Cooperation of E2F-p130 and Sp1-pRb Complexes in Repression of the Chinese Hamster dhfr Gene

In mammalian cells reiterated binding sites for Sp1 and two overlapping and inverted E2F sites at the transcription start site regulate the dhfr promoter during the cell growth cycle. Here we have examined the contributions of the dhfr Sp1 and E2F sites in the repression of dhfr gene expression. In serum-starved cells or during serum stimulation, the Chinese hamster dhfr gene was not derepressed by trichostatin A (TSA), an inhibitor of histone deacetylases (HDAC). Immunoprecipitation experiments showed that HDAC1 and hypophosphorylated retinoblastoma protein (pRb) are associated with Sp1 in serum-starved CHOC400 cells. In transfection experiments, reporter plasmids containing the reiterated dhfr Sp1 sites were stimulated 10-fold by TSA, while a promoter containing four dhfr E2F sites and a TATA box was responsive to E2F but was completely unaffected by TSA. HDAC1 did not coprecipitate with p130-E2F DNA binding complexes, the predominant E2F binding activity in cell extracts after serum starvation, suggesting that p130 imposes a TSA-insensitive state on the dhfr promoter. In support of this notion, recruitment of GAL4-p130 to a dihydrofolate reductase-GAL4 reporter rendered the promoter insensitive to TSA, while repression by GAL4-pRb was sensitive to TSA. Upon phosphorylation of pRb and p130 after serum stimulation, the Sp1-pRb and p130-E2F interactions were lost while the Sp1-HDAC1 interaction persisted into S phase. Together these studies suggest a dynamic model for the cooperation of pRb and p130 in repression of dhfr gene expression during withdrawal from the cell cycle. We propose that, during initial phases of cell cycle withdrawal, the binding of dephosphorylated pRb to Sp1-HDAC1 complexes and complexes of E2F-1 -to -3 with DP results in transient, HDAC-dependent suppression of dhfr transcription. Upon withdrawal of cells into G0, recruitment of p130 to E2F-4‐DP-1 complexes at the transcription start site results in a TSA-insensitive complex that cooperates with Sp1-HDAC-pRb complexes to stably repress dhfr promoter activity in quiescent cells. E2F plays an important role in the regulation of genes required for cell cycle progression and entry into the S phase (reviewed in references 2, 12, and 19). The mammalian E2F protein family contains at least six members (E2F-1 to -6) that can associate with the two members of the DP protein family (DP-1 and DP-2), resulting in multiple heterodimers capable of binding E2F sequences. Control of E2F activity during the cell cycle is complex: expression of E2F-1 to -3 is low in quiescent cells and increases during growth stimulation, while E2F-4 and E2F-5 accumulate in quiescent cells or during differentiation (2, 12, 19). Various E2F-DP heterodimers also preferentially bind different members of the pRb tumor suppressor protein family: E2F-1 to -3 tend to associate with pRB, while E2F-4 and -5 are most often found in complexes with p107 and p130 (12). E2F-pRb family member complexes are dynamic during the cell cycle, with association and dissociation regulated by cyclin-dependent and other protein kinases. Moreover, members of the E2F and pRb families have been reported to be controlled by nuclear localization, phosphorylation, protein stability, and transcriptional regulation by E2F itself (reviewed in references 2, 12, and 19).