Mediator is a global transcriptional co-regulatory protein complex that plays a central role in the activation and repression of RNA polymerase II transcribed genes. The highly conserved composition and function of Mediator, as well as the intricacies of its mechanism, make Saccharomyces cerevisiae an ideal model for incisive molecular studies. The current paradigm for Mediator function is that it serves as a direct intermediary between DNA-bound activators and the general transcription machinery. However, yeast genetic studies have revealed two important functional properties of the complex that are not compatible with this simple model. The first is Mediator's ability to repress transcription of a large number of genes via a possible link to chromatin structure. The second is the additional co-activator complexes and specific core promoter sequences required for Mediator function in activated transcription. Although ably studied by yeast genetic techniques over the past 10 to 15 years, very little is known about the direct mechanisms used by Mediator to facilitate these functions. Our proposed research meets a critical need by using biochemical experiments to reveal the mechanistic basis of these novel functional properties of Mediator. The long-term goal of our research program is to obtain a complete molecular understanding of the 'communication'between activation or repression signals and the core transcription machinery in eukaryotic cells. Our proposed studies of yeast Mediator center on the use of purified proteins and nucleic acids to elucidate mechanisms. They also marshal the substantial genetic, genomic and proteomic resources in yeast to understand these mechanisms in a physiological context. Our central hypothesis is that Mediator facilitates repression and co-operates with other co-activators through mechanisms that are distinct from serving as a direct intermediary between activators and RNA Polymerase II.
The specific aims of the current research project will answer two key questions. First, how are Mediator-facilitated and chromatin-based transcription repression linked? We propose biochemical and genetic experiments to elucidate a mechanism of repression that involves direct interactions between Mediator and chromatin. Second, how do SAGA, TFIID, and core promoter sequence affect Mediator activated transcription? We have designed biochemical experiments to determine how and if Mediator directly works with different co-activators to stimulate high levels of activated transcription in a cooperative manner. These studies are essential to understand the full range of transcriptional mechanisms used to regulate cellular and developmental processes.

Public Health Relevance

Therapeutic strategies that manipulate the expression of certain genes have been proposed for diseases as wide ranging as cancer, hemoglobinpathies (such as sickle-cell anemia), Alzheimer's disease, and infections by single celled eukaryotes such as pathogenic fungi. A limitation and frustration in the implementation of these strategies has been the inability to specifically target certain genes due to a lack of appreciation for the complex mechanisms of transcriptional regulation in eukaryotic cells. By determining how a central component of the transcription machinery facilitates both positive and negative regulation, our proposed research will add an important new consideration to efforts designed to manipulate gene expression.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics A Study Section (MGA)
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Sledjeski, Darren D
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Dartmouth College
Schools of Medicine
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Lindsay, Allia K; Morales, Diana K; Liu, Zhongle et al. (2014) Analysis of Candida albicans mutants defective in the Cdk8 module of mediator reveal links between metabolism and biofilm formation. PLoS Genet 10:e1004567
Zhang, Anda; Liu, Zhongle; Myers, Lawrence C (2013) Differential regulation of white-opaque switching by individual subunits of Candida albicans mediator. Eukaryot Cell 12:1293-304
Zhu, Xuefeng; Zhang, Yongqiang; Bjornsdottir, Gudrun et al. (2011) Histone modifications influence mediator interactions with chromatin. Nucleic Acids Res 39:8342-54
Zhu, Xuefeng; Liu, Beidong; Carlsten, Jonas O P et al. (2011) Mediator influences telomeric silencing and cellular life span. Mol Cell Biol 31:2413-21
Bjornsdottir, Gudrun; Myers, Lawrence C (2008) Minimal components of the RNA polymerase II transcription apparatus determine the consensus TATA box. Nucleic Acids Res 36:2906-16
Baidoobonso, Shamara M; Guidi, Benjamin W; Myers, Lawrence C (2007) Med19(Rox3) regulates Intermodule interactions in the Saccharomyces cerevisiae mediator complex. J Biol Chem 282:5551-9
Nair, Dhanalakshmi; Kim, Yeejin; Myers, Lawrence C (2005) Mediator and TFIIH govern carboxyl-terminal domain-dependent transcription in yeast extracts. J Biol Chem 280:33739-48
Guidi, Benjamin W; Bjornsdottir, Gudrun; Hopkins, Daniel C et al. (2004) Mutual targeting of mediator and the TFIIH kinase Kin28. J Biol Chem 279:29114-20
Auty, Roy; Steen, Hanno; Myers, Lawrence C et al. (2004) Purification of active TFIID from Saccharomyces cerevisiae. Extensive promoter contacts and co-activator function. J Biol Chem 279:49973-81
Myers, Lawrence C; Lacomis, Lynne; Erdjument-Bromage, Hediye et al. (2002) The yeast capping enzyme represses RNA polymerase II transcription. Mol Cell 10:883-94