The Retinoblastoma Gene Product RB Stimulates Spl-Mediated Transcription by Liberating Spl from a Negative Regulator

Abstract

Studies have demonstrated that the retinoblastoma susceptibility gene product, RB, can either positively or negatively regulate expression of several genes through cis-acting elements in a cell-type-dependent manner. The nucleotide sequence of the retinoblastoma control element (RCE) motif, GCCACC or CCACCC, and the Spl consensus binding sequence, CCGCCC, can confer equal responsiveness to RB. Here, we report that RB activates transcription of the c-jun gene through the Spl-binding site within the c-jun promoter. Preincubation of crude nuclear extracts with monoclonal antibodies to RB results in reduction of Spl complexes in a mobility shift assay, while addition of recombinant RB in mobility shift assay mixtures with CCL64 cell extracts leads to an enhancement of DNA-binding activity of Spl. These results suggest that RB is directly or indirectly involved in Spl-DNA binding activity. A mechanism by which RB regulates transactivation is indicated by our detection of a heat-labile and protease-sensitive Spl negative regulator(s) (Spl-I) that specifically inhibits Spl binding to a c-jun Spl site. This inhibition is reversed by addition of recombinant RB protein, suggesting that RB stimulates Spl-mediated transactivation by liberating Spl from Spl-I. Additional evidence for Spl-I involvement in Spl-mediated transactivation was demonstrated by cotransfection of RB, GAL4-Spl, and a GAL4-responsive template into CV-1 cells. Finally, we have identified Spl-I, a ~20-kDa protein(s) that inhibits the Spl complexes from binding to DNA and that is also an RB-associated protein. These findings provide evidence for a functional link between two distinct classes of oncoproteins, RB and c-Jun, that are involved in the control of cell growth, and also define a novel mechanism for the regulation of c-jun expression.