The C Terminus Of MINT Forms Homodimers And Abrogates MINT-mediated Transcriptional Repression

Cell-cell interactions regulate cell proliferation and differentiation and play an important control role in the development, maturing, aging, and illness. Protein-protein interactions and protein-nucleic acid interactions are essential elements of cell-cell interaction. Studies on interactions of protein-protein or transcription will be important to understand the function and constitute of signaling pathway, and the native characteristics of life. The Notch signaling pathway plays a pivotal role in cell fate specification in development by mediating cell-cell interaction and is highly conserved during evolution from worm through man. The Notch signaling pathway has an important regulative function in the development of eye, pancreas, tooth, generation and regeneration of the nervous system. The Notch is named after wing margin notch in Drosophila mutation analysis. Notch receptors and ligands are highly conserved in structure and function from Caenorhabditis elegans through human evolutionarily. The Notch signaling pathway plays an element role in the regulative network.It was found that RAM7 (RBP-Jkappa associated molecule 7) could interact with RBP-J (Recombination signal binding protein-J kappa, a key effect element in the Notch signaling pathway) and compete with Notch intracellular domain (NIC) in the studies of molecule elements and regulative functions of Notch signaling pathway. The RAM7 was proven to be Msx2 (Muscle segment homeobox 2)-interacting nuclear target protein (MINT), which participated in craniofacial skeletal and neural development with Msx2. MINT protein is cleaved into approximately 110 kDa (Kilo Dalton, N-terminal) and 250 kDa (C-terminal) fragments shown by Western blot analysisof fractionated cell extracts. MINT has three N-terminal RNA recognition motifs (RRMs) and four nuclear localization signals (NLS) with amino acid sequence analysis. The Drosophlia homologue of MINT, Spen ( Split ends) ,has been reported to negatively regulate transcription by binding to Msx2, RAR(retinoic acid receptor) and RBP-J. In the regulative network, MINT can interact with NCOR/SMRT (Nuclear receptor co-repressor /silencing mediator for retinoid and thyroid hormone receptor), recruiting HDACs (Histone deacetylases) to play the repression function. MINT deficient mice were embryonic lethal. The preliminary analysis of the knockout mice showed that MINT is required for the embryonic and neural development, and B cell differentiation. MINT protein associates with many signaling pathway molecules in the nuclear. It is not clear that whether MINT functions only as a scaffold protein or participates in the transcriptional. regulation.To study the function and mechanism of MINT-mediated transcription repression in the Notch/RBP-J signaling pathway, yeast two-hybrid assay was used to screen proteins that interact with MINT. From 4×10~6 yeast clones transformed with the bait plasmid and a cDNA library of E9 mouse embryo, the MINT protein and the other proteins were identified. For further analysis, BD-MINT-F6 and AD-MINT-F6 plasmids were constructed, and the self-interaction of MINT through C-terminus was confirmed with yeast two-hybrid assay.To confirm the interaction between MINT proteins, His-MINT-F6-C and GST-MINT-F6-C were constructed. GST pull-down assays were carried out after the purification of proteins. It was found that His-MINT-F6-C could interact with GST-MINT-F6-C physically in vitro by Western blot analysis. Subsequently, the C-terminus of MINT (MINT-F6) were truncated as MINT-F6-N, MINT-F6-C-N, and MINT-F6-C-C. It was found that the integrity of the SPOC domain (Spen paralog and ortholog C-terminus domain) was indispensable to self-interaction of MINT.Then, we examined those self-interactions of MINT in cells. The Co-immunoprecipitation and the mammalian cell two-hybrid assay were performed. pCMV-FLAG-MINT-F6 and pCMV-Myc-MINT-F6 plasmids were constructed and transformed accordingly. The cultured cells were harvested after 48 hours and the interaction was detected with Western blot assay. GAL4-DBD-MINT-F6 and VP16-MINT-F6-C were constructed and transfected with TK MH 100x4 Luc in COS7 and HEK293 cells. The cultured cells were harvested after 48 hours and the interaction was detected with the luciferase (LUC) activity. It was shown that MINT could self-interact in cells.