Rjt. Rodenburg et al., A FUNCTIONAL SP1 BINDING-SITE IS ESSENTIAL FOR THE ACTIVITY OF THE ADULT LIVER-SPECIFIC HUMAN INSULIN-LIKE GROWTH-FACTOR-II PROMOTER, Molecular endocrinology, 11(2), 1997, pp. 237-250
The human gene encoding insulin-like growth factor II contains four pr
omoters (P1-P4) that are differentially activated in various tissues d
uring development, Expression of insulin-like growth factor II in adul
t 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 rel
ative to the transcription start site contains a high affinity Sp1 bin
ding site, This was demonstrated by electrophoretic mobility shift ass
ays 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 t
he 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 S
p1 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 exchang
ed by a functional TATA box. Activation of P1 by transactivators CCAAT
/enhancer binding protein-beta and hepatocyte nuclear factor-3 beta is
strongly impaired after mutation of the Sp1 binding site. These resul
ts demonstrate that the specific presence of a binding site for the ub
iquitously expressed transcription factor Sp1 is of eminent importance
for efficient activation of P1 by liver-enriched transactivators.