IN-VITRO TRANSCRIPTION OF THE RAT INSULIN-LIKE GROWTH-FACTOR-I GENE

Although the liver is the major source of circulating insulin-like gro wth factor-I (IGF-I), relatively little is known about the regulation of IGF-I gene transcription in this tissue. Since transcripts are init iated largely in exon 1, we established an in vitro transcription syst em to evaluate activation of transcription via the major exon 1 initia tion site. Transcription of a G-free cassette reporter was directed by rat IGF-I genomic fragments, and the adenovirus major late promoter w as used as an internal control. Tissue specificity was demonstrated by a 60-90% decrease in transcripts with spleen extracts as compared wit h liver. 54 base pairs (bp) of upstream sequence were sufficient to di rect IGF-I gene transcription, and activity increased 5-fold with 300 bp of upstream sequence. DNase I footprinting revealed four protected regions between -300 and -60 bp; binding was confirmed by gel shift an alysis, and tissue specificity was demonstrated by reduced shifts with spleen extracts. The necessity of transcription factor binding to suc h sites was established by competition analysis, which revealed a spec ific decrease in IGF-I transcription in the presence of a competing fr agment. Use of this in vitro transcription system should permit analys is of the function of individual transcription factors involved in reg ulation of IGF-I gene expression.