Our long-term goal is to define molecular mechanisms important for the regulation of cell proliferation and apoptosis in the exocrine pancreas which is a crucial step for better understanding normal morphogenesis and pancreatic cancer. Toward this end, we are studying the role of growth-factor inducible C2H2 zinc finger transcription factors in the regulation of these phenomena. Zinc finger proteins play a crucial role in organogenesis in several mammalian tissues, and mutations in some of these genes give rise to neoplastic transformation. However, their presence and function in the exocrine pancreas remain to be elucidated. The mechanistic experiments outlined in this proposal will test the central hypothesis that a novel TGFbeta-inducible zinc finger protein TIEG is a transcription factor involved in the regulation of apoptosis and/or the cell cycle in pancreatic cells. We have isolated a TIEG cDNA from a rat pancreas library and demonstrated that this gene is an early response target for TGFbeta in exocrine pancreatic cell populations. Interestingly, although its biochemical properties have not yet been determined, sequence analysis of the deduced TIEG protein reveals the presence of several motifs that are characteristic of transcription factors. Because TGFbeta induces both apoptosis and cell cycle arrest in pancreatic cell populations, TIEG is a good candidate to participate in these phenomena. Indeed, we have recently shown that the overexpression of TIEG in pancreatic cell populations induces apoptosis. Thus, in this proposal, we hypothesize that: 1) TIEG functions as a sequence-specific transcription factor, 2) the apoptotic effects of TIEG depend on the activity of this protein as a transcription factor, and 3) the overexpression of TIEG induces cell cycle arrest prior to apoptosis. These hypotheses will be addressed in the following specific aims: 1) Determine the nuclear localization and transcriptional regulatory activity of TIEG, 2) Determine the DNA binding sequence(s) for TIEG, and 3) Characterize the mechanisms involved in TIEG-induced apoptosis (transcriptional activity and cell cycle arrest). We propose to use state-of-the-art molecular techniques in combination with well- established functional assays to approach these aims. We are optimistic that the successful completion of this proposal will significantly advance our understanding on the role of zinc finger proteins in pancreatic cell physiology and begin to fill a gap in the existing knowledge in this underrepresented area of pancreatic research. Furthermore, this information will be crucial as a theoretical framework for future studies on the role of zinc finger transcription factors in pancreatic development and cancer.
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