The diffuse neuroendocrine system includes pancreatic islets, gastrointestinal and respiratory neuroendocrine cells, thyroid C cells, adrenal medulla cells, and pituitary cells. They share common phenotypes and signaling pathways with neuronal cells. A set of transcription factors were identified, which were activated at different stages of neuroendocrine cell development. Studies aimed at the molecular mechanisms of how these transcription factors interact with each other to confer their regulatory pathway are important for neuroendocrine cell growth and differentiation. The long-term goal of this grant proposal is to understand the role of a novel zinc-finger transcription factor, IA-1, in pancreatic endocrine and neuronal cell development. The IA-1 gene is expressed primarily in fetal pancreas, fetal brain, and tumors of neuroendocrine origin. Preliminary data indicated that the IA-1 gene is developmentally regulated and functions as a DNA-binding transcriptional repressor. Using a selected and amplified random oligonucleotide binding assay and bacterially expressed GST-IA-1 carboxyl-terminal protein, we identified the consensus IA- 1 binding site. The IA-1 protein binding was confirmed by competitive electrophoretic mobility shift assay. Studies on the IA-1 promoter revealed a DNasel footprint protected site between -90 to -66 bp and this site is an almost perfect match to the IA-1 consensus binding site. Therefore, it appears that IA-1 may autoregulate itself. Further sequence analysis and competition binding assays indicated that NeuroD/beta2 represents another downstream target gene for IA-1. The NeuroD/beta2 gene encodes a basic helix-loop- helix transcription factor that is crucial for the development of pancreatic endocrine and neuronal cells. These results establish the importance of IA-1 to neuroendocrine cell development. Therefore, in this proposal we aim to study: 1) the mechanisms of how the IA-1 gene regulates NeuroD/beta2 gene expression, 2) whether the IA-1 gene plays an important role in neuroendocrine development in vivo, and 3) to characterize the regulatory elements in the IA-1 gene that confer a tissue-specific expression. To achieve these aims, we will use in vitro biochemical assays, transient mammalian cell transfections, yeast two-hybrid system, in vivo IA-1 gene knockout animal model, and yeast one-hybrid system. Results from this study should help us to define the interplay between the IA-1 zinc-finger transcription factor and other neuroendocrine-specific transcription factors in pancreatic endocrine and neuronal development.
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