RETINOBLASTOMA PROTEIN IN LUNG AND OTHER TISSUES

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Rb is a tumor suppressor that is required for movement of cells from G1 to S phase of the cell cycle. It is now thought that Rb is either mutated or that the pathway that regulates Rb activity is disrupted in tumors, resulting in the loss of Rb function in virtually all tumor cells. Rb also functions in normal biologic processes, which is evident from the embryonic lethal phenotype, observed in Rb knockout mice. Recent studies have suggested roles for Rb in formation of skeletal muscle and CNS, and for lung branching morphogenesis. In the previous grant period we have examined the structure/function of Rb in some detail. Two major findings arose from these studies. The first has to do with the structure of the repressor domain of Rb. We found that the central so called pocket region of Rb (the target of most inactivating mutations of the Rb gene in tumors) is the repressor motif. There are two highly conserved regions, designated A and B, within the pocket. We found that these two regions interact with one another to form the repressor motif. And more importantly, this A-B interaction is disrupted by the hyper-phosphorylation catalyzed by G1 cdks, which blocks Rb function. Recently, the Rb pocket has been crystallized, and we will use this crystal structure along with our results on Rb structure/function to direct a targeted mutational analysis of structures in the Rb pocket. A second finding during the previous grant period was that Rb can repress transcription through two distinct mechanisms. When targeted to cell cycle promoters, it an bind surrounding transcription factors and block their activity. Additionally, Rb represses transcription by recruiting histone deacetylase to the promoter. Gene promoters can be wound into nucleosomes by histone octamers-this inhibits binding of transcription factors. Nucleosome assembly is inhibited by acetylation of the core histones, and histone deacetylase removes the acetyl groups, promoting nucleosome assembly and repressing transcription. We propose additional studies to further examine the mechanism of action and the role of Rb in cultured cells and transgenic mice.