The molecular mechanisms by which eukaryotes regulate gene expression and chromatin structure are important for basic scientific knowledge that is necessary for understanding many complex biological phenomena including development and human disease. This proposal addresses two related issues of broad significance: the molecular mechanisms of transcriptional repression by the evolutionarily related yeast Cyc8-Tup1 and human TLE corepressors, and the relationship between global patterns of histone modifications to transcription and maintenance of epigenetic states. To do this, we will combine chromatin immunoprecipitation, genetic, genomic, and biochemical approaches to carry out the following projects. First, we will address the mechanisms and relative importance of three general models for how Cyc8-Tup1 mediates repression: interaction with the Mediator complex that associates with RNA polymerase II; direct effects on chromatin; steric interference. We will determine the mechanistic step(s) inhibited by Cyc8-Tup1, analyze whether Cyc8-Tup1 is sufficient to recruit Mediator and cooperates with or inhibits activators from recruiting Mediator, identify direct targets of the Tup1 repression domain within the Mediator complex, and address whether Cyc8-Tup1 recruits histone deacetylases and how it mediates long-range effects on chromatin. Second, we will test the model that Eaf3 controls the global pattern of histone acetylation in yeast cells via an interaction between Eaf3 chromodomains and methylated histones in the coding region that causes preferential deacetylation by the Rpd3 complex. We will also test the idea that an unappreciated level of transcription throughout the yeast genome accounts for the apparent paradox that the Pall transcriptional elongation complex is required for genome-wide methylation of histone H3 at lysines 4 and 79. Third, we will test whether histone deposition during S phase occurs by the conventional model involving H3-H4 tetramers or a newly proposed model involving H3-H4 dimers, an issue with major implications for how epigenetic states are maintained through cell division. In addition, we will directly address the relationship and mechanism between transcriptional activity and histone dissociation, and measure the rate of transcription-associated (i.e. non-replicative) nucleosome deposition. Fourth, as part of our continuing collaboration with Tom Gingeras at Affymetrix that involves tiled microarrays, we will define, in an unbiased manner and on a whole-genome scale, the physiological targets of human TLE proteins in HL60 cells undergoing retinoic acid-induced differentiation. These results will be linked to detailed maps of RNA transcripts, histone modifications, chromatin-modifying activities, and transcriptional regulatory factors that will be performed on the identical samples in work not covered by this proposal. We will use RNAi to identify genes affected by the various TLE proteins, and will address the mechanism of repression by TLE proteins by using derivatives of the well characterized B-interferon promoter.

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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Carter, Anthony D
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Harvard University
Schools of Medicine
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Fan, Xiaochun; Lamarre-Vincent, Nathan; Wang, Qian et al. (2008) Extensive chromatin fragmentation improves enrichment of protein binding sites in chromatin immunoprecipitation experiments. Nucleic Acids Res 36:e125
Joshi, Amita A; Struhl, Kevin (2005) Eaf3 chromodomain interaction with methylated H3-K36 links histone deacetylation to Pol II elongation. Mol Cell 20:971-8
Cawley, Simon; Bekiranov, Stefan; Ng, Huck H et al. (2004) Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs. Cell 116:499-509
Reid, Juliet L; Moqtaderi, Zarmik; Struhl, Kevin (2004) Eaf3 regulates the global pattern of histone acetylation in Saccharomyces cerevisiae. Mol Cell Biol 24:757-64
Geisberg, Joseph V; Struhl, Kevin (2004) Cellular stress alters the transcriptional properties of promoter-bound Mot1-TBP complexes. Mol Cell 14:479-89
Ng, Huck Hui; Xu, Rui-Ming; Zhang, Yi et al. (2002) Ubiquitination of histone H2B by Rad6 is required for efficient Dot1-mediated methylation of histone H3 lysine 79. J Biol Chem 277:34655-7
Geisberg, Joseph V; Moqtaderi, Zarmik; Kuras, Laurent et al. (2002) Mot1 associates with transcriptionally active promoters and inhibits association of NC2 in Saccharomyces cerevisiae. Mol Cell Biol 22:8122-34
Ng, Huck Hui; Feng, Qin; Wang, Hengbin et al. (2002) Lysine methylation within the globular domain of histone H3 by Dot1 is important for telomeric silencing and Sir protein association. Genes Dev 16:1518-27
Proft, Markus; Struhl, Kevin (2002) Hog1 kinase converts the Sko1-Cyc8-Tup1 repressor complex into an activator that recruits SAGA and SWI/SNF in response to osmotic stress. Mol Cell 9:1307-17
Deckert, J; Struhl, K (2001) Histone acetylation at promoters is differentially affected by specific activators and repressors. Mol Cell Biol 21:2726-35

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