The goal of the proposed research is to identify pancreatic transcription factors that establish and maintain the developmental program specific for the exocrine tissue compartment of the pancreas. We will begin (Specific Aim 1) by identifying the DNA-binding transcription factors that mediate the acinar cell activity of the transcriptional enhancer of the gene for PTFl-p48, a critical transcriptional activator of acinar specific genes. To do this we will define the minimal functional enhancer and then map nuclear protein binding sites by DNase I footprinting in vitro and computer-based searches for evolutionarily conserved binding sequences characteristic of specific transcription factor families. We will confirm the relevance of the footprints and consensus binding sites by analyzing the effects of mutating the sites on the activity of the enhancer. We will identify candidate DNA-binding transcription factors of the exocrine pancreas by screening all members of seven major transcription factor families for expression in the developing and the mature exocrine pancreas by an RT-PCR protocol (Specific Aim 2). We will match individual pancreatic factors with potential p48 enhancer binding sites through the known consensus binding sequences of the family to which they belong. We will verify the match between factor and the enhancer site by testing whether the nucleotide sequence requirements for binding match the sequence requirements for activity in the enhancer and whether the factor can activate a reporter gene through the element in transfected cells. We will screen additional candidates by examining whether their expression occurs at an embryonic stage consistent with a developmental regulatory role. Finally, we will test the role of candidate factors by forced expression of dominant-negative and constitutively active chimeric forms of the factors in embryonic pancreatic rudiments capable of morphogenesis in culture (Specific Aim 3).
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