Charaterization of the P4 promoter region of the human insulin-like growth factor II gene

Abstract

The human insulin-like growth factor II (IGF-II) gene contains four promoters (P1, P2, P3 and P4). In order to determine the mechanism by which the P4 promoter is controlled, the human IGF-II P4 promoter was analyzed in cell lines. DNA sequence analysis of the human IGF-II P4 promoter gene showed that the P4 promoter region contains a TATA-like sequence and several G + C rich regions which are essential for transcription. Analysis of the transcription initiation site by S1 nuclease mapping revealed two transcription start sites; both are located immediately 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 the coding region for chloramphenicol acetyltransferase (CAT). These constructs were transfected into HepG2, PLC/PRF/5, G401 and A549 cells, and were examined for Cat activity. All transfected cells showed a similar profile of CAT activity. Sequences responsible for putative enhancer 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 base pairs upstream of the start site contains positive regulatory activity. Gel mobility shift assay showed that Sp1 and another proteins imght be involved in positive regulation of the human IGF-II P4 promoter.