CHARACTERIZATION OF THE P4 PROMOTER REGION OF THE HUMAN INSULIN-LIKE GROWTH FACTOR-II GENE

The human insulin-like growth factor II (IGF-II) gene contains four pr omoters (P1, P2, P3 and P4). In order to determine the mechanism by wh ich the P4 promoter is controlled, the human IGF-II P4 promoter was an alyzed in cell lines. DNA sequence analysis of the human IGF-II P4 pro moter gene showed that the P4 promoter region contains a TATA-like seq uence and several G + C rich regions which are essential for transcrip tion. Analysis of the transcription initiation site by S1 nuclease map ping revealed two transcription start sites; both are located immediat ely behind TATA-like sequence. To determine the location of sites that may be important for the function of the human IGF-II P4 promoter, we constructed chimeric genes of the human IGF-II P4 promoter fused to t he coding region for chloramphenicol acetyltransferase (CAT). These co nstructs were transfected into HepG2, PLC/PRF/5, G401 and A549 cells, and were examined for CAT activity. All transfected cells showed a sim ilar profile of CAT activity. Sequences responsible for putative enhan cer and silencer regions were identified and the 5'flanking sequences of the human IGF-II P4 promoter contain negative regulatory regions (- 213 to -174). The 53-base pair fragment located between 111 and 59 bas e pairs upstream of the start site contains positive regulatory activi ty. Gel mobility shift assay showed that Sp1 and another proteins migh t be involved in positive regulation of the human IGF-II P4 promoter.