Nuclear protein binding and functional activity of a variant insulin gene found in non-insulin-dependent diabetes mellitus.
In a subset of patients with non-insulin-dependent diabetes mellitus an 8-base pair (bp) repeat was found from -322 to -315 in the 5'-flanking region of the insulin gene. This 8-bp repeat is inserted into a sequence that is highly homologous to a sequence motif, called PISCES (pancreatic islet cell-specific enhancer sequences), found within cell-specific enhancer elements of the rat insulin I (Ins-E1, from -332 to -285), rat glucagon (Glu-G3) and rat somatostatin (SMS-UE) genes. The PISCES motif confers pancreatic islet-specific activity and is recognized by an islet-specific transcription factor (PISCES-BP). The consequences on functional activity and on protein binding of the 8-bp repeat sequence in the human insulin promoter was investigated. When fused to a reporter gene and transiently transfected into an insulin-producing islet cell line, the 8-bp repeat decreased basal transcriptional activity of the human insulin promoter (from -366 to +42) whereas the induction of promoter activity by cAMP was unaffected. The isolated rat Ins-E1 element was sufficient to confer basal transcriptional activity to a minimal promoter; the corresponding fragments of the normal and variant human insulin genes (from -329 to -288), however, were not. Using nuclear extracts in an electrophoretic mobility shift assay, it was found that PISCES-BP recognizes rat Ins-E1, but PISCES-BP binding to the corresponding normal and variant human insulin promoter fragments was not detectable and weak, respectively. However, a nuclear protein was found that binds to the variant but not normal human sequence. These data suggest that the 8-bp repeat in the variant human insulin promoter found in patients with non-insulin-dependent diabetes mellitus allows the binding of a nuclear protein that interferes with promoter function.
Yildiz
N
N
Department of Biochemical Pharmacology, University of Göttingen, Germany.
Diedrich
T
T
Knepel
W
W
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D014644:Genetic Variation; D005934:Glucagon / Q000235:genetics; D006801:Humans; D007328:Insulin / Q000096:biosynthesis / Q000235:genetics*; D007515:Islets of Langerhans / Q000502:physiology / Q000503:physiopathology*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D013004:Somatostatin / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D007328:Insulin; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013004:Somatostatin; D005934:Glucagon