The Epigenetic Mechanism Of Regulating Myoblasts Differentiation By Suv39h1

Muscle cell differentiation is an important issue of Developmental Biology and it is currently the most studied problems. Muscle cell differentiation is a very complex molecular mechanisms related to multiple muscle-related gene family of transcriptional regulatory factors, signaling pathways and epigenetic aspect. Muscle regulatory factors family(MRFs) and Myocyte Enhancer Factor 2(MEF2) family play the important roles in muscle differentiation program. Recently studies show that histone-modifying enzymes are also involved and played an important role in the regulation mechanism of muscle differentiation. However, the present studies were focused on the relationship between MyoD and histone-modifying enzymes. The mechanism of muscle cells differentiation regulated by histone-modifying enzymes and others Myo D downstream regulatory factors is still unclear.The purpose of this research is to analyse porcine histone methyltransferase Suv39h1 gene expression patterns in the muscle tissue of Meishan. Analysis of biological function of histone methyltransferase Suv39h1 in porcine muscle satellite cells. Elaborated the regulatory mechanism of muscle cells differentiation by Suv39h1 interacts with MEF2. These studies provide insight into the mechanism of muscle differentiation. The results in this study are as follows:1. Swine histone methylase Suv39h1 and heterochromatin protein HP1 cloningWe isolated Suv39h1 and HP1 full length coding region from longissimus muscle tissue of Meishan swine and predicted their conserved domains by bioinformatic methods, finding these two proteins have typical conserved domain structure: Suv39h1 has the chromo domain and SET domain. HP1 has the chomo domain and chromo shadow domain.2. Analysis expression pattern of Suv39h1 in Meishan muscle tissuesThe expression pattern in Meishan pig tissues were respectively analyzed by semi quantitative PCR and realtime PCR. The results show that the Suv39h1 expressed in heart, liver, spleen, lung, kidney, brain, cerebellum, rectum, soleus, shoulder fat, back fat, biceps femoris, semitendinosus, masseter, and longissimus dorsi muscles. The expression of Suv39h1 was no significant difference between the biceps femoris, semitendinosus,masseter and longissimus dorsi muscles in embryonic day 65, 3 days, 35 days, 90 days, 150 days and 180 days after birth.3. Analysis of effects of Suv39h1 in porcine skeletal muscle satellite cellsSkeletal muscle satellite cells were separately transfected p IRES-Suv39h1 vector or Suv39h1 interference RNA. After culturing 48 h, cell was analyzed by flow cytometry. The results showed that overexpression of Suv39h1 might inhibit the cell DNA synthesis in the S phase, which resulting the cell accumulation in G0/G1 phase. Skeletal muscle satellite cells which was transfectd pIRES-Suv39h1 vector was stained by Edu. The results showed that overexpression of Suv39h1 could reduce the number of cells in S phase. Skeletal muscle satellite cells were separately transfected pIRES-Suv39h1 vector or Suv39h1 interference RNA, after cells was maintained in differentiation medium in 8 days. The immunofluorescence was performed to detect cell differentiation late marker protein MyHC. The results from immune experiments showed that overexpression of Suv39h1 or interference Suv39h1 might reduce the number of pig skeletal muscle satellite cells with expression MyHC than control cells.4. Investigate the regulation mechanism of Suv39h1 during the muscle cells differentiation(1) Effects of histone methyltransferase Suv39h1 on myoblast proliferationC2C12 myoblasts were separately transfected p IRES-Suv39h1 vector or Suv39h1 interference RNA. After culturing 48 h, cell was analyzed by flow cytometry. The results showed that overexpression of Suv39h1 might inhibit the cell DNA synthesis in the S phase, which resulting the cell accumulation in G0/G1 phase. C2C12 myoblasts which was transfectd p IRES-Suv39h1 vector was stained by EdU. The results showed that overexpression of Suv39h1 could reduce the number of cells in S phase.(2) Effects of histone methyltransferase Suv39h1 on myoblast differentiationC2C12 myoblasts were separately transfected p IRES-Suv39h1 vector or Suv39h1 interference RNA, after cells was maintained in differentiation medium in 8 days. The immunofluorescence was performed to detect cell differentiation late marker protein MyHC. The results from immune experiments showed that overexpression of Suv39h1 or interference Suv39h1 might reduce the number of C2C12 myoblasts with expression MyHC than control cells. In C2C12 myoblasts overexpressing or Suv39h1 interference, 2% horse serum were cultured for 0 days, 2 days, 4 days and 6 days, then realtime PCR was performed to analyze the expression patterns of the early or late marker gene during C2C12 myoblasts differentiation. The results showed that the expression patterns of Myf5 in C2C12 myoblasts with overexpression of Suv39h1 compared in C2C12 myoblasts with interference of Suv39h1 was different. There was no significant change of Mef2 c and My HC ompared with the control group.(3) Histone methyltransferase Suv39h1 through interacting with MEF2 C to inhibit MEF2 C downstream target genes transcription activationC2C12 myoblasts were treated by 2% horse serum differentiating for 0 days, 2 days, 4 days and 6 days to stain with Suv39h1, HP1 and MEF2 C antibody. The results showed that Suv39h1, HP1 and MEF2 C have been found in the nucleus during C2C12 myoblasts differentiation. The results of mammalian two-hybrid assay showed that the interaction between Suv39h1 and MEF2 C in undifferentiated C2C12 cells, which might form Suv39h1-MEF2 C complex. Suv39h1 might decrease the transcriptional activity of MEF2-dependent gene effectively.(4) The regulation mechanism of Mef2 c promoterOverexpression HP1 could decrease the transcriptional activity of Mef2 c promoter and down-regulated the expression of Mef2 c.In summary, the conclusions of our research:Histone methyltransferase Suv39h1 regulates pig skeletal muscle satellite cells and C2C12 myoblasts of cell cycle. Overexpression Suv39h1 can inhibit the differentiation of pig skeletal muscle satellite cells and C2C12 myoblasts. The interaction between Suv39h1 and MEF2 C in undifferentiated C2C12 cells, which might form Suv39h1-MEF2 C complex. Histone methyltransferase Suv39h1 might decrease the transcriptional activity of MEF2-dependent gene effectively.