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A Functional Sp1 Binding Site Is Essential for the Activity of the Adult Liver-Specific Human Insulin-Like Growth Factor II Promoter

Laboratory for Physiological Chemistry Graduate School of Developmental Biology Utrecht University Utrecht, The Netherlands

The human gene encoding insulin-like growth factor II contains four promoters (P1–P4) that are differentially activated in various tissues during development. Expression of insulin-like growth factor II in adult liver tissue is directed by P1, which is activated by liver-enriched members of the CCAAT/enhancer binding protein family of transcription factors. In the present report we show that the region around -48 relative to the transcription start site contains a high affinity Sp1 binding site. This was demonstrated by electrophoretic mobility shift assays using nuclear extracts from Hep3B hepatoma cells and with specific antibodies directed against Sp1. Competition electrophoretic mobility shift assays revealed that the Sp1 binding site of P1 and a consensus Sp1 binding site bind Sp1 with comparable efficiencies. Mutation of the Sp1 binding site results in an 85% decrease in P1 promoter activity in transient transfection assays using two different cell lines, COS-7 and Hep3B. Investigation of P1 mutants in which the spacing of the Sp1 binding site and the transcription start site was increased showed that the role of the Sp1 binding site in regulation of P1 is position dependent. Interestingly, the Sp1-responsive element cannot be exchanged by a functional TATA box. Activation of P1 by transactivators CCAAT/enhancer binding protein-ß and hepatocyte nuclear factor-3ß is strongly impaired after mutation of the Sp1 binding site. These results demonstrate that the specific presence of a binding site for the ubiquitously expressed transcription factor Sp1 is of eminent importance for efficient activation of P1 by liver-enriched transactivators.

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