GENOMIC STRUCTURE AND REGULATION OF THE PROMOTER OF THE RAT INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-2 GENE

We describe the complete genomic organization of the rat insulin-like growth factor binding protein-2 (rIGFBP-2) gene. This single-copy gene spans over 36 kilobases (kb) and is split into four exons of 475, 224, 141, and 472 nucleotides (nt), and three introns of 32 kb, 686, and 1793 nt, respectively. A single transcription start site (-90) was mapped by S1 protection assay and primer extension. The putative promoter of the rIGFBP-2 gene does not possess TATA or CAAT elements; however, it contains three GC-rich regions located 37, 57, and 81 nt 5' of the cap site. Deletion analysis of the 0.6-kb region of the upstream sequences and transfection of these constructs into BRL-3A and Chinese hamster ovary cells were used to localize possible cis-acting elements. The three GC boxes enhanced chloramphenicol acetyltransferase and luciferase transcription almost to the same level as the XbaI-NsphI (-579 to +1) fragment and displayed synergism and orientation dependence. In addition a similar positive effect on luciferase transcription has been obtained by cotransfecting these fragments with varying amounts of Sp1 expression vector into Drosphila cells that lack endogenous Sp1. In vitro gel mobility shift assays demonstrated that box 1 (GGGCGG), box 2 (GGGAGG), and box 3 (GGGAAGG) bind to SpI with variable affinities and display cooperativity. A protein that gave a similar DNA binding pattern was present in nuclear extracts of BRL-3A cells. To ascertain that this binding was due to Sp1, competition analysis using consensus or aberrant Sp1 elements and a polyclonal Sp1 antiserum to inhibit DNA binding were performed. These in vivo and in vitro data demonstrated that Sp1 plays an important role in the regulation of the expression of rIGFBP-2.