Regulation of RNA polymerase II (pol II) transcription controls all cellular processes. As a result, the external control of this process is an attractive target for therapeutic agents. This proposal focuses on the mechanisms by which the activation potential of diverse regulatory factors bound to a promoter translates into increased activity of the core pol II transcription machinery on its target gene. Transcription initiation proceeds in two stages, the relief of inhibition by chromatin followed by the interaction of polymerase and accessory factors with the promoter to start transcription. Transcriptional repressors and activators regulate initiation at each of these steps. The proposed research will address the role of Saccharomyces cerevisiae Mediator, a global co-activator found in all eukaryotic cells, in regulation of the assembly step of transcription initiation. The long range goal of our research is to use a multi-disciplinary approach to investigate transcriptional activation and repression on a molecular level in a reconstituted system that also incorporates chromatin. Since the underlying mechanisms of transcription and its regulation are fundamentally conserved from yeast to man, our studies will provide general insight into gene expression. Emerging mammalian genetic studies, which associate different forms of Mediator with disease states, accentuate the imperative need to understand the fundamental mechanisms of Mediator complexes. Our studies are based on a highly purified yeast transcription system in which Mediator is required for activators to stimulate the reaction. The results of the in vitro system are subject to validation by yeast genetics. Our current goal is to carefully dissect the mechanism of yeast Mediator. The specific objectives of this research are: First, to ascertain the function of the highly conserved core subunits of all metazoan Mediators by analyzing the yeast homologs. We will start by depleting Med7p, the most widely utilized subunit, from yeast and using genomic, biochemical and genetic methods to assess the role of this protein. Second, to purify factors that modulate Mediator activity and reconstitute the physiological interplay of these factors with Mediator in the highly purified yeast transcription system. In one case a factor that specifically inhibits Mediator facilitated activated transcription and the antagonist of this inhibitor will be characterized. In the other case we will determine why Mediator is required for all transcription in vivo by biochemically characterizing a general repressor of transcription antagonized by Mediator.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Cell Development and Function Integrated Review Group (CDF)
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Tompkins, Laurie
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Dartmouth College
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Liu, Zhongle; Myers, Lawrence C (2015) Fungal mediator tail subunits contain classical transcriptional activation domains. Mol Cell Biol 35:1363-75
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
Liu, Zhongle; Myers, Lawrence C (2012) Med5(Nut1) and Med17(Srb4) are direct targets of mediator histone H4 tail interactions. PLoS One 7:e38416
Zhang, Anda; Petrov, Kostadin O; Hyun, Emily R et al. (2012) The Tlo proteins are stoichiometric components of Candida albicans mediator anchored via the Med3 subunit. Eukaryot Cell 11:874-84
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
Zhu, Xuefeng; Zhang, Yongqiang; Bjornsdottir, Gudrun et al. (2011) Histone modifications influence mediator interactions with chromatin. Nucleic Acids Res 39:8342-54
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

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