The mammalian Retinoblastoma (RB) family including pRB, p107 and p130 represses E2F transcription through mechanisms that are not fully understood. The transforming proteins SV40 large T antigen (T Ag) and Adenovirus E1A inactivate all members of the RB family and overcome the growth inhibition imposed by their repression of E2F activity. Mammalian p130 binds specifically to a 5-subunit complex called DREAM that contains RBBP4, LIN9, LIN37, LIN52 and LIN54. Similar protein complexes have been identified in association with RB homologues in fly and worm. The DREAM complex bound specifically to p130/E2F4 on chromatin during G0/G1 and was unable to bind to p107 or pRB. T Ag disrupted p130 binding to DREAM. In contrast, p107 but not p130 or pRB can bind to the NuRD (Nucleosome Remodeling and histone Deacetylase) complex containing HDAC1 indicating that p130 and p107 form two entirely distinct complexes to repress E2F dependent gene transcription. The mammalian DREAM and NuRD complexes as well as RB, E2F and DP are homologues of the C. elegans synMuvB gene products. Genetic studies of worms led to the identification of 3 classes (A, B, and C) of synthetic multivulva (synMuv) genes that when combined serve to repress Ras signaling. The synMuvC class genes form the NuA4 complex containing TRRAP, EP400, TIP60, and EPC homologues. Notably, the NuA4 complex is also a transformation target of Adenovirus E1A and SV40 T Ag. This application proposes that p130 and p107 together with DREAM and NuRD form a class of mammalian genes equivalent to synMuvB that repress oncogenic signaling by inhibiting E2F dependent transcription. In addition, inactivation of a mammalian synMuvB gene combined with inactivation of a mammalian synMuvC gene would result in unregulated Ras signaling and cellular transformation. In this manner, Adenovirus E1A and SV40 T Ag inactivate both synMuvB (p107 and p130) and synMuvC (EP400) to transform cells. To test this model, the following specific aims are proposed.
Specific Aim 1. Perform a structure and function analysis of the p130-DREAM complex. Determine the contribution of DREAM complex to p130-mediated repression of E2F activity.
Specific Aim 2. Characterize the p107-NuRD complex. Determine if 107 recruits NuRD to E2F dependent promoters by chromatin immunoprecipitation of individual promoters and global analysis using microarrays.
Specific Aim 3. Determine if DREAM and NuRD complexes function as tumor suppressors in cellular transformation. Test the hypothesis that DREAM and NuRD function as tumor suppressors using transformation assays with SV40 large T antigen, activated RAS, and shRNA-mediated knockdown against selected candidate tumor suppressors. The Retinoblastoma (RB) family including pRB, p107 and p130 act as tumor suppressor genes to inhibit cancer development. The viral transforming proteins SV40 large T antigen (T Ag) and Adenovirus E1A inactivate all members of the RB family and overcome the growth inhibition imposed by their repression of E2F activity. We will test if two protein complexes termed DREAM and NuRD that bind to p130 and p107 are also inactivated by T Ag and determine if DREAM and NuRD also serve as tumor suppressors.
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