How enhancers and promoters communicate is one of the important questions in the field of eukaryotic gene regulation. Despite provocative models from textbooks and review articles, very little is genuinely known of the mechanism. We were the first lab to show a direct interaction between TFIID and Mediator co-activators, and demonstrate that this interaction was required for Pol II preinitiation complex assembly in vitro. Based on these studies, we hypothesize that the key interaction driving Pol II preinitiation complex assembly and function is activator-regulated interaction between Mediator at the enhancer and TFIID at the promoter. We will leverage the knowledge and biochemical/genomewide technologies acquired from studies over the last three funding cycles to comprehensively identify proteins forming the TFIID-Mediator interface in vitro and determine if these interactions are central to gene expression and promoter-enhancer looping in cells. Our studies will employ biochemical and biological assays developed to analyze how the murine embryonic stem cell activator Esrrb activates transcription in vivo in murine embryonic stem cells (mESCs) and in vitro in extracts. This system will serve as a model and focal point for understanding the TFIID-Mediator interface.
In Aim 1, we will identify the interface between Esrrb, Mediator and TFIID using crosslinking mass spectrometry (XL-MS). We will validate and further study these surfaces using protein-protein interaction assays and mutagenesis.
In Aim 2, we will identify the TFIID-Mediator co-activator surface in vivo by systematic RNAi knockdown of co-activator subunits, individually and then in pairwise combinations, followed by RNA-seq to identify overlapping effects. We will perform genomewide ChIP-seq of TFIID and Mediator under mock and knockdown conditions to probe effects and correlate with the results from Aim 1.
Aim 3 examines whether the TFIID-Mediator interface is necessary or not for promoter-enhancer looping in vivo using 4C. Our results will provide a detailed mechanism of eukaryotic gene activation using powerful state-of-the-art biochemical and biological approaches.

Public Health Relevance

Aberrant gene regulation is associated with many forms of cancer and other diseases. We will employ state-of-the-art biochemical and genomewide methodologies to understand the mechanisms involved in turning genes on and off with an emphasis on a core complex of proteins termed TFIID and Mediator, whose regulation in central to gene activity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM074701-16
Application #
10084168
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
2005-08-01
Project End
2022-01-31
Budget Start
2021-02-01
Budget End
2022-01-31
Support Year
16
Fiscal Year
2021
Total Cost
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
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Sun, Fei; Chronis, Constantinos; Kronenberg, Michael et al. (2018) Promoter-Enhancer Communication Occurs Primarily within Insulated Neighborhoods. Mol Cell :
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Pradhan, Suman K; Su, Trent; Yen, Linda et al. (2016) EP400 Deposits H3.3 into Promoters and Enhancers during Gene Activation. Mol Cell 61:27-38
Xue, Yong; Van, Christopher; Pradhan, Suman K et al. (2015) The Ino80 complex prevents invasion of euchromatin into silent chromatin. Genes Dev 29:350-5
Sridharan, Rupa; Gonzales-Cope, Michelle; Chronis, Constantinos et al. (2013) Proteomic and genomic approaches reveal critical functions of H3K9 methylation and heterochromatin protein-1? in reprogramming to pluripotency. Nat Cell Biol 15:872-82
Chen, Xiao-Fen; Lehmann, Lynn; Lin, Justin J et al. (2012) Mediator and SAGA have distinct roles in Pol II preinitiation complex assembly and function. Cell Rep 2:1061-7
Lehmann, Lynn; Ferrari, Roberto; Vashisht, Ajay A et al. (2012) Polycomb repressive complex 1 (PRC1) disassembles RNA polymerase II preinitiation complexes. J Biol Chem 287:35784-94

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