Combinatorial control of gene expression is a fundamental mechanism for integrating diverse regulatory pathways within a cell. It is founded on the principles of cooperativity and synergy. Unique arrays of activators bind cooperatively to enhancers and promoters and assemble into nucleoprotein complexes termed enhanceosomes. Enhanceosomes then activate transcription synergistically via interaction with coactivator complexes. The enhanceosome and coactivator complexes are emerging as cellular organizing centers, which integrate signals from a wide range of inputs to generate specific regulatory responses. Knowledge of the mechanisms underlying these important steps in gene control will provide broad insights into biological regulation. ? ? This proposal focuses largely on the biochemical mechanisms of activator-coactivator interactions and their role in assembling transcription complexes. We hypothesize that a higher order assembly composed of activators and two coactivators, termed the DA (TFIID and TFIIA) and Med (Mediator) complexes, serves as a key intermediate in transcription initiation.
In Aim #1 we test how this so-called DAMed complex is involved in the mechanism of transcriptional synergy and action at a distance by using cell-free mammalian systems and two model activators, GAL4-VP16 and ZEBRA.
Aim #2 explores the mechanism and relevance of DAMed complex phosphorylation in initiation.
Aim #3 diverges slightly from the main theme of the proposal to examine a prototypical example of specificity by the abundant architectural protein HMGB1 in assembly of ZEBRA-containing enhanceosomes. ? ? Our studies are designed to leverage the vast amount of knowledge on the GAL4-VP16 and ZEBRA systems to understand the detailed mechanism of the mediator and enhanceosome in gene control. Both topics are relatively new discoveries, whose understanding will have broad implications on diverse areas of biology. Detailed knowledge of mechanism is necessary to apply concepts derived from studying gene expression to disease-oriented problems. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM057283-05
Application #
6619283
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Tompkins, Laurie
Project Start
1998-07-01
Project End
2006-04-30
Budget Start
2003-05-01
Budget End
2004-04-30
Support Year
5
Fiscal Year
2003
Total Cost
$286,057
Indirect Cost
Name
University of California Los Angeles
Department
Biochemistry
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Johnson, Kristina M; Wang, Jin; Smallwood, Andrea et al. (2004) The immobilized template assay for measuring cooperativity in eukaryotic transcription complex assembly. Methods Enzymol 380:207-19
Johnson, Kristina M; Carey, Michael (2003) Assembly of a mediator/TFIID/TFIIA complex bypasses the need for an activator. Curr Biol 13:772-7
Johnson, Kristina M; Wang, Jin; Smallwood, Andrea et al. (2002) TFIID and human mediator coactivator complexes assemble cooperatively on promoter DNA. Genes Dev 16:1852-63
Mitsouras, Katherine; Wong, Ben; Arayata, Charina et al. (2002) The DNA architectural protein HMGB1 displays two distinct modes of action that promote enhanceosome assembly. Mol Cell Biol 22:4390-401
Ellwood, K B; Yen, Y M; Johnson, R C et al. (2000) Mechanism for specificity by HMG-1 in enhanceosome assembly. Mol Cell Biol 20:4359-70
Ellwood, K; Huang, W; Johnson, R et al. (1999) Multiple layers of cooperativity regulate enhanceosome-responsive RNA polymerase II transcription complex assembly. Mol Cell Biol 19:2613-23
Wang, J; Ellwood, K; Lehman, A et al. (1999) A mathematical model for synergistic eukaryotic gene activation. J Mol Biol 286:315-25
Ellwood, K; Chi, T; Huang, W et al. (1998) Cooperative assembly of RNA polymerase II transcription complexes. Cold Spring Harb Symp Quant Biol 63:253-61