Section: Research

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Scot Wolfe, Ph.D.

Academic Role: Assistant Professor

Faculty Appointment(s) In:
   Biochemistry and Molecular Pharmacology
   Program in Gene Function and Expression

Other Affiliation(s):
   Interdisciplinary Graduate Program

Creating Novel Transcription Factors for Targeted Gene Regulation

My research focuses on the development of novel transcription factors that can be tailored to regulate individual genes in the context of a genome. To be effective, these transcription factors must incorporate a domain designed to bind tightly and specifically to the target site within genomic DNA without significant binding to closely related sequences. The DNA-binding domain will be linked to activation/repression domains or a DNA modification domain to control transcription of the target gene. The ultimate goal is to make proteins that can serve as biochemical tools for the study gene function in vivo. These transcription factors could also find use as reagents for gene therapy.

A dimeric Cys2His2 zinc finger protein will serve as the DNA recognition module in these proteins. The DNA-binding specificity of zinc fingers can be changed using rational design or selection methods; consequently these proteins can be made to recognize any desired DNA sequence (such as a target sequence in a gene of interest). Recently, I developed a dimeric zinc finger system, which binds cooperatively to DNA and which should have many advantages for the recognition of extended DNA sequences in vivo. [Recognition of more than 16 base pairs is probably necessary to specify a unique site in the human genome.] These dimeric zinc finger proteins have been characterized biochemically, as well as structurally. Like natural zinc finger proteins, these dimers can recognize a wide variety of sequences, and due to their design they should have important advantages over natural zinc finger systems in the level of sequence discrimination that can be achieved.

Heterodimeric zinc finger proteins with novel sequence specificities are being selected by phage display to recognize DNA sequences in genes in various disease and model systems. In conjunction with transcriptional profiling using DNA arrays, these transcription factors should allow us to define the size of the DNA binding site that must be specified to ensure unique addressing of a DNA-binding protein within a genome. These zinc finger proteins will also be characterized in vitro to correlate sequence specificity and affinity with in vivo efficacy. X-ray crystallography will be used to define the molecular basis of sequence discrimination by these dimeric zinc finger proteins. In combination, these studies should provide a much more detailed understanding of the key attributes necessary for sequence discrimination in the context of chromosomal DNA.

Office: LRB 619
Phone: 508-856-3953
E-mail: Scot.Wolfe@umassmed.edu
Keywords: Protein-DNA recognition, Gene Therapy, Structural Biology, Biochemistry, Gene Regulation

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