Study On The Interaction Of KyoT2 With PIAS1 In Vitro And In Vivo And The Construction Of SUMOylation System In Vitro

Notch proteins act as receptors in an evolutionarily conserved signaling pathway that controls differentiation and proliferation in response to ligands expressed on neighboring cells. Ligand binding to the extracellular region of Notch receptors causes activation of a signal by triggering a series of proteolytic cleavages that release the intracellular portion of Notch (NIC) from the plasma membrane. NIC then migrates to the nucleus where it activates the transcriptionof target genes. The primary target of NIC in the nucleus is the highly conserved DNA-binding transcription factor, CSL (also known as RBP-J, CBF1, Suppressor of Hairless (Su)H, and Lag-1). In addition to NIC, RBP-J also mediates transactivation of Epstein-Barr (EB) virus nuclear antigen (EBNA)2. In mammals, the Notch signaling pathway is critical for embryonic development, as targeted disruption of Notch1 or its downstream transcription factor RBP-J in mice resulted in disorganization of somites and delayed closure of neurotube, which finally leads to early embryonic lethality.In the absence of NIC or (EBNA)2, RBP-J represses transcription by virtue of interactions with a number of co-repressors, including SMRT/N-CoR (silencing mediator forretinoic acid and thyroid hormone receptors/nuclear repressorco-repressor), CIR (CBF1-interacting co-repressor), and KyoT2. 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. 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), such as PcG proteins complex HPC2 and RING1, to RBP-J through its LIM domains. LIM domains, have been shown to function as protein-protein interaction interface by a large mount of data. Base on our previous work, we supposed that KyoT2 also can recruit other molecule to regulate the RBP-J-mediated transcription through its LIM domains.PIAS1 was isolated in a yeast two hybrid screening using KyoT2 as a bait protein in previous work, we investigated the physical interaction of KyoT2 with PIAS1 in this study. We also constructed the SUMOylation system in vitro and laid a foundation for the further study on target proteinsSUMOylation testification.The main results are as follows:1. Study on the interaction of KyoT2 with PIAS 1 in vitro and in vivo.Firstly, we showed that KyoT2 interacted with PIAS1 through its LIM1...