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Function Research Of Mouse Embryonic Stem Cell Highly Expressed Gene MN6A1

Posted on:2009-05-29Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y B LiuFull Text:PDF
GTID:1100360245483094Subject:Medical Genetics
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Introduction: A mouse putative N6-DNA methyltransferase gene (mN6A1) (GenBank No AY456393), was cloned by suppression subtractive hybridization, beginning with the EST (CN304553) in our previous study, which was highly expressed in Undifferentiated human ES cells. It corresponds to gene 5830445C04Rik, a novel gene named mN6amtl. Bioinformatics analysis indicates that this gene contains the DNA methytransferase domain (N6-Mtase) and the protein methytransferase domain (HemK-rel-arch), therefore it should be a methytransferase and its homologous genes in yeast (Mtq2p) and bacteria (PrmC) have been approved to be the members of HemK methyltransferase family, which can methylate polypeptide chain release factors 1 (eRF1) in yeast and RF1 and RF2 in bacteria. The only published paper showed that the gene in mouse was located in nucleus by passive diffusion due to its small molecular weight, and that the activity of N-6 adenine-specific DNA methyltransferase was not detected.1. Expression of gene mN6A1 in differentiation ofmouse embryonic stem cell and embryonic development andmethylation status in CpG island of its upstreamAIM: To detect the methylation status of CpG island upsteam of the gene mN6A1 in ESC and its expression in development of mouse embryo. Methods: Bisulfite sequencing PCR was used to detect the methylation status of CpG island upsteam of the gene mN6A1. RT-PCR were used to detect expression of gene mN6A1 in 2-cell , 4-cell , 8-cell phase and blastula phase. Real-time PCR were used to detect its expression in spontaneous differentiaon of ESCs into embryoid body (EB) on the day of 0, 2, 4, 6, 8 and 10. Results: No methylation of 5 CpG dinucleotides in CpG island upsteam of the gene mN6A1 were detected in ESC. In development of mouse embryo, expression of mN6A1 gene was not detected by RT-PCR in 2-cell and 4-cell phase and was higher in blastula phase than that in 8-cell phase. Real-time PCR showed that the change trend of expression of mN6A1 is similar to those of gene mNaong and mSox2 which decrease at the second day and increase at the fourth day sharply, then decrease on following days, except for the tenth day in which expression amount of mN6A1 is increase instead of decrease. Conclusion: It was concluded that gene mN6A1 is related to differentiaon of ESCs.2 . Passive diffusion is not the mechanism of entering nucleus for protein mN6A1AIM: Previous study showed that the mN6A1 protein is located in nuclear and gave an explanation of passive diffusion of smaller mN6A1 molecule (23kD). Before this publication, our experiment also certified that mN6A1 protein is located in nuclear, but the model of nuclear entry was not regarded as passive diffusion. For further research its model of nuclear entry, following experiments were performed. Methods: Prepare the antibody of anti-mN6A1 and FITC conjugated anti-rabbit IgG and performed immuno-fluorescent staining on cell P19,GC-1 and C2C12. Plasmid pCMV-Myc-mN6A1 was constructed and transfected into P19 cell and immuno-fluorescent staining was performed. Plasmid pEGFP-c3-mN6A1 was constructed and transfected into P19 cell and fluorescent signals were detected. The proportion of cells containing the signals in nucleus was compared between transfection 30 hour and transfection 72 hour. Mutation plasmid pCMV-Myc-mN6A1-106 was constructed, which lack 400 bp in ORF of mN6A1, and transfected into P19 cell then performed immuno-fluorescent staining. Results: Immuno-fluorescent staining on cell P19, GC-1 and C2C12 with anti-mN6A1 antibody indicate that protein mN6A1 located in nucleus. The fusion protein EGFP-mN6A1 and Myc-tagged-mN6A1 were found both in cytoplasm and in nuclear for most cells, mainly in nuclear for a few cells. The proportion of cells containing the stronger signals in cytoplasm or nucleus has no distinct differences between transfection 30 hours and transfection 72 hours. The protein Myc-tagged-mN6A1-106 were also detected mainly in cytoplasm or in nucleus .Conclusion: The mechanism of entering nucleus of protein mN6A1 should not be passive diffusion and may need other factors help. Finally, because the mutation protein Myc-tagged-mN6A1-106 can also go into nuclear, so factors involved in nuclear entry of protein mN6A1 may act with N terminal 35 amino acids. 3. RNAi-mediated knock-down of mN6A1 reduces cell proliferation and decreases protein translation and mechanismresearchAims: To investigate the effect of mN6aAl on cell proliferation, apoptosis and protein translation and whether mN6A1 can methylate eRF1 protein. Methods: Plasmid pAVU6+27-mN6A1 for RNA interference (RNAi) mediated knock-down were constructed and co-transfected into P19 cells with plasmid pCMV-Myc-mN6A1 to detect the efficiency of RNAi through detect the expression of myc-tagged-mN6A1 with western Blot. Co-transfected plasmid pAVU6+27-mN6A1 and pEGFP-C3/mN6A1 into P19 cells to detect the efficiency of RNAi through detect the expression of pEGFP-C3-mN6A1. Respectively transfected plasmid pAVU6+27-mN6A1 and pCMV-Myc-mN6A1 into P19 cell, and cell cycle and apoptosis were analyzed by flow cytometry and apoptosis analysis kit. To investigate its function in protein translation, the phRL-TK vector (for expression of Renilla Luciferase reporter gene) was co-transfected with above plasmids or with pEQ842 (for expression of protein human eRF1) into P19 cells, and expression of luciferase was detected by siRus Luminoter. To investigate the binding of protein N6mA1 to eRF1, plasmid pCMV-Myc-mN6A1 was transfected into P19 cells, followed by co-immunoprecipitation with anti-Myc antibody and protein-A-agarose beads, and by western blot certification with antibody anti-eRF1. The pull-down experiment was performed too, in which total protein of P19 cells were mixed with MBP-mN6A1 fusion protein (prokaryotic expression) and amylose resin followered by Western blot certification with antibody anti-eRF1. To investigate whether mN6A1 protein can methylate eRF1 protein, in vitro protein mathylation reactions were performed with eRF1 protein, eRF3 protein and MBP-mN6A1 fusion protein, which were prepared by prokaryotic expression with plasmids pEQ814, pEQ609, and pMAL-p2x-mN6A1, respectively, together with methyl donor ~3H-SAM. The methylation of eRF1 was detected by liquid scintillation counter and mass spectra. In in vivo experiments, methylation status of eRF1 were detected by mass spectra in P19 cells thransfected with plasmids pCMV-Myc-mN6A1 or pAVU6+27-mN6A1. Results: The cell proliferation decreases in mouse P19 cells transfected with pAVU6+27-mN6A1, whereas has no significant change in those cells transfected with pCMV-Myc-mN6A1. The expression of luciferase was reduced by RNAi for mN6A1, whereas increased by the over-expression of mN6A1 or eRF1. Furthermore, co-expression of mN6A1 protein and eRF1 protein leads to further increase in expression of luciferase, compared with expression of mN6A1 or eRF1 alone. The binding of mN6A1 protein to eRF1 protein was confirmed by pull-down and co-immunoprecipitation experiments, in which eRF1 protein in total protein of P19 cells was dragged down by MBP-mN6A1 fusion protein (prokaryotic expression) or by myc-tagged mN6A1 protein with confirmation by western blot. No methylation of eRF1 protein catalysed by prokaryotically expressed N6mAl protein or eukaryotically expressed myc-tagged N6mA1 protein has yet been confirmed by liquid scintillation counter and mass spectra. Conclusions: The knocked-down of mN6A1 gene leads to decrease in cell proliferation in P19 cells and mN6A1 protein participates in the protein translation. Though the binding of mN6A1 protein and eRf1 protein was confirmed, whether mN6A1 protein can methylate eRf1 protein still needs to be certified.
Keywords/Search Tags:ESC differentiaon, embryonic development, expression, methylation, sub-cellular localization, passive diffusion, mutation plasmid, RNAi, cell proliferation, protein translation
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