Cell growth regulation of the hamster dihydrofolate reductase gene promoter by transcription factor Sp1.

AU: Noé, V., Chen, C., Alemany, C., Nicolás, M., Caragol, I., Chasin, L.A., and Ciudad, C.J.

SO: Eur. J. Biochem (1997) 249:13-20

The dihydrofolate reductase (dhfr) promoter contains cis -acting elements for the transcription factors Sp1 and E2F. Given the strong properties of Sp1 in activating the dhfr promoter, we have evaluated the contribution of Sp1 to the cell growth regulation of the dhfr gene. Using gel mobility assays performed with DNA probes from the hamster dhfr gene minimal promoter and nuclear extracts from cultured hamster cells (CHO K1) we show that the binding of Sp1 to the dhfr promoter is cell growth phase regulated. Accordingly, DHFR transcription and mRNA levels in K1 cells also increase upon serum stimulation. Cytological detection of Sp1 by immunofluorescence reveals a decrease of this protein in the process leading to the Go state, and an increase upon serum stimulation of quiescent cells. These results were confirmed by Western analysis. It is concluded that Sp1 progressively binds to the hamster dhfr promoter after stimulation of cell proliferation, which can account for the transcriptional regulation of the dhfr gene during the cell cycle. The role of Sp1 in the specific control of DHFR during the cell cycle was confirmed in vivo using cell lines derived from dhfr negative cells transfected with dhfr minigenes carrying either the wild type or mutated Sp1 or E2F binding sites in the dhfr minimal promoter.