Structure and Function of a Human Insulin-like Growth Factor-I Gene Promoter

Sung-Woon Kim, Rosemarie Lajara*, and Peter Rotwein Metabolism Division Departments of Internal Medicine and Genetics (P.R.) Washington University School of Medicine St. Louis, Missouri 63110

effects on growth, development, and intermediary metabolism in many vertebrate species (1 -3). Despite the relative simplicity of its protein structure, the IGF-I gene and its mRNAs are complicated. In humans and rats the single copy gene is large [>70 kilobases (kb)] and contains at least six exons that are variably expressed in IGF-I mRNAs (ranging in length from -1-7.5 kb) and encode two protein precursors (reviewed in Ref. 4). Several mechanisms have been demonstrated to play a role in the generation of this diverse population of mRNA species. In the human gene, alternative premRNA processing involving exons 5 and 6 is responsible for the production of transcripts that encode peptides with distinct COOH-termini (IGF-IB and IGF-IA, respectively) and different 3'-untranslated regions (5, 6), while variable polyadenylation within exon 6 accounts for short and long IGF-IA mRNA species (7). It also has been shown that differential use of exons 1 and 2 is the mechanism by which IGF-I mRNAs with distinct 5'-untranslated regions and different NH2-terminal coding domains are produced (8,9). Similar mechanisms appear to be responsible for the generation of multiple IGF-I mRNAs in the rat (10-13) and probably in other mammalian species (14, 15). Despite these advances, the structures of the human and rat genes are incomplete (6, 8, 9, 13, 16, 17). No gene promoter has been characterized, and the mechanisms by which GH and other trophic agents alter IGF-I gene expression remain poorly defined. As part of a series of studies undertaken to address the evolution of IGFs, we recently reported that the relatively simple chicken IGF-I gene contained heterogeneous 5'- and 3'-ends and also identified by both structure and function the chicken IGF-I gene promoter (18). We now have characterized the analogous human promoter. As was seen in the chicken gene, this human IGF-I promoter lacks typical proximal regulatory elements and, consequently, activates transcription over a relatively dispersed region of exon 1, leading to IGF-I mRNAs with heterogeneous 5'-ends.

We have identified and characterized a promoter regulatory region for the human insulin-like growth factor-l (IGF-I) gene. The 5'-ends of IGF-I mRNAs were first mapped to a series of sites within a 158nucleotide portion of exon 1 that was found to be structurally similar to the recently delineated transcription start region of the chicken IGF-I gene. In both species multiple initiation sites are probably a reflection of the absence of typical transcriptional regulatory signals, such as a TATA or CCAAT box, in the proximal promoter, although neither gene sequence resembles a GC-rich housekeeping promoter, which also controls a dispersed initiation region. To test promoter function, hybrid genes were constructed linking human IGF-I DNA to a promoterless reporter plasmid. Fusion genes containing from 385-4300 nucleotides of the IGF-I 5'- flanking region enhanced luciferase activity after transfection into the IGF-l-producing SK-N-MC cell line. A plasmid with 1630 nucleotides of 5'-DNA gave maximal activity and directed accurate initiation of the hybrid gene, while the same promoter fragment inserted in the reverse orientation was inert. Although cognate recognition sequences were identified for several transcription factors in the 1630 nucleotides 5' to the transcription start region, no sites were found that resembled the putative GH response element recently mapped to the proximal promoter of the rat Sp/2.1 gene. This study underscores the diversity of mechanisms contributing to IGF-I gene expression by demonstrating that heterogeneous transcription initiation accounts for IGF-I mRNAs with different 5'-ends and provides a starting point for elucidating the ways in which GH and other trophic agents induce IGF-I gene transcription. (Molecular Endocrinology 5: 1964-1972,1991)

INTRODUCTION RESULTS Insulin-like growth factor-l (IGF-I) is a highly conserved 70-residue circulating peptide that exerts prominent

Characterization of Transcription Start Sites in Exon 1

0888-8809/91/1964-1972S03.00/0 Molecular Endocrinology Copyright © 1991 by The Endocrine Society

By a combination of cDNA-cloning experiments and ribonuclease protection assays we showed recently 1964

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Human IGF-I Gene Promoter

1965

that the chicken IGF-I gene contained a transcription initiation region that was dispersed over 74 nucleotides of its most 5' exon (18). We used a similar approach to determine the beginning of the human gene. Primer extension analysis first was performed using a polymerase chain reaction (PCR)-based approach (19). Human liver RNA was reverse transcribed with a specific oligonucleotide primer complementary to a region within exon 3. The cDNA was "tailed" at its 5'-end, and PCR was performed according to the 5' rapid identification of cDNA ends (RACE) protocol (19), using oligo(dT) and 5'-adaptor primers and oligonucleotides complementary to different regions within exon 1. Five types of cDNA were subsequently identified by DNA sequencing. All ended within exon 1, but at discrete sites 245385 nucleotides 5' to the exon-intron junction (Table

Table 1. Human IGF-I Gene: Mapping the 5'-Ends by cDNA Cloning Length of Exon 1 (nucleotides)

385 334 320-321

298 245

No. of Clones

7 3 7 3 2"

See Fig. 6. Isolated by standard cDNA cloning.

Map Position8

+1 +52 +65-66

+88 +141

j

Structure and function of a human insulin-like growth factor-I gene promoter.

We have identified and characterized a promoter regulatory region for the human insulin-like growth factor-I (IGF-I) gene. The 5'-ends of IGF-I mRNAs ...
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