Deletion Analysis of the DNA-Binding Domains of HNF-3 Factors
Institution
Morehead State University
Faculty Advisor/ Mentor
David K. Peyton
Abstract
The Alpha-fetoprotein gene in rodents is regulated by a promoter and three enhancer elements. Members of the Forkhead Box (Fox) family of transcription factors actively bind to the upstream regulatory elements of the AFP gene. One member, HNF-3b (now called FoxA2), can bind to the promoter and repress activity when assayed in tissue culture cells. A PCR-generated clone of HNF-3b that contained only the DNA-binding domain (DBD) of the protein was effective in repressing activity, in spite of lacking the transactivation domain. To localize the critical region of the DBD that was sufficient to confer repression, a series of mutants were generated that had successive deletions from either the amino, carboxy, or both ends of the protein. The deletion mutants were subsequently cloned into a mammalian expression vector (pTARGET) and then assayed for their DNA-binding activity by electrophoretic mobility shift assay. The mutants were also co-transfected with an AFP promoter reporter construct to determine if the deletion mutants retained the repressive capacity of the full-length DBD clone. Our data indicate that a significant portion of the full-length DBD can be removed without destroying binding ability or repressive activity. Our analysis has now been extended to include the HNF-3 alpha factor. Currently we are in the process of generating a new series of clones to analyze this related but functionally distinguishable transcription factor.
Deletion Analysis of the DNA-Binding Domains of HNF-3 Factors
The Alpha-fetoprotein gene in rodents is regulated by a promoter and three enhancer elements. Members of the Forkhead Box (Fox) family of transcription factors actively bind to the upstream regulatory elements of the AFP gene. One member, HNF-3b (now called FoxA2), can bind to the promoter and repress activity when assayed in tissue culture cells. A PCR-generated clone of HNF-3b that contained only the DNA-binding domain (DBD) of the protein was effective in repressing activity, in spite of lacking the transactivation domain. To localize the critical region of the DBD that was sufficient to confer repression, a series of mutants were generated that had successive deletions from either the amino, carboxy, or both ends of the protein. The deletion mutants were subsequently cloned into a mammalian expression vector (pTARGET) and then assayed for their DNA-binding activity by electrophoretic mobility shift assay. The mutants were also co-transfected with an AFP promoter reporter construct to determine if the deletion mutants retained the repressive capacity of the full-length DBD clone. Our data indicate that a significant portion of the full-length DBD can be removed without destroying binding ability or repressive activity. Our analysis has now been extended to include the HNF-3 alpha factor. Currently we are in the process of generating a new series of clones to analyze this related but functionally distinguishable transcription factor.