Screening Differentiation Related GCN5 Recruitment Proteins During Differentiation Of Mesenchymal Stem Cells Into Cradiac Like Myocytes

ObjectiveScreening and analysing the recruitment of protein complexes with GCN5 during the process of mesenchymal stem cells differentiating into cardiac myocytes have been designed to identify unknown proteins that were recruited by histone acetyltransferase GCN5. This experiment screens GCN5 recruitment proteins for regulation. We will further study differentiating signal transduction pathway of stem cells, as well as the specific biological target for intervention to improve the ratio of differentiation of stem cells on the basis of experiments.Methods and materials1. MSCs can differentiate into cardiomyocyte-like cells after 5-azaC induction treatment. Immunofluorescence cytochemistry identify the expression of myocardial cell structure and function of proteins cTnt, MHC, and Cx43. Real-time PCR analysis identify sequential expression of cardiac-specific genes GATA4 and MEF2C after 5-azaC induction treatment.2. Immunofluorescence cytochemistry detects position of GCN5 in the cell. Western blot analysis determines the nucleoprotein extraction time according to the expression levels of GCN5-immunoprecipitation.3. Co-immunoprecipitation - polyacrylamide gel electrophoresis - Coomassie brilliant blue staining separates and purifys GCN5-binding protein. Na-chip high-performance liquid chromatography electrospray ionization tandem mass spectrometry streaming technology (HPLC-CHIP-NanoSpray-ESI-MS/MS) used to identify GCN5-binding proteins.4. ExpAsy, EMBL-EBI and the Intact databases are used to bioinformatics analyse the results of the above-mentioned MS analysis. Screening and analyzing differentiation-related protein factors from the point of differentiation of myocardial cells and stem cell respectively.5. Western-blot verify parts of the differentiation-related proteins of myocardial. Cell proliferation was evaluated by MTT assay and cell cycle by flow cytometry.6. Expression of P21,P53,SOX9 was analysed by real-time PCR. CHIP strategy was used for analyzing binding ability between P21 promoter and GCN5 together with acetylated histones at the GCN5 site on the P21 gene to verify that GCN5 regulation stem cell cycle and proliferation characteristics of MSCs induced by 5-aza.C Results1. The significant increasing expression of cardiac-specific genes, cardiac structure and function proteins and the appearance of cardiomyocyte-like cells in morphological changes after MSCs induced by 5-azaC indicate that 5-azaC can induce MSCs to differentiate into cardiomyocyte-like cells.2. In this study, immune cells fluorescence results showed that the induction group and the uninduced group of GCN5 location has always been in the nucleus. Western blotting results showed that the induction group and the non-induced group of GCN5 expression was no significant difference.3. The result of SDS-PAGE-COIP-MS/MS analysis showed that GCN5 played roles in changing status of chromosomes to regulate transcriptional activity of gene transcription.4. Bioinformatic analysis showed that part of the GCN5 protein complex is not only involved in 5-azaC-induced differentiation of MSCs cardiomyocyte-like cells in the process of transcription regulation,but also is involved in the process of in vitro differentiation of MSCs in G0/G1 phase cell cycle regulation and cell proliferation characteristics. GCN5 physically interacts with the following proteins which can be categorized as: DNA-binding protein of Sp1/KLF, transcriptional co-activators of PGC-1αand Rb1 and components of transcription elongation complex, and finally signaling pathway component of protein Akt. And part of GCN5 interaction proteins ( ATP-dependent chromatin remodeling complex: Arid1b and Smarca5 ; transcription initiation complex:BRF1 ; transcription factor:Sox9 and Erf; zinc finger protein, such as zfml)have biological functions of cell cycle and proliferation.5. Verification test results: (1) Western blot validation showed that IP-MS/MS effectively screened part of the protein groups associated with myocardial differentiation; (2) MTT assay and cell cycle by flow cytometry domenstrates:The proportion of G0/G1 phase MSCs and expression of P21 gene reached the highest at 3 days after 5-azaC-induced, then gradually decreasing. Cell proliferation index PI value and the G2/S phase cell ratio were the opposite to the above.6. GCN5 and recruitment protein factors form the transcription elongation complex. It receives extracellular signal. Then it transfers information by nuclear actin and Akt in nuclei-cytoplasm, which regulate gene transcription with Sp1 binding site. 5-aza raised the combination of GCN5-protein-complexes and the P21 gene promoter in MSCs, vitro differentiation process regulates G0/G1 phase of cell cycle and cell proliferation.Conclusions1. GCN5-containing complex has been recruited by transcriptional activators to the gene promoter region to regulate gene transcription. GCN5 protein complex have biological functions of regulating stem cell differentiation, cell cycle and proliferation.2. High concentration of 5-azaC increases G0/G1 phase arrest for MSCs. G0/G1 phase arrest decreased and cell proliferation increased as the concentration of 5-azaC decreasing. 5-azaC raised the combination of GCN5-protein-complexes and the P21 gene promoter in vitro differentiation of MSCs through regulating G0/G1 phase of cell cycle and cell proliferation.