CELL-GROWTH REGULATION OF THE HAMSTER DIHYDROFOLATE-REDUCTASE GENE PROMOTER BY TRANSCRIPTION FACTOR SP1

The dihydrofolate reductase (DHFR) gene (dhfr) promoter contains cis-a cting elements for the transcription factors Sp1 and E2F. Given the ab ility of Sp1 to activate the dhfr promoter, we have evaluated the cont ribution of Sp1 to the cell-growth regulation of the dhfr gene, Using gel-mobility assays performed with DNA probes from the minimal promote r of the hamster dhfr gene 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 increase upon serum stimulation. Cytological detec tion of Sp1 by immunofluorescence reveals a decrease of this protein i n the process leading to the G0 state, and an increase upon serum stim ulation of quiescent cells, These results confirmed by western blot an alysis. It is concluded that Sp1 progressively binds to the hamster dh fr promoter after stimulation of cell proliferation, which can account for the transcriptional regulation of the dhfr gene during the cell c ycle. The role of Sp1 in the specific control of dhfr during the cell cycle was confirmed in vivo using cell lines derived from dhfr-negativ e cells transfected with dhfr plasmids carrying either the wild-type o r mutated Sp1-binding or E2F-binding sites in the dhfr minimal promote r.