Common carp insulin-like growth factor-I gene: complete nucleotide sequence and functional characterization of the 5′-flanking region

Abstract

Insulin-like growth factor-I (IGF-I) plays an important role in the growth and development of fish. To understand the molecular mechanism which controls the transcription of the IGF-I gene in common carp, we have cloned and completely sequenced the IGF-I gene and the 5′-flanking region from a local tropical fish, the common carp ( Cyprinus carpio), and characterized its promoter activity by transfection into human embryonic kidney (293GHR) cells which express the human growth hormone (GH) receptor. The common carp gene is the smallest IGF-I gene known so far, spanning approximately 13 kb, and is consisted of five exons and four introns. The sequence of the gene is consistent with the single type of IGF-I cDNA that we have isolated previously from a common carp liver cDNA library. The expression pattern of IGF-I is similar between juvenile carp and adult carp. While liver was found to be the major site of IGF-I gene expression in common carp, the expression levels in other tissues are relatively low. Like many other IGF-I gene promoters, there are no apparent TATA box and CCAAT box upstream of the transcription initiation site. However, sequence analysis of the common carp IGF-I promoter region identified several consensus liver-enriched transcription factor binding sites, including HNF-1α, HNF-3β, C/EBP, and one STAT5. We have analyzed the promoter activity of the 5′-flanking region of the common carp IGF-I gene by performing luciferase reporter assays in transfected 293GHR cells. Addition of human GH to the transfected cells led to an increased expression of the reporter gene, indicating that the cloned genomic fragment possessed promoter activity. This was confirmed by the lack of promoter activity of a construct in which the putative promoter was cloned in a reverse orientation upstream of the reporter gene. The liver-specific transcription factor, hepatic nuclear factor (HNF)-1α, was also found to be involved in the regulation of the common carp IGF-I transcription. Transfection results from a set of deletion mutants helped to map the locations of the responsive elements on the promoter responsible for the GH effect and for the interaction with HNF-1α. These observations provide information for delineating the transcriptional regulation of IGF-I gene expression in common carp.