Nuclear protein binding and functional activity of a variant insulin gene found in non-insulin-dependent diabetes mellitus

 

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Summary

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-El, 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-El 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-El, 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.