Protein-protein interactions among transcription factors play critical roles in the regulation of gene expression in normal and disease states. This application proposes to study protein-protein interactions mediated by Cys2His2 zinc finger proteins, the most abundant class of transcription factors in humans. In particular, we are focusing our studies on dimerization zinc finger (DZF) domains found in members of the Ikaros family of transcription factors and related homologues. DZF domains provide a model system for studying Cys2His2 zinc finger-mediated protein-protein interactions. The long-term goals of this proposal are: to gain a detailed biophysical understanding of how Cys2His2 zinc fingers mediate specific interactions with a wide variety of different proteins; to utilize this knowledge to devise strategies to enhance, disrupt, or predict such interactions; and to engineer synthetic zinc fingers with novel interaction specificities that could serve as useful modules for creating artificial regulatory circuits with potential applications in biotechnology and therapeutics.
The specific aims of this application are: 1) to define and model the Ikaros, Pegasus, and Hunchback DZF domain interfaces using mutational analysis, genetic selection methods, and molecular graphics tools; 2) to determine high-resolution structures of dimeric Ikaros, Pegasus, and Hunchback DZF domain protein-protein complexes using X-ray crystallography; 3) to use genetic selection to identify synthetic, obligate heterodimeric DZF domains and to test the use of DZF domains as """"""""interaction modules"""""""" for assembling heterologous protein complexes in eukaryotic cells. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM072621-04
Application #
7387433
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Tompkins, Laurie
Project Start
2005-04-01
Project End
2010-03-31
Budget Start
2008-04-01
Budget End
2010-03-31
Support Year
4
Fiscal Year
2008
Total Cost
$298,678
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199
Sander, Jeffry D; Maeder, Morgan L; Reyon, Deepak et al. (2010) ZiFiT (Zinc Finger Targeter): an updated zinc finger engineering tool. Nucleic Acids Res 38:W462-8
Sander, Jeffry D; Zaback, Peter; Joung, J Keith et al. (2007) Zinc Finger Targeter (ZiFiT): an engineered zinc finger/target site design tool. Nucleic Acids Res 35:W599-605
Meng, Xiangdong; Thibodeau-Beganny, Stacey; Jiang, Tao et al. (2007) Profiling the DNA-binding specificities of engineered Cys2His2 zinc finger domains using a rapid cell-based method. Nucleic Acids Res 35:e81
Giesecke, Astrid V; Fang, Rui; Joung, J Keith (2006) Synthetic protein-protein interaction domains created by shuffling Cys2His2 zinc-fingers. Mol Syst Biol 2:2006.2011
Wright, David A; Thibodeau-Beganny, Stacey; Sander, Jeffry D et al. (2006) Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly. Nat Protoc 1:1637-52
Meng, Xiangdong; Smith, Robin M; Giesecke, Astrid V et al. (2006) Counter-selectable marker for bacterial-based interaction trap systems. Biotechniques 40:179-84