting with Tup1. Hda1 binds to Tup1 in vitro and an HDA1 deletion results in hyperacetylation of histones at several Tup1-controlled genes (Wu et al., 2001). Hyperacetylation of Tup1-repressed genes also is seen when Tup1 is deleted (Bone and Roth, 2001;Davie et al., 2002). ...

The general transcriptional repressor Tup1 is responsible for the regulation of a large, diverse set of genes in Saccharomyces cerevisiae, and functional homologues of Tup1 have been identified in many metazoans. The crystal structure for the C-terminal portion of Tup1 has been solved and, when sequences of Tup1 homologues from fungi and metazoans were compared, a highly conserved surface was revealed. In this article, we analyze five point mutations that lie on this conserved surface. A statistical analysis of expression microarrays demonstrates that the mutant alleles are deficient in the repression of different subsets of Tup1-regulated genes. We were able to rank the mutant alleles of TUP1 based on the severity of their repression defects measured both by the number of genes derepressed and by the magnitude of that derepression. For one particular class of genes, the mutations on the conserved surface disrupted recruitment of Tup1 to the repressed promoters. However, for the majority of the genes derepressed by the Tup1 point mutants, recruitment of Tup1 to the regulated promoters is largely unaffected. These mutations affect the mechanism of repression subsequent to recruitment of the complex and likely represent a disruption of a mechanism that is conserved in fungi and metazoans. This work demonstrates that the evolutionarily conserved surface of Tup1 interacts with two separate types of proteinssequence-specific DNA-binding proteins responsible for recruiting Tup1 to promoters as well as components that are likely to function in a conserved repression mechanism.