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Scot Wolfe, Ph.D.Academic Role: Associate Professor
Faculty Appointment(s) In:
Other Affiliation(s):
Creating artifical DNA-binding domains for targeted gene regulation and gene modification
Protein-DNA recognition - Our research on protein-DNA recognition is focused primarily on two of the most abundant families of DNA-binding domains in metazoans:
We have recently performed the first comprehensive analysis of homeodomain specificities in a metazoan (D. melanogaster – fruit fly) in collaboration with Michael Brodsky (UMMS-PGFE) & Gary Stormo (Wash. U) (Noyes et al., Cell 2008). Using this information we can build simple qualitative models of recognition that allow the design of homeodomains with novel DNA-binding specificity. This dataset can also be used to broadly predict the specificity of family members from other species (see ural.wustl.edu/flyhd). We continue to build upon this work to understand fundamental aspects of DNA-recognition for the homeodomain and zinc finger families with the goal of broadly and accurately predicting the specificity of naturally-occurring family members in all species. These studies will also provide a valuable resource for understanding specificity determinants within each family for rationally engineering the specificity of these DNA-binding domains.
B1H selection systems - We continue to develop a bacterial one-hybrid system for rapidly characterizing the DNA-binding specificities of sequence-specific transcription factors, both naturally-occurring and engineered. Using this technology we intend to characterize all of the sequence-specific transcription factors in the D. melanogaster genome in collaboration with the laboratory of Michael Brodsky (UMMS – PGFE). This dataset will be used to unravel transcription factor regulatory networks within the fly in collaboration with Saurabh Sinha (UI-Urbana Champaign). We have already begun building computational tools to allow the scientific community to identify cis-regulatory modules using clusters of phylogenetically conserved binding sites for the ~15% of the TFs in the fly genome that we have characterized to date (GenomeSurveyor - biotools.umassmed.edu/genomesurveyor).
ZFNs in Zebrafish - We have utilized our selection technology to create zinc finger nucleases that recognize specific genes in the Zebrafish genome in collaboration with Nathan Lawson (UMMS – PGFE). Zinc finger nucleases (ZFNs) are tailor-made restriction endonucleases that can generate a double-stranded break at a specific DNA sequence defined by the specificity of the attached zinc fingers. Using this technology we have made the first targeted gene knockouts in the zebrafish (Meng et al., Nat. Biotech 2008). We continue to develop these DNA-targeting and cleavage tools with the goal of creating an accessible resource for model organism communities that will allow them to disrupt, or modify, a desired gene in any model organism. This technology should revolutionize reverse genetic approaches in most model organisms and may allow the straightforward creation of tailor-made human disease models with profound implications for the development of treatments for a variety of diseases.
PublicationsNoyes, M.B., Christensen, R.G., Wakabayashi, A., Stormo, G.D., Brodsky, M.H., and Wolfe, S.A. (2008) Analysis of homeodomain specificities allows the family-wide prediction of preferred recognition sites. Cell, 133: 1277-1289. Meng, X., Noyes, M.B., Zhu, L.J., Lawson, N.D., and Wolfe, S.A. (2008) Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Nat. Biotechnol., 26: 695-701. Meng, X., Thibodeau-Beganny, S., Jiang, T., Joung, J.K., and Wolfe, S.A. (2007) Profiling the DNA-binding specificities of engineered Cys2His2 zinc finger domains using a rapid cell-based method. Nucleic Acids Res., 35: e81. Meng, X. and Wolfe, S.A. (2006) Identifying DNA sequences recognized by a transcription factor using a bacterial one-hybrid system. Nature Protoc., 1: 30-45. Meng, X., Smith, R.M., Giesecke, A.V., Joung, J.K., and Wolfe, S.A. (2006) A counter-selectable marker for bacterial-based interaction trap systems. BioTechniques, 40: 179-184. Meng, X., Brodsky, M.H., and Wolfe, S.A. (2005) A bacterial one-hybrid system for determining the DNA-binding specificity of transcription factors. Nature Biotechnol., 23: 988-994. Wolfe, S.A., Grant, R.A., and Pabo, C.O. (2003) Structure of a designed dimeric zinc finger protein bound to DNA. Biochemistry, 42: 13401-13409. Wolfe, S.A., Grant, R.A., Elrod-Erickson, M., and Pabo, C.O. (2001) Beyond the "recognition code": structures of two Cys2His2 zinc finger/TATA box complexes. Structure, 9: 717-723. Wolfe, S.A, Ramm, E.I., and Pabo, C.O. (2000) Combining structure-based design with phage display to create new Cys2His2 zinc finger dimers. Structure, 8: 739-750. Wolfe, S.A., Nekludova, L., and Pabo, C.O. (2000) DNA recognition by Cys2His2 zinc finger proteins. Ann. Rev. Biophys. Biomol. Struct., 29: 183-212. Wolfe, S.A., Greisman, H.A., Ramm, E.I., and Pabo, C. O. (1999) Analysis of zinc fingers optimized via phage display: evaluating the utility of a recognition code. J. Mol. Biol., 285: 1917-1934. Pomerantz, J.P., Wolfe, S.A., and Pabo, C.O. (1998) Structure-based design of a dimeric zinc finger protein. Biochemistry, 37: 965-970. Erlanson, D.A., Wolfe, S.A., Chen, L., and Verdine, G.L. (1997) Selective base-pair destabilization enhances binding of a DNA methyltransferase. Tetrahedron, 53: 12041-12056. Wolfe, S.A., Zhou, P., Dötsch, V., Chen, L., You, A., Ho, S.H., Crabtree, G.R., Wagner, G., and Verdine, G.L. (1997) Unusual Rel-like architecture in the DNA-binding domain of the transcription factor NFATc. Nature, 385: 172-176. Wolfe, S.A., Ferentz, A.E., Grantcharova, V., Churchill, M.E.A.,and Verdine, G.L. (1995) Modifying the helical structure of DNA by design: recruitment of an architecture-specific protein to an enforced DNA bend. Chem. Biol., 2: 213-221. Wolfe, S.A. and Verdine, G.L. (1993) Ratcheting torsional stress in duplex DNA. J. Am. Chem. Soc., 115: 12585-12586. Potential Rotation ProjectsSelection of dimeric zinc finger proteins with novel DNA-binding specificity The DNA-binding specificity of a dimeric zinc finger chimera - created using a combination of structure-based design and phage display - will be altered to recognize new sequences that occur in various genes. A bacterial two-hybrid system will be used to facilitate the selection of these proteins. The resulting proteins will be examined in vitro with regards to specificity and affinity, and they will then be tested in vivo to determine their ability to alter gene expression. This project will provide training in a cutting-edge selection methodology and experience with gel-shift assays. Depending upon the timeframe, it could lead to cell culture experience, and transcriptional profiling to examine the specificity of these proteins in vivo. Selection of dimeric zinc finger proteins that preferentially heterodimerize Recently I created a dimeric zinc finger protein using a combination of structure-based design and phage display. This protein is a fusion between the leucine zipper of GCN4 and the zinc fingers of zif-268. The junction between these two domains was optimized by phage display in the context of a homodimer. Currently this system needs to undergo further analysis and optimization. The dimerization potential of the leucine zipper needs to be attenuated, and also optimized to prefer heterodimerization over homodimerization. This will involve truncation of the leucine zipper to find the minimal element necessary for cooperative DNA recognition. Subsequently, this region must be optimized to preferentially heterodimerize with another partner over the homodimerization with another copy of itself. Two methods of selection will be explored to achieve this goal: phage display and a bacterial two-hybrid system. The resulting proteins will be examined in vitro and in vivo with regards to specificity. This project will provide training in a cutting-edge selection methodology and depending upon the timeframe, it could lead to crystallization trials of the selected proteins to facilitate structural studies by X-ray crystallography. (The structure of the homodimer has been solved bound to DNA at a 1.5Å resolution.) Selection of dimeric zinc finger proteins that bind DNA in a drug-dependent manner A drug regulatable transcription factor that could be targeted to any gene of interest would provide a powerful new tool for researchers studying gene function in vivo. Building upon the dimeric zinc finger protein system described above, a drug dependent dimerization element will be substituted for the leucine zippers. This will involve examining the compatibility of structures of drug-based dimerization systems with the structure of the dimeric zinc finger protein. Based on computer modeling, prototypes of various fusions will be constructed and tested. Promising leads will then be optimized by phage display or using a bacterial two-hybrid system. The resulting proteins will be examined in vitro and in vivo with regards to drug dependent-DNA recognition. This project will provide training in basic computer modeling of proteins and in a cutting-edge selection methodology. Depending upon the timeframe, it could lead to cell culture experience and transcriptional profiling when examining the function of these proteins in vivo. Laboratory PersonnelJoseph McNulty, Postdoctoral Fellow Academic BackgroundScot Wolfe received his B.S. in Chemistry and Biology from Caltech in 1990, and his Ph.D. from Harvard in Chemistry in 1996. From 1996-2001 he was a post-doctoral fellow at Massachusetts Institute of Technology where his work was supported in part by the Leukemia and Lymphoma Society. In 2001, Dr. Wolfe joined the faculty of UMMS.
Office: LRB 619
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My research program is focused on three inter-related areas:
364 Plantation Street Worcester, MA 01605