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J Biol Chem, Vol. 273, Issue 30, 19339-19347, July 24, 1998

Positive Regulation of the Human Macrophage Stimulating Protein Gene Transcription
IDENTIFICATION OF A NEW HEPATOCYTE NUCLEAR FACTOR-4 (HNF-4) BINDING ELEMENT AND EVIDENCE THAT INDICATES DIRECT ASSOCIATION BETWEEN NF-Y and HNF-4

Atsuhisa Ueda^§, Fumihiko Takeshita^§, Shigeo Yamashiro, and Teizo Yoshimura

From the  Immunopathology Section, Laboratory of Immunobiology, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 and ^§ The First Department of Internal Medicine, Yokohama City University School of Medicine, Yokohama 236, Japan

We previously reported that the transcription of the human macrophage stimulating protein (MSP) gene was positively regulated by the binding of NF-Y to the CAATT sequence in the promoter region of this gene. Here we confirmed our previous results and further characterized the MSP promoter. Luciferase assay with deletion constructs showed the importance of the region, +32 to +39, for the promoter activity in Hep3B cells. Two nuclear protein-DNA probe (+15 to +40) complexes, C1 and C2, were detected by electrophoretic mobility shift assay. C2 was specific to hepatoma cells and contained hepatocyte nuclear factor-4 (HNF-4). DNase I footprinting with recombinant HNF-4 located another HNF-4-binding site in the distal region, 89 to 54. Mutations in the CAATT or the proximal HNF-4-binding site significantly reduced the promoter activity in Hep3B cells and HNF-4-transfected HeLa cells, whereas mutations in the distal HNF-4-binding site had no effect. The close proximity between the CAATT and the proximal HNF-4-binding site suggested that a direct contact between NF-Y and HNF-4 might be important. Protein-protein interaction between the A-subunit of NF-Y and HNF-4 was detected by a yeast two-hybrid system. The binding of in vitro translated HNF-4 to immobilized NF-YA and in vitro translated NF-YA to immobilized HNF-4 was also detected. These results suggest the binding of HNF-4 to the proximal HNF-4-binding site directs the basal transcription of the MSP gene, and the maximal promoter activity may depend on the direct association between HNF-4 and NF-Y.

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