The promoter of the human gene for insulin-like growth factor binding protein-1. Basal promoter activity in HEP G2 cells depends upon liver factor B1.

A Suwanichkul,M L Cubbage,D R Powell

doi:10.1016/s0021-9258(17)45344-0

A Suwanichkul, M L Cubbage + Show 1 more

Open Access

https://doi.org/10.1016/s0021-9258(17)45344-0

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Abstract

Characterization of the human insulin-like growth factor binding protein-1 (IGFBP-1) promoter was initiated to facilitate study of developmental and hormonal factors regulating IGFBP-1 production. The region immediately 5' to the IGFBP-1 mRNA capsite is typical of a eukaryotic promoter, with a TATA sequence beginning 28 base pairs (bp) and a CCAAT promoter element beginning 72 bp upstream from this capsite. A 1.3-kilobase insert containing the IGFBP-1 capsite and 1205 bp of this putative IGFBP-1 promoter region directs expression of the reporter gene chloramphenicol acetyltransferase (CAT) in an orientation-specific manner in transfected HEP G2 cells, and the capsite identified for the CAT mRNA is identical to that identified for native IGFBP-1 mRNA. These observations suggest that the 1.3-kilobase insert contains the IGFBP-1 promoter. This promoter was further characterized by deletion analysis, site-directed mutagenesis, gel mobility shift assays, and DNaseI protection assays. These studies identify the CCAAT box region as the major cis element involved in basal IGFBP-1 promoter activity in HEP G2 cells, demonstrate that increased basal promoter activity is associated with the binding of at least one HEP G2 nuclear factor to the CCAAT box region, and indicate that the DNA binding factor(s) responsible for increased basal promoter activity is related to liver factor B1. These observations suggest that liver B1 is the major trans-acting factor stimulating basal IGFBP-1 promoter activity in HEP G2 cells.

Highlights

Characterization of the human insulin-like growth factor binding protein- 1 (IGFBP- 1) promoter was initiated to facilitate study of developmental and hormonal factors regulating IGFBP-1 production
IGFBP-1 promoter activity in HEP G2 cells, demonstrate that increased basal promoter activity is associated with the binding of at least one HEP G2 nuclear factor to the CCAAT box region, and indicate that the DNA binding factor(s) responsible for increased basal promoter activity is related to liver factor Bl
Much evidence suggests that the promoter region of the human IGFBP-1 chromosomal gene is located in the 5’ flanking region of DNA beginning 165 bp upstream from the first nucleotide of the IGFBP-1 translation start codon. (i) Primer extension of native IGFBP-1 mRNA places the mRNA capsite at this position

Summary

PROCEDURES

Phage EMBL (Clontech, Palo Alto, CA) was isolated and subcloned into pGEM-3 (Promega, Madison, WI) as described previously [17]. The 1205.bp IGFBP-1 promoter insert in p1205CAT was digested progressively from the 5’ end using exonuclease III, with the resulting plasmids named for the number of remaining nucleotides 5’ to the mRNA capsite. These plasmids, and the control plasmids pCAT(An) and p1205CATa, were transfected into HEP G2 cells; for each transfection, the pSVZA/L plasmid was cotransfected to control for transfection efficiency. The 1205-bp promoter fragment in p1205CAT was subjected to site-directed mutagenesis of the CCAAT box to either CCGTT (in pCCGTT) or TTAAT (in pTTAAT, see “Experimental Procedures”); these plasmids were transfected into HEP. CM, nona cetylated [“C]chloramphenicol; AC, two forms of monoacetylated chloramphenicol

G A T C PE

RESULTS

BPIL NF-I nnr-ln

DISCUSSION

Methods