Strong evidence indicates that N-CoR(nuclear receptor co-repressor) and a related factor SMRT (silencing mediator of retinoid and thyroid hormone receptors) are key factors in mediating repression by unliganded TR and RAR and by many other transcription factors including Mad/Max, BCL6/LAZ3 and ETO. Thus, N-CoR and SMRT are likely to play fundamental roles in development, differentiation and tumorigenesis through their ability to mediate transcription repression by a variety of transcription factors. How N-CoR mediates transcription repression is not yet fully understood. The repression mediated by N-CoR and SMRT appears to depend on histone deacetylase (HDAC) activity and involves multiple histone deacetylases including HDAC1/2, HDAC4, HDAC5 and HDAC7. We demonstrated that in HeLA nuclear extracts N-CoR resides exclusively in multi-protein complexes of 1.5-2 mDa in size. The N-CoR complexes contain HDAC3 but not mSin3A. Furthermore, the majority of N-CoR and SMRT protein appear to associated with HDAC3, since a HDAC3 antibody could deplete both N-CoR mediate repression, we believe that it is essential to isolate N-CoR complexes and identify the proteins that associate with N-CoR. Thus, in Specific Aim 1, we propose to purify N-CoR complexes by a combination of convention and antibody-affinity chromatography and identify the general exhibit only marginal enzymatic activity, we hypothesize that the association of HDAC3 with other components in N-CoR complexes would be essential for maintaining a complete HDAC3 activity in the N-CoR complexes.
In Specific Aim 2, we propose to study the protein-protein interactions that lead to the formation of N-CoR complexes and the protein-protein interactions that modulate HDAC3 activity.
In Specific Aim 3, we propose to study the functional significance of the N-CoR complexes and the associated HDACs in the repression of unliganded TR/RXR by using an in vitro chromatin-based transcription system and an Xenopus oocyte model system.
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