Role of the LXCXE binding site in Rb function

Oncoproteins from DNA tumor viruses such as adenovirus E1a, simian virus 40 T antigen, and human papillomavirus E7 contain an LSCXE sequence, which th ey use to bind the retinoblastoma protein (Rb) and inhibit its function. Ce llular proteins such as histone deacetylases 1 and 2 (HDAC1 and -2) also co ntain an LXCXE-like sequence, which they use to interact with Rb. The LXCXE binding site in Rb was mutated to assess its role in Rb function. These mu tations inhibited binding to HDAC1 and -3, which each contain an LXCXE-like sequence, but had no effect on binding to HDAC3, which lacks an LXCXE-like sequence. Mutation of the LXCXE binding site inhibited active transcriptio nal repression by Rb and prevented it from effectively repressing the cycli n E and A gene promoters. In contrast, mutations in the LXCXE binding site did not prevent Rb from binding and inactivating E2F. Thus, the LXCXE mutat ions appear to separate Rb's ability to bind and inactivate E2F from its ab ility to efficiently recruit HDAC1 and -2 and actively repress transcriptio n. In transient assays, several of the LXCXE binding site mutants caused an increase in the percentage of cells in G(1) by flow cytometry, suggesting that they can arrest cells, However, this effect was transient, as none of the mutants affected cell proliferation in longer-term assays examining bro modeoxyuridine incorporation or colony formation. Our results then suggest that the LXCXE binding site is important for full Rb function. Mutation of the LXCXE binding site does not inhibit binding of the BRG1 ATPase componen t of the SWI/SNF nucleosome remodeling complex, which has been shown previo usly to be important for Rb function. Indeed, overexpression of BRG1 and Rb in cells deficient for the proteins led to stable growth inhibition, sugge sting a cooperative role for SWI/SNF and the LXCXE binding site in efficien t Rb function.