The retinoblastoma (RB) gene product has been shown to restrict cell proliferation, promote cell differentiation, and inhibit apoptosis. Loss of RB function can induce both p53-dependent apoptosis and p53-independent apoptosis; little is known about the mechanisms of RB-regulated p53-independent apoptosis. Here we show that RB specifically activates transcription of the survival gene bcl-2 in epithelial cells but not in NIH 3T3 mesenchymal cells. This transcriptional activity is mediated by the transcription factor AP-2. By monitoring protein-DNA interactions in living cells using formaldehyde cross-linking and chromatin immunoprecipitation, we show that endogenous RB and AP-2 both bind to the same bcl-2 promoter sequence. In addition, we demonstrate that RB and AP-2 also bind to the E-cadherin gene promoter in vivo, consistent with regulation of this promoter by both AP-2 and RB in epithelial cells. This study provides evidence that RB activates bcl-2 and E-cadherin by binding directly to the respective promoter sequences and not indirectly by repressing an inhibitor. This recruitment is mediated by a transcription factor, in this case AP-2. For the first time, our results suggest a direct molecular mechanism by which RB might inhibit apoptosis independently of p53. The results are discussed in a context where RB and Bcl-2 contribute under nonpathological conditions to the maintenance of cell viability in association with a differentiated phenotype, contributing to the tumor suppressor function of RB and playing important roles in normal development.The harmonious development of any organism requires a fine equilibrium between the processes of cell proliferation, differentiation, and apoptosis. These different functions are highly regulated and interdependent. Many of the genes involved in the regulation of these functions function as oncogenes or tumor suppressor genes.The retinoblastoma (RB) gene is an important tumor suppressor, and its protein product has been shown to restrict cell proliferation, promote cell differentiation, and inhibit apoptosis (for reviews, see references 15, 28, and 53).RB can act as either a negative or a positive regulator of transcription. In the context of cell proliferation, it acts as a negative regulator. The most widely accepted hypothesis proposes that RB represses transcription through the E2F family of transcription factors, partly through masking their activation domains and partly by recruiting a histone deacetylase to promoters that are repressed during the G 1 phase of the cell cycle (for a review, see reference 37). It has also been suggested that repression and activation of E2F-responsive genes may occur through distinct E2F heterodimers (46). In contrast to cell proliferation, when RB promotes differentiation, it regulates the activity of several transcription factors in a positive manner (for a review, see reference 35). However, the precise molecular mechanism for this activity of RB has not yet been elucidated. Similarly, RB also inhibits apoptosis, but it is not...

We have previously shown that RB plays an important role in the maintenance of the epithelial phenotype. p21 is also involved in several terminal dierentiation systems including keratinocytes. We report here that p21 is an RB target gene in epithelial cells, but not in ®broblasts where RB is unable to transactivate p21 transcriptional expression. In epithelial cells, when RB family factors were inactivated by SV40 T antigen (LT), p21 expression was strongly repressed, whereas its expression was not aected when the cells were transformed by a mutated LT leaving RB active but inactivating p53. Moreover, retransformation by RB of LT transformed epithelial cells totally restored p21 expression. By cotransfection experiments and using deletions and point mutations of the p21 promoter, we show that the minimal region required for the RB-mediated transcriptional activation maps to a GC-rich region located between 783 and 774. This region is shown to interact speci®cally with the transcription factor Sp1 and Sp3. Thus for the ®rst time, we show a positive transcriptional relationship between RB and p21 in epithelial cells. Since p21 keeps RB in a hypophosphorylated state important for its transcriptional activity during dierentiation, our results imply an auto-loop of regulation between RB and p21 that may be essential for the maintenance of the dierentiation state. We propose that this transcriptional relationship might be necessary of their roles in cell cycle arrest and in several dierentiation pathways. Oncogene (2001) 20, 962 ± 971.