Genomic structure and promoter characterization of the gene encoding the ErbB ligand betacellulin

Abstract

Betacellulin (BTC) belongs to the epidermal growth factor (EGF) family of peptide ligands that are characterized by a six-cysteine consensus motif (EGF-motif) that forms three intra-molecular disulfide bonds, crucial for binding the ErbB receptor family. A variety of in vitro studies have identified BTC as an important factor in the growth and/or differentiation of pancreatic islet cells. The molecular mechanisms that regulate the transcription of the BTC gene however have not been delineated. As an initial step, we have characterized the genomic structure of the mouse BTC (mBTC) gene. mBTC cDNA was used as a probe to screen a mouse 129/SVJ genomic bacterial artificial chromosome (BAC) library. Three positive clones containing the entire gene were isolated. DNA sequence analysis identified six exons (1–6) and five introns (A–E); a structure conserved among the EGF family. PCR analysis showed that introns A–E are approximately 7.8, 8.9, 3.8, 1.4 and 1.4 kb in length, respectively. The EGF-motif is encoded by exons 3 and 4 with an intron (intron C) disrupting the coding sequence between the second and third disulfide loops. All exon–intron boundaries are consistent with the “gt-ag” rule. Multiple transcription start sites and one poly(A) site, located 18 bp downstream of a polyadenylation signal sequence, were identified by 5′- and 3′-RACE, respectively. Approximately 2.6 kb of 5′-flanking region was sequenced and was shown to lack consensus TATA and CCAAT boxes, but was found to contain several putative cis-acting regulatory elements. These included consensus binding sites for transcription factors HNF3β, USF, Nkx2–5, AP-4, and Sp1. Functional promoter analysis of the 5′-flanking region in COS-7 cells, using 5′-deletion fragments (−168/+335; −635/+335; −732/+335; −1175/+335; −1698/+335) cloned into a promoterless firefly luciferase reporter vector, identified basal promoter activity and both positive and negative cis-acting elements.