Functional Analysis of the Rat Insulin-Like Growth Factor I Gene and Identification of an IGF-I Gene Promoter

Abstract

Insulin-like growth factor I (IGF-I) mediates many of the systemic growth-promoting effects of growth hormone and also functions as a locally acting growth stimulator. In mammals, IGF-I gene expression is complicated, as the gene is transcribed and processed into multiple mRNAs (ranging in length from less than 1 to nearly 7.5 kb) that encode at least two protein precursors. As a step toward understanding the regulation of IGF-I, we report the complete organization of the rat IGF-I gene, including identification of the structural determinants for all IGF-I mRNA species, and an initial functional analysis of its promoters. The gene is composed of 6 exons distributed over nearly 80 kb of chromosomal DNA and is structurally heterogeneous. Several transcription start sites were identified within IGF-I exons 1 and 2, adjacent to presumptive promoters 1 and 2, respectively, and at least three polyadenylation sites were mapped to exon 6. To test promoter function, fusion genes were constructed linking fragments of IGF-I DNA to a reporter plasmid. Chimeric genes containing at least 395 bp of DNA from the 5'-flanking region of exon 1 enhanced luciferase activity after transfection into the IGF-I-producing SK-N-MC cell line, while fusion plasmids containing up to 1,300 bp of DNA from the 5'-flanking region of exon 2 were inactive. Relative levels of IGF-I mRNAs containing exons 1 or 2 varied among different rat tissues, although in response to acute or chronic growth hormone treatment both classes of transcripts were induced coordinately in rat liver. These observations represent the first thorough characterization of a mammalian IGF-I gene, and provide a starting point for defining the mechanisms by which growth hormone and other trophic factors regulate IGF-I gene expression.

Transcriptional regulation of IGF-I expression in skeletal muscle

“…The exon 1 promoter includes four TSS located within ϳ0.36 kb of exon 1 (2). TSS 3 appears to be the major site of transcription initiation in most tissues, including rat skeletal muscles that were analyzed by ribonuclease protection assay techniques (17,30). The results of the present study are consistent with TSS 3 as the most active TSS in skeletal muscle, with TSS 2 transcripts expressed at much lower levels.…”

“…1A) consist of six exons, with distinct promoter regions regulating transcription initiation for both exons 1 and 2 (17). Exons 1 and/or 2 encode untranslated alternate leader sequences, whereas exons 3 and 4 encode the mature peptide domains A, B, C, and D. Exon 4 also encodes a small portion of an extension (E) domain that serves as a signaling peptide, whereas exons 5 and 6 each encode alternate E domains (35).…”

“…Circulating IGF-I in adult rats is primarily synthesized in the liver, with hepatic transcription initiated by both exon 1 and 2 promoters (30). In contrast, local IGF-I transcription appears to be regulated by the exon 1 promoter in most other tissues, including skeletal muscle (17,30).…”

“…1) reveals the complexity of the rat IGF-I gene, which lacks typical promoter regulatory elements such as TATA boxes, CCAAT boxes, and GC-rich regions (17,34). Adding further to this complexity, previous in vitro studies have identified two major and two minor exon 1 transcription start sites (TSS) in the ϳ0.36-kb promoter region located within exon 1 (34).…”

“…TSS 3 was identified as the primary TSS in several types of nonmuscle cells in vitro, because deletion of this site caused an ϳ1.5-to 9-fold decrease in promoter activity (34). Moreover, ribonuclease protection assays indicate that TSS 3 also appears to be the main TSS in skeletal muscle (17).…”