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J Biol Chem, Vol. 274, Issue 43, 30943-30949, October 22, 1999

Sp1 and NF-Y Are Necessary and Sufficient for Growth-dependent Regulation of the Hamster Thymidine Kinase Promoter

From the Institute of Molecular Biology, University of Vienna, Vienna BioCenter, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria

Thymidine kinase (TK) genes from different species are growth- and cell cycle-regulated in a very similar manner; still, the promoter regions of these genes show little homology to each other. It was previously shown that the murine TK gene is growth-regulated by Sp1 and E2F. Here we have characterized cis-regulatory elements in the hamster promoter that are essential and sufficient to confer efficient and serum-responsive expression. The TK promoter was isolated from baby hamster kidney cells. DNase I protection experiments revealed a protected region from positions 24 to 99 relative to the transcription start site. Within this region, binding sites for the transcription factor Sp1 and a CCAAT box, which interacts with the transcription factor NF-Y, were identified. An E2F-like sequence was found not to bind protein, and its removal did not affect promoter activity. This was supported by the observation that cotransfection of a hamster TK reporter gene construct with E2F-1 does not lead to transactivation of the promoter. A 122-base pair region that contains a single Sp1 site, a CCAAT box, and a TATA element was found to be sufficient for serum-responsive expression of a reporter gene. Mutations that inactivate any one of these three elements caused a strong reduction or a loss of promoter activity.

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