Identification of a key regulatory element for the basal activity of the human insulin-like growth factor II gene promoter P3

Abstract

Transcription of the human insulin-like growth factor II (IGF-II) gene is under the control of four promoters (P1-P4) that are differentially active during growth and development. Promoter 3 (P3) is the most active promoter during fetal development as well as in most adult tissues. P3 is also the most active promoter in tumour tissues and cell lines expressing IGF-II. Transient transfections of HeLa and Hep3B cells with truncated promoter constructs revealed that the region between -289 and -183 relative to the transcription start site supports basal promoter activity in both cell lines. Footprint experiments showed that the region between positions -192 and -172 (P3-4) is the only element bound by nuclear proteins. P3-4 is bound by five proteins, of which three proteins (proteins 3, 4 and 5) bind specifically and are expressed at the same levels in HeLa and Hep3B cells. Electrophoretic mobility shift assays and differential footprint experiments revealed the presence 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 concluded that Box A is essential for P3 activity. Box A is part of a region 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 functional 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.

Dual role for transcription factor AP-2 in the regulation of the major fetal promoter P3 of the gene for human insulin-like growth factor II

“…The following oligonucleotides were provided by Pharmacia (Uppsala, Sweden) : M3upper, 5h-CCGGGTAATCAGGGCCC-CCAGCCCGCACCCCCCGCCCGCTCTTGGC-3h ; M3lower, 5h-CCGAGCCAAGAGCGGGGCGGGGGTGCGGGCTG-GGGGCCCTGATTAC-3h. Other oligonucleotides used, P3-4, P3-4M1 and P3-4M3, were as described previously [6]. The AP-2 consensus oligonucleotide pair 5h-GATCGAACTGAC-CGCCCGCGGCCCGT-3h was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).…”

“…1300 nt upstream of the transcription start site of exon 5 and consists of 70-80 % GC base pairs. Transient transfection assays have revealed that the region between k289 and the transcription start site (j1) supports basal activity in HeLa and Hep3B cells [5,6]. Four elements were identified in this proximal promoter region that were bound by nuclear factors from HeLa cells in footprint analysis.…”

“…P3-1 (k90 to k63) contains an inverted CCAAT box ; P3-2 (k131 to k103) encompasses an Sp1 binding site. P3-3 (k146 to k128) binds an unknown protein ; P3-4 (k193 to k172) is composed of two protein-binding sites, box A (k193 to k188) bound by IGF-II promoter-binding protein (IPBP)4\5, and box B (k183 to k172) bound by IPBP3 [6,7]. Element P3-4 also contains a consensus sequence for AP-2 ; here we examine the putative binding of AP-2 to the IGF-II promoter P3 and its function in transcription of P3.…”

Identification of a Novel Cis Element Required for Cell Density-dependent Down-regulation of Insulin-like Growth Factor-2 P3 Promoter Activity in CaCo2 Cells

“…The absence of WT-1 is associated with overexpression of IGF-2 in Wilms' tumor (20). Other transcription factors that bind the proximal segment of the P3 promoter downstream of position Ϫ288 include p53 (34), the P3-4 cis element-binding proteins (35,36), and AP2 (22, 31) (reviewed in Ref. 37).…”

“…A representative autoradiograph of DNA footprint of a total of 2 autoradiographs from 2 experiments with day-5 samples is shown. A labeled DNA ladder was co-run with the DNase-treated samples as a size marker, and the nt sequence of the DNase-protected site was determined by Maxam and Gilbert GϩA track method, as published previously (31,35). A 14-bp region (Ϫ1084/Ϫ1070) with the indicated sequence was specifically protected from DNase 1 digestion (containing the putative P3-D binding site) in the presence of day-5 samples ( lanes 1 and 2).…”