Gastrin, a peptide which both regulates acid secretion and can modulate mucosal growth, is expressed in enteroendocrine cells of the gut and islet endocrine tissue of the pancreas. Pancreatic gastrin expression occurs during the phase of endocrine stem cell commitment and proliferation, which is followed by attenuation and silencing of transcription coincident with islet cell terminal differentiation. The overall goal of these studies is to determine the transcriptional mechanisms of tissue-specific and developmental control of gastrin expression in pancreatic islets. Preliminary studies have identified multiple cis-regulatory elements in the gastrin 5' flanking sequence which are important for transcriptional activity in transfected islet cell lines. Each of the elements appears to bind one or more islet nuclear proteins with sequence specificity.
Aim 1. Identify and characterize the gene encoding the CACC binding protein which activates gastrin transcription. The CACC element is a significant positive activator of gastrin transcription and no similar CACC cis-regulatory element or binding protein has been described for endocrine or exocrine genes of the pancreas. The cDNA encoding a 70 kDa CACC binding protein is being cloned. The gene sequence, structure and function of its in vitro translated product and pattern of tissue-specific and developmental expression will be determined of this apparently novel transcription factor.
Aim 2. Functionally define the Homeodomain-like element (HD) and identify its sequence specific binding proteins. Analysis of the gastrin HD element will involve scanning point mutagenesis in reporter plasmids to determine those nucleotides critical for activity. HD sequence specific DNA-binding proteins will be purified from islet cell nuclear extracts by ion-exchange and oligonucleotide affinity chromatography, gel mobility shift assays, and Southwestern binding.
Aim 3. Characterize the gastrin promoter E-box element and its binding proteins. Multiple islet nuclear proteins, some of the Helix-Loop-Helix type, bind to the gastrin E-box element. One islet nuclear protein with binding specificity for the gastrin E-box and not other E-box elements will be further characterized by affinity purification, gel mobility shift and footprinting assays. Elucidation of transcriptional regulatory mechanisms of gastrin expression may provide greater insight into mechanisms of islet neogenesis and gastrinoma neoplasia.
| Tillotson, L G; Lodestro, C; Hocker, M et al. (2001) Isolation, maintenance, and characterization of human pancreatic islet tumor cells expressing vasoactive intestinal peptide. Pancreas 22:91-8 |
| Tillotson, L G (1999) RIN ZF, a novel zinc finger gene, encodes proteins that bind to the CACC element of the gastrin promoter. J Biol Chem 274:8123-8 |
| Nakata, H; Wang, S L; Chung, D C et al. (1998) Oncogenic ras induces gastrin gene expression in colon cancer. Gastroenterology 115:1144-53 |
