Transcriptional activation often requires protein cofactors in addition to the general transcription machinery which includes TFIIB, TFIID, TFIIF, TFIIH and RNA polymerase II (pol II). At present, there are three general cofactors (TAF/IIs, USA, and Mediator) commonly thought to be critical for activator function. Although many studies have been conducted to identify the components and functional properties of these general cofactors, the relative contribution of each general cofactor in gene activation and the mechanism by which each cofactor function remain unclear. To address this important issue, we have developed a TAF/II- independent activation system reconstituted with only recombinant general transcription factors (TBP, TFIIB, TFIIE, and TFIIF), a recombinant cofactor (PC4), and epitope-lagged multi-protein complexes (TFIIH and pol II). In this system, PC4 is the only cofactor required for Gal4-VP16-mediated activation, thereby providing a unique in vitro functional assay to evaluate the role of each general cofactor in transcriptional activation. Since TAF/II-independent activation mediated by Gal4-VP16 can be recapitulated with only PC4 and components of the general transcription machinery, we hypothesize that some general transcription factors may play a role analogous that TFIIH can significantly affect activator function in our TAF/II-independent activation system and that TFIIH exhibit several activities also found in TFIID have prompted us to investigated whether TFIIH can indeed functionally substitute for TAF/IIs. Accordingly, we will perform order-of-addition and functional recruitment assays to address the role of general transcription factors and PC4 in TAF/II-independent activation. Although TAF/II-independent activation represents a new paradigm in eukaryotic gene regulation, many activators such as estrogen receptor (ER) strictly rely on TAF/IIs for activated transcription. Since a highly purified ER-dependent in vitro activation system has also been established in our laboratory, we will dissect the role of TAF/IIs in regulating the step of pre-initiation complex assembly and explore the hypothesis that ligand-dependent activation mediated by ER is likely due to the recruitment of Mediator which interacts with ER in a ligand- dependent manner. Collectively, these studies will shed light on the role and functional redundancy or transcriptional or transcriptional synergism of general cofactors in activator-dependent transcription.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM059643-05
Application #
6636321
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Tompkins, Laurie
Project Start
2000-03-01
Project End
2004-02-29
Budget Start
2003-03-01
Budget End
2004-02-29
Support Year
5
Fiscal Year
2003
Total Cost
$172,125
Indirect Cost
Name
Case Western Reserve University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Chakravarty, Kaushik; Hanson, Richard W (2007) Insulin regulation of phosphoenolpyruvate carboxykinase-c gene transcription: the role of sterol regulatory element-binding protein 1c. Nutr Rev 65:S47-56
Chakravarty, Kaushik; Wu, Shwu-Yuan; Chiang, Cheng-Ming et al. (2004) SREBP-1c and Sp1 interact to regulate transcription of the gene for phosphoenolpyruvate carboxykinase (GTP) in the liver. J Biol Chem 279:15385-95
Hu, Xinli; Chen, Yixin; Farooqui, Mariya et al. (2004) Suppressive effect of receptor-interacting protein 140 on coregulator binding to retinoic acid receptor complexes, histone-modifying enzyme activity, and gene activation. J Biol Chem 279:319-25
Wu, Shwu-Yuan; Zhou, Tianyuan; Chiang, Cheng-Ming (2003) Human mediator enhances activator-facilitated recruitment of RNA polymerase II and promoter recognition by TATA-binding protein (TBP) independently of TBP-associated factors. Mol Cell Biol 23:6229-42
Zhou, Tianyuan; Chiang, Cheng-Ming (2002) Sp1 and AP2 regulate but do not constitute TATA-less human TAF(II)55 core promoter activity. Nucleic Acids Res 30:4145-57
Hou, Samuel Y; Wu, Shwu-Yuan; Chiang, Cheng-Ming (2002) Transcriptional activity among high and low risk human papillomavirus E2 proteins correlates with E2 DNA binding. J Biol Chem 277:45619-29
Zhou, T; Chiang, C M (2001) The intronless and TATA-less human TAF(II)55 gene contains a functional initiator and a downstream promoter element. J Biol Chem 276:25503-11
Wu, S Y; Chiang, C M (2001) TATA-binding protein-associated factors enhance the recruitment of RNA polymerase II by transcriptional activators. J Biol Chem 276:34235-43
Fukuda, A; Yamauchi, J; Wu, S Y et al. (2001) Reconstitution of recombinant TFIIH that can mediate activator-dependent transcription. Genes Cells 6:707-19