RETINOBLASTOMA PROTEIN ASSOCIATES WITH SP1 AND ACTIVATES THE HAMSTER DIHYDROFOLATE-REDUCTASE PROMOTER

The dihydrofolate reductase (dhfr) promoter is powerfully activated by the transcription factor Sp1. It has been suggested that Sp1 is a pot ential target for transcriptional regulation by the cell cycle regulat or retinoblastoma protein (Rb), and so we have explored this possibili ty using the hamster dhfr gene as a model. By the use of DNA probes fr om the hamster dhfr gene promoter, containing the most proximal GC box (minimal promoter), and nuclear extracts from cultured hamster cells (CHO K1), we show that polyclonal and monoclonal antibodies against Rb supershift the binding of Sp1. Nuclear extract immunoprecipitation wi th anti- Rb followed by Western analysis using anti-Sp1 also shows tha t Rb is complexed to Sp1. Complementary Immunoprecipitation/WB analysi s shows both forms of Rh protein in the anti-Sp1 immunoprecipitates. M oreover, nuclear extract immunodepletion of Rb abolishes Sp1 gel-shift . The interaction between Rb and Sp1 is maintained in all the phases o f the cell cycle. Transient overexpression of Rb in dhfr negative cell s co-transfected with a dhfr minigene driven by its minimal promoter i ncreases DHFR activity and potentiates transcription when overexpressi ng Sp1. Both effects are severely reduced when the co-transfections ar e performed with a homologous dhfr minigene containing a single point mutation in the GC box. Thus, the activation by Rb of the dhfr gene ma y be exerted through Sp1. Stable transfectants of pCMVRb in K1 cells s how an increase in both mRNA and DHFR activity. It is concluded that S p1 is physically associated with Rb, and that this association increas es Sp1-mediated transcription of the hamster dhfr gene.