| The Notch signaling pathway plays a pivotal role in cell fate specification in development by mediating cell-cell interaction and is conserved during evolution from worm through man. In Drosophila, loss of function mutation of the Notch receptor or other essential molecules of the Notch signaling pathway during early embryogenesis leads to abnormal cell fate determination in numerous differentiation steps. In mammals, the Notch signaling pathway is also critical for embryonic development, as targeted disruption of Notch 1 or its downstream transcription factor RBP-J (recombination signal binding protein-Jk) in mice resulted in disorganization of somites and delayed closure of neurotube, which finally leads to early embryonic lethality. More recently, conditional gene knock-out mice of Notch 1 and RBP-J have been constructed by Radtke et al and Han et al, respectively. Phenotype analyses of these mice have demonstrated that disruption of Notch 1 or RBP-J in adult mice caused abnormal differentiation of common lymphoid progenitors, resulting in blockade of T cell fate determination and induction of B cell differentiation in thymus where normally most T lymphocytes but few B lymphocytes are generated. Therefore Notch signaling pathway also plays a critical role in theregulation of cell differentiation in adults. Therefore, elucidating the functions and molecular mechanisms of Notch signaling pathway have important implications for understanding embryonic development and stem celldifferentiation as well as related human disorders.Notch is a family of type I transmembrane proteins. When Notch is triggered by direct interaction with its ligands, the Delta family proteins expressed on neighboring cells, the intracellular domain of the Notch receptor (NIC) is released from the membrane after receptor cleavage executed by a-r -secretase-like protease. NIC translocates to nucleus and associates with RBP-J through its N-terminal RAM (RBP-J association molecule) domain, and transactivates promoters harboring RBP-J-binding sites, which leads to expression of genes associated cell differentiation. Therefore, RBP-J is the major downstream effector of Notch signaling pathway. In addition to NIC, RBP-J also mediates transactivation of Epstein-Barr (EB) virus nuclear antigen (EBNA) 2.On the other hand, in the absence of transactivators such as NIC or EBNA2, RBP-J suppresses transcription of promoters recognized by RBP-J. In mammals, some reports indicated that histone deacetylases HDACs and MINT (MSX2-interacting nuclear target protein) suppress the RBP-J-mediated transactivation by competing for binding sites with NIC or EBNA2. However, the molecular mechanisms of RBP-J-mediated transcriptional repression are unclear.KyoT2, a LIM domain protein, inhibits transactivation of promoters containing RBP-J recognition sites by NIC and EBNA2. However, evidence suggested that in addition to work as a competitor for binding sites on RBP-J, KyoT2 may also regulate the RBP-J-mediated transactivation by recruiting other cosuppressor(s) to RBP-J through its LIM domains, which have been shown to function as protein-protein interaction interface.In order to assess this assumption, we investigated the physical and functional interaction of KyoT2 with PcG proteins HPC2 and RING1 as well as between KyoT2 and KBP1 (KyoT2 binding protein 1) in this study. We also investigated their effect on RBP-J-mediated transcriptional activity.The main results are as follows:1.RING1 inhibits transactivation of RBP-J by Notch through interaction with LIM protein KyoT2.Firstly, we showed that RINGl and KyoT2 interacted through the C-terminal fragment of RINGl and the LIM domains of KyoT2 but not through the N-terminal fragment of RINGl by yeast hybrid system. Secondly, GST-pull down and co-immunoprecipitation as well as mammalian cell two-hybrid assays furtherly showed that RINGl and KyoT2 possessed physical and physiological interactions both in vitro and in vivo. Meanwhile, by using a co-immunoprecipitation assay we also showed, th...
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