TRANSCRIPTIONAL REGULATION OF THE RAT INSULIN-LIKE GROWTH-FACTOR-I GENE INVOLVES METABOLISM-DEPENDENT BINDING OF NUCLEAR PROTEINS TO A DOWNSTREAM REGION

Insulin-like growth factor-I (IGF I) gene transcription is mediated la rgely via exon 1. In an initial search for regulatory regions, rat hep atocytes were transfected with IGF-I constructs. Since omission of dow nstream sequences led to reduced expression, we then used in vitro tra nscription to evaluate potential metabolic regulation via downstream r egions. With templates including 219 base pairs of downstream sequence , transcriptional activity was reduced 70-90% with hepatic nuclear ext racts from diabetic versus normal rats. However, activity was comparab le with templates lacking downstream sequences. The downstream region contained six DNase I footprints, and templates with deletion of eithe r region III or V no longer provided reduced transcriptional activity with nuclear extracts from diabetic rats. Nuclear protein binding to r egions III and V appeared to be metabolically regulated, as shown by r educed DNase I protection and activity in gel mobility shift assays wi th nuclear extracts from diabetic rats. Southwestern blotting probes c orresponding to regions III and V recognized a similar to 65-kDa nucle ar factor present at reduced levels in diabetic rats, These findings i ndicate that a downstream region in exon 1 may be important for both I GF-I expression and metabolic regulation, Altered concentration or act ivity of a transcription factor(s) binding to this region may contribu te to reduced IGF-I gene transcription associated with diabetes mellit us.