The major goal of this proposal is to reveal molecular mechanisms underlying formation and function of critical transcriptional assemblies essential to embryonic stem (ES) cells and cells with induced pluripotency (induced pluripotent stem (iPS) cells). Using bioinformatics and high throughput experimental methods, we will prepare defined domains of critical transcriptional factors controlling cell pluripotency and analyze them in their functional associations. Thousands of assemblies will be evaluated by biochemical and biophysical methods to identify the critical ones to be targeted by X-ray crystallography.
We aim for determination of three-dimensional structures for about 100 stable multi-component transcriptional assemblies. Each of them will represent a partial image of complicated transcriptional machinery controlling the specific transcriptional landscape of pluripotent cells. We expect that thoughtful analyses of these structures will enable us to establish the proper connections between these partial images and reconstruct a general model for function of critical participants of this transcriptional machinery. We will justify this model and the observed regulatory interactions within identified transcriptional complexes in mutational and functional studies using iPS cells. The experiments are to be done at multiple sites: The Methodist Hospital Research Institute (Houston), Department of Biochemistry and Biophysics at UCSF, the Gladstone Institute of Cardiovascular Disease (UCSF) and X-ray crystallography by the PSI labs. The proposed structural and functional studies will propel our general knowledge of the basic mechanisms controlling cell fate, including those underlying self renewal, differentiation and pathogenesis of cancer. The results of this research will also provide more efficient molecular tools allowing precise control over cell programming and reprogramming. The accumulated structural and functional data would be immediately available to biochemical and clinical researchers, and therefore, would have a major impact on stem cell research as well as regenerative medicine.

Public Health Relevance

The proposed studies on embryonic and reprogrammed stem cells will reveal parts of the complex mechanisms controlling cell fate, including those underlying self renewal, differentiation, and development of cancer. The results of our research will provide more efficient molecular tools allowing precise control over cell programming and reprogramming. The images and concepts that we produce will have immediate impact on regenerative medicine as well as stem cell and cancer research.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01GM094614-05
Application #
8690904
Study Section
Special Emphasis Panel (ZGM1-CBB-0 (BC))
Program Officer
Preusch, Peter
Project Start
2010-09-30
Project End
2015-06-30
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
5
Fiscal Year
2014
Total Cost
$1,101,240
Indirect Cost
$222,150
Name
University of California San Francisco
Department
Biochemistry
Type
Schools of Medicine
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
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Miyaoka, Yuichiro; Chan, Amanda H; Judge, Luke M et al. (2014) Isolation of single-base genome-edited human iPS cells without antibiotic selection. Nat Methods 11:291-3
Bhattacharya, Suparna; Lou, Xiaohua; Hwang, Peter et al. (2014) Structural and functional insight into TAF1-TAF7, a subcomplex of transcription factor II D. Proc Natl Acad Sci U S A 111:9103-8
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