| Notch gene and its encoded signalling receptor is evolutionarily conserved in the structrue and the function from Caenorhabditis elegans to human. In Drosophlia Notch is typically involved in binary cell fate decisions including neurogenesis, myogenesis and hematopoiesis. In mammals, Notch regulates development of hematopoietic system, muscle, pancreas, and many other tissures. The intracellular domain has four distinct regions: the RAM domain, the ankyrin repeats, a transcriptional activator domain (TAD) and the PEST (proline-, glutamate-, serine-, threonine-rich) sequence. Two nuclear localisation sequences are present prior to and following the ankyrin repeats. Delta, Jagged and Serrate have been identified as Notch ligands. The transcription factor Suppressor of Hairless [Su (H)] is the major downstream effector of Notch signaling pathway.Notch interaction with its ligands induces the cleavage of its intracellular domain (IC), and the Notch IC translocates to the nucleus and binds to RBP-J, the mammalian homolog of Su(H), to transactivate transcription of target genes such as E(spl) (Enhancer ofsplit), Hesl (Hairy and enhancer of split) and Hes5Four Notch receptors and their ligands are differentially and redundantly expressed in a variety of vertebrate tissues. On the other hand, the RBP-J protein is ubiquitously expressed, and commonly activated by all of four Notch receptors. Thus RBP-J is an essential mediator of Notch signaling pathway. The targeted disruption of mouse RBP-J revealed that homozygous null mutants die before 10.5 days postcoitum (dpc) with various abnormalities, including growth retardation and defects in the central nervous system and somites.In order to assess the function of RBP-J, a yeast two-hybrid screening using RBP-J as a bait protein was performed and a nuclear matrix protein, MINT was isolated. MINT (Msx2-interacting nuclear target protein) can interact with RBP-J and MSX2 (the murine homologue of Drosophlia muscle-segment homeobox ) , and represses their transcription activation activity. Identified as encoding protein that bind Msx2, MINT, human gene termed as SHARP (SMRT/HDAC1 Associated Represser Protein), is a large nascent polypeptideof 3576 amino acids and has three N-terminal RNA recognition motifs (RRMs) and four nuclear localization signals. Western blot analysis of fractionated cell extracts reveals that mature MINT is cleaved into approximately 110 kDa (N-terminal) and approximately 250 kDa (C-terminal) fragments. MINT, the Drosophlia homologue of Spen ( Split ends) has been reported to negatively regulate transcription by binding to MSX2, RAR(retinoic acid receptor) and RBP-J. MINT deficient mice were embryonic lethal. The preliminary analysis of theknockout mice show that MINT is required for the embryonic and neural development.To search proteins that associate with the mouse MINT protein and regulate Notch signaling in nuclei, and to study the function and mechanism of MINT-mediated transcription repression, yeast two-hybrid assay was used to screen proteins that interact with a fragment of MINT (F5, amino acids 2226-2959). From 4x106 yeast clones transformed with the bait plasmid and a cDNA library of 9 dpc mouse embryo, fifty-one were positive for nutritional screening and p-galactosidase assay. Restriction digestion identified 10 independent positive clones and these were analyzed by DNA sequencing These clones represent 3 correctly fused cDNA fragments, which are MINT, alpha A-crystallin-binding protein I (AlphaA-CRYBPl), and nuclear receptor co-repressor 1 (N-CoRl), respectively. We further analyzed the sites by which MINT interacts with N-CoRl. The results show that N-CoRl can also interact with another fragment F6 (amino acids 2960-3576). Further screening proteins that interact with anther fragment F6, we isolated the cDNA of IgM heaven chain contant region, ubiquitin- conjugating enzyme E2L6 and a new protein.N-CoRl involving a corepressor complex (including NCoR, SMRT, and HDAC) can mediate the transcriptional repression of...
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