IDENTIFICATION OF A KEY REGULATORY ELEMENT FOR THE BASAL ACTIVITY OF THE HUMAN INSULIN-LIKE-GROWTH-FACTOR-II GENE PROMOTER P3

Transcription of the human insulin-like growth factor II (IGF-II) gene is under the control of four promoters (P1-P4) that are differentiall y active during growth and development. Promoter 3 (P3) is the most ac tive promoter during fetal development as well as in most adult tissue s. P3 is also the most active promoter in tumour tissues and cell line s expressing IGF-II. Transient transfections of HeLa and Hep3B cells w ith truncated promoter constructs revealed that the region between -28 9 and -183 relative to the transcription start site supports basal pro moter activity in both cell lines. Footprint experiments showed that t he region between positions -192 and -172 (P3-4) is the only element b ound by nuclear proteins. P3-4 is bound by five proteins, of which thr ee proteins (proteins 3, 4 and 5) bind specifically and are expressed at the same levels in HeLa and Hep3B cells. Electrophoretic mobility s hift assays and differential footprint experiments revealed the presen ce of two protein-binding regions within the P3-4 element. Proteins 4 and 5 bind box A (-193 to -188), whereas box B (-183 to -172) is bound by protein 3. From transcription experiments in vitro it can be concl uded that Box A is essential for P3 activity. Box A is part of a regio n 11 dG residues long and is protected by proteins 4 and 5 that bind a contiguous set of six dG residues. DNA-binding of proteins 4 and 5 to box A requires the presence of Zn2+ ions. Thus structural and functio nal analysis reveals that the P3-4 element is a key regulatory element of P3 that contains two separate binding sites for proteins essential for the basal activity of IGF-II P3.