RB-1 was the first tumor suppressor gene to be identified and it has served as a prototype for this class of cancer-related genes. pRB is believed to prevent appropriate cell proliferation, in large part, by repressing E2F-dependent transcription. pRB antagonizes E2F-dependent activation in two general ways: pRB binds to E2F proteins and inhibits their ability to activate transcription;in addition, when bound to E2F, pRB recruits complexes that modify chromatin structure and actively repress transcription. These two activities of pRB are mediated via distinct binding sites. Currently it is unclear how many genes are controlled by pRB-mediated recruitment of repressor complexes, as opposed to the more direct inhibition of activator E2Fs. It is also uncertain which of the many cellular functions ascribed to pRB depend on its ability to recruit repressor complexes to DNA. Indeed there is very little information about which repressor complexes are required at E2F-controlled genes, and precisely which of these repressors are recruited by pRB. Using homologous recombination we have generated a knock-in allele of Rb that specifically lacks the LXCXE-binding cleft (Rb delta/LXCXE}. The mutant protein retains the ability to interact with E2F and to block E2F-mediated activation but it lacks the ability to interact with several proteins, including several repressor complexes. Our preliminary experiments with RB/delta/LXCXE/deltaLXCXE MEFs show that these cells are defective for certain types of pRB-dependent cell cycle arrest, but not for others. Since the mutant protein is expressed from the natural Rb promoter and is present at normal levels we can be certain that the phenotypes of these cells reflect physiological functions of pRB. We plan to take advantage of these cells to discover which genes are controlled by this specific element of pRB action, and to identify the proteins that pRB recruits to exert its effects. In doing so we will discover whether pRB recruits the same types of complexes to all its target promoters, or whether it is needed for the recruitment of different complexes at different promoters. By examining cells as they respond to different types of cell cycle arrest signals we will also discover whether pRB recruits different types of repressor complexes in different contexts.
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