Insulinlike Growth Factor 1 Gene Variation in Vertebrates

Abstract

IGF1-a small, single-chain, secreted peptide in mammals-is essential for normal somatic growth and is involved in a variety of other physiological and pathophysiological processes. IGF1 expression appears to be controlled by several different signaling mechanisms in mammals, with GH playing a key role by activating an inducible transcriptional pathway via the Jak2 protein kinase and the Stat5b transcription factor. Here, to understand aspects of Igf1 gene regulation over a substantially longer timeline than is discernible in mammals, Igf1 genes have been examined in 21 different nonmammalian vertebrates representing five different classes and ranging over ∼500 million years of evolutionary history. Parts of vertebrate Igf1 genes resemble components found in mammals. Conserved exons encoding the mature IGF1 protein are detected in all 21 species studied and are separated by a large intron, as seen in mammals; the single promoter contains putative regulatory elements that are similar to those functionally mapped in human IGF1 promoter 1. In contrast, GH-activated Stat5b-binding enhancers found in mammalian IGF1 loci are completely absent, there is no homolog of promoter 2 or exon 2 in any nonmammalian vertebrate, and different types of "extra" exons not present in mammals are found in birds, reptiles, and teleosts. These data collectively define properties of Igf1 genes and IGF1 proteins that were likely present in the earliest vertebrates and support the contention that common structural and regulatory features in Igf1 genes have a long evolutionary history.

Figure 1.

Organization of the chicken Igf1 gene and mRNAs. (A) Map of the chicken Igf1 locus, including chromosomal coordinates. Exons appear as boxes, and introns and flanking DNA appear as horizontal lines. The enlargement shows the Igf1 promoter (P) and exons 1, 1a, and 2. A scale bar is indicated. (B) Diagram of chicken Igf1 mRNAs. Coding regions are in color, and 5′ and 3′ untranslated regions are in white. A_N represents the polyadenylic acid tail at the 3′ end of the transcript. A scale bar is shown. (C) Diagram of chicken IGF1 protein precursors, illustrating the derivation of each segment from different Igf1 exons. Mature, 70‒amino acid IGF1 is in blue, portions of the signal peptide are in gray and black, and components of E peptide are in shades of red. AA, amino acid; Chr, chromosome; nt, nucleotide.

Figure 2.

Comparison of selected vertebrate Igf1 genes. Schematics of chicken Igf1 and eight other Igf1 genes and loci are shown. Exons are illustrated as boxes, introns and flanking DNA as horizontal lines, and ECRs 1 to 7 as orange ovals. A scale bar is indicated. The percentage of nucleotide identity with different parts of chicken Igf1 is listed for each gene (red for exons, black for promoters). Ch, Chinese; nd, no identity detected; X, Xenopus.

Figure 3.

Comparison of additional vertebrate Igf1 genes. Maps of tetraodon Igf1 and nine other selected Igf1 genes and loci are shown. Exons are illustrated as boxes, and introns and flanking DNA are shown as horizontal lines. A scale bar is indicated. The percentage of nucleotide identity with different parts of chicken Igf1 is listed for each gene (red for exons, black for promoters). nd, no identity detected.

Figure 4.

Igf1 promoters in nonmammalian vertebrates. (A) Location of alignment of terrestrial vertebrate Igf1 promoters with the chicken Igf1 promoter. Length and percentage of identity are in parentheses for each species. (B) Location of alignment of aquatic vertebrate Igf1 promoters with the tetraodon Igf1 promoter. Length and percentage of identity are in parentheses for each species. No sequence alignments could be detected for the cave fish, coelacanth, or lamprey. X, Xenopus.

Figure 5.

Alignments of vertebrate IGF1 proteins. (A) Amino acid sequences of IGF1 from eight terrestrial vertebrates, coelacanth, and human in single-letter code. Differences among species are indicated, with identities depicted by dots. Dashes indicate no residue and have been placed to maximize alignments. (B) Amino acid sequences of IGF1 from 11 ray-finned fish and lamprey in single-letter code. Differences among species are indicated, with identities depicted by dots. Blue text indicates amino acid identities between the chicken in (A) and the tetraodon in (B), and red text depicts differences. Dashes indicate no residue and have been placed to maximize alignments. (C) Cladogram of mature IGF1 in vertebrates. The numbers at the nodes indicate fractional differences, with 0 representing the highest levels of similarity. The length of each branch approximates the evolutionary distance.

Figure 6.

Alignments of vertebrate IGF1 signal peptides. (A) Amino acid sequences of IGF1 signal peptides from eight terrestrial vertebrates, coelacanth, and human in single-letter code. Differences are indicated, identities are depicted by dots, and a dash indicates no residue. The # next to the chicken and turkey indicates that a shorter IGF1 signal peptide of 25 amino acids is encoded by an alternative transcript in these two avian species (see Figs. 1 and 2). (B) Amino acid sequences of IGF1 signal peptides from 11 ray-finned fish and lamprey in single-letter code. Differences are indicated, with identities depicted by dots. A dash indicates no residue. Blue text depicts amino acid identities between the chicken in (A) and the tetraodon in (B), and red text indicates differences. The asterisk in front of the cave fish heading denotes the presence of an alternate signal peptide with the following 17 extra N-terminal amino acids: MTSKNKLLFVAWRRPAG. (C) Cladogram of the IGF1 signal peptide in vertebrates. The numbers at the nodes indicate fractional differences, with 0 representing the highest levels of similarity. The length of each branch approximates the evolutionary distance. Note that the IGF1 signal peptide in the coelacanth is more similar to that of other fish than is the mature IGF1 and that the lamprey is the outgroup in both comparisons (see Fig. 5C)

Figure 7.

Alignments of vertebrate IGF1 E peptides. (A) Amino acid sequences of C-terminal common E (16 amino acids) and E_A peptides (19 residues) in eight terrestrial vertebrates, coelacanth, human, 11 ray-finned fish, and lamprey in single-letter code. Differences are indicated, with identities depicted by dots. The number of amino acids is listed in parentheses if it differs from 16 residues for the common E region or from 19 residues for E_A peptides. (B) Amino acid sequences of C-terminal E_B peptides in 11 ray-finned fish, lamprey, and human in single-letter code. Differences are indicated, with identities depicted by dots. A dash indicates no residue. The number of amino acids is listed in parentheses. Blue text depicts amino acid identities between the chicken in (A) and the tetraodon in (B), and red text indicates differences. The asterisk in front of the cave fish heading depicts the presence of an alternate E_B peptide with 26 fewer N-terminal amino acids. AA, amino acid.

Figure 8.

Comparison of Igf1 promoter elements. (A) Schematic of human IGF1 gene promoter 1 and exon 1. Bent arrows indicate transcription start sites in exon 1, and the location of the ATG codon is labeled. Coding DNA is in black and noncoding DNA in white. The locations of binding segments for transcription factors HNF-1, C/EBPα/β, HNF-3, and C/EBPδ are shown. The presence of sites identical to the human sequences in different species is indicated by + for each transcription factor site, and absence is depicted by −. Altered nucleotides within similar sites are shaded in gray.