Insulin promoter factor-1 gene mutation linked to early-onset type 2 diabetes mellitus directs expression of a dominant negative isoprotein.
The homeodomain transcription factor insulin promoter factor-1 (IPF-1) is required for development of the pancreas and also mediates glucose-responsive stimulation of insulin gene transcription. Earlier we described a human subject with pancreatic agenesis attributable to homozygosity for a cytosine deletion in codon 63 of the IPF-1 gene (Pro63fsdelC). Pro63fsdelC resulted in the premature truncation of an IPF-1 protein which lacked the homeodomain required for DNA binding and nuclear localization. Subsequently, we linked the heterozygous state of this mutation with type 2 diabetes mellitus in the extended family of the pancreatic agenesis proband. In the course of expressing the mutant IPF-1 protein in eukaryotic cells, we detected a second IPF-1 isoform, recognized by COOH- but not NH2-terminal-specific antisera. This isoform localizes to the nucleus and retains DNA-binding functions. We provide evidence that internal translation initiating at an out-of-frame AUG accounts for the appearance of this protein. The reading frame crosses over to the wild-type IPF-1 reading frame at the site of the point deletion just carboxy proximal to the transactivation domain. Thus, the single mutated allele results in the translation of two IPF-1 isoproteins, one of which consists of the NH2-terminal transactivation domain and is sequestered in the cytoplasm and the second of which contains the COOH-terminal DNA-binding domain, but lacks the transactivation domain. Further, the COOH-terminal mutant IPF-1 isoform does not activate transcription and inhibits the transactivation functions of wild-type IPF-1. This circumstance suggests that the mechanism of diabetes in these individuals may be due not only to reduced gene dosage, but also to a dominant negative inhibition of transcription of the insulin gene and other beta cell-specific genes regulated by the mutant IPF-1.
Stoffers
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DA
Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Harvard Medical School, and Howard Hughes Medical Institute, Boston, Massachusetts 02114, USA.
Stanojevic
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V
Habener
J F
JF
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D003924:Diabetes Mellitus, Type 2 / Q000209:etiology*; D006367:HeLa Cells; D018398:Homeodomain Proteins; D006801:Humans; D007340:Insulinoma / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D011817:Rabbits; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000235:genetics*
D018398:Homeodomain Proteins; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein