Identification of core sequences involved in metabolism-dependent nuclear protein binding to the rat insulin-like growth factor I gene

In the liver, most insulin-like growth factor I (IGF-I) transcripts origina te in exon 1, where important cis-regulatory regions are located downstream from the major transcription initiation sites. Within these regions, we ha ve attempted to identify sequences which are involved in the decrease in IG F-I gene transcription associated with diabetes mellitus. The function of d ifferent genomic templates was assessed by in vitro transcription, which re vealed a consistent 50-80% decrease in the activity of nuclear extracts fro m streptozotocin-diabetic as compared with normal rats. The disparity in tr anscriptional activity between normal and diabetic nuclear extracts was red uced with templates containing 11-bp mutations within DNase I protected reg ions III or V (+ 42 and + 129 bp, respectively, from the major transcriptio n initiation site), but a mutation between regions IV and V had little effe ct. Within region III, gel mobility shift analysis and methylation interfer ence studies indicated that DNA-protein interactions involve a GCGC core se quence. In region V, gel mobility shift studies and uracil interference ana lysis revealed interactions involving a TTAT core. While gel mobility shift analysis and transient transfection studies indicate that the GCGC core se quence in region III recognizes C/EBP, the AT-rich sequence in region V is likely to recognize a protein with homeodomain characteristics. Identificat ion of the nuclear factor(s) interacting with regions III and V, downstream from exon 1 initiation sites, will be important for understanding the mech anism of reduced IGF-I gene transcription due to diabetes mellitus.