Study On The Mechanism Of Transcriptional Regulation By Methylation Of MyoD1 Promoter

Myogenic differentiation 1(MyoD1)is a significant member of the myogenic regulatory factors(MRFs)family.As a nuclear phosphoprotein,it is involved in many physiological regulatory pathways of animal growth and development in the form of transcription factors.Its main functions are to promote the differentiation of myogenic cells into myoblasts in the early stage of embryonic development and to participate in the regulation of muscle growth and repair of injured muscles during individual growth and development.MyoD1 is an indispensable regulatory factor in animal growth and development,and it is also a major candidate gene in the improvement of livestock and poultry breeds.In this study,Guizhou Guanling cattle and Weining cattle were used as research objects.Firstly,the relationship between the methylation probability of the MyoD1 gene promoter and its gene expression level was detected and analyzed.Secondly,both methylated and non-methylated MyoD1 gene promoter double luciferase reporter vectors were constructed and transfected into Guanling cattle primary myoblast cells to detect and analyze the effect of methylation on the activity of MyoD1 gene promoter.MyoD1 gene was overexpressed and interfered in Guanling cattle primary myoblast cells,and then the influence of overexpression or underexpression of MyoD1 on promoter methylation state was detected and analyzed.Finally,MyoD1 and myostatin(MSTN)genes were overexpressed in Guanling cattle primary myoblast cells,respectively,to verify the mutual regulation between MyoD1 and MSTN.The main research results are as follows:1 Correlation analysis of MyoD1 promoter methylation and its transcriptional level.The methylation level of CpG island in the promoter region of MyoD1 gene in Guanling cattle and Weining cattle was detected by bisulfite sequencing PCR(BSP),and the correlation analysis with the mRNA expression level detected by qRT-PCR was conducted to clarify the relationship between the methylation probability of MyoD1 gene promoter and its gene expression level.The results showed that the methylation probability of the MyoD1 gene promoter first CpG island in the heart,liver,small intestine,longissimus dorsi,thigh muscle,adipocytes were 30.00%,31.43%,36.67%,28.10%,31.43% 33.81%,respectively.Weining cattle were 29.05%,30.95%,36.67%,28.57%,32.86% and 32.38%,respectively.qRT-PCR results showed that the relative expression levels of the MyoD1 gene in heart,liver,small intestine,longissimus dorsi,thigh muscle,adipose tissue were 7.40±0.381,4.87±0.384,0.36±0.028,37.89±4.644,2.50±0.048,0.81±0.138.Weining cattle were 8.52±0.836,4.06±0.270,0.29±0.033,24.29±2.056,2.27±0.219,1.00 ± 0.064.The correlation coefficient between the expression level of the MyoD1 gene and the methylation probability of the promoter on the first CpG island was-0.710(P=0.01),while the correlation coefficient between the expression level of the MyoD1 gene and the methylation probability of the two sites on the first CpG island was-0.737(P=0.006).The results showed that MyoD1 gene expression level was negatively correlated with promoter methylation level.2 Study on the effect of promoter methylation of MyoD1 on its initiation activity.Firstly,the primary myoblast cells of Guanling cattle were isolated and cultured,and the promoter sequence of the MyoD1 gene was amplified by PCR,and the pGL3-MyoD1-Pro double luciferase reporter vectors were constructed,both methylated and non-methylated.The methylation probability of the promoter on the constructed methylated reporter vector was 95.24%±0.67%,identified by BSP sequencing.After transfected into Guanling cattle primary myoblast cells,the relative luciferase activity of the control group was 0.039±0.007,that of the un-methylated group was 2.994±0.237,and that of the methylated group was 0.685±0.048.The results showed that the activity of the MyoD1 gene promoter decreased after hypermethylation.3 Study on the influence of MyoD1 expression level on promoter methylation.The pEGFP-N3-MyoD1 overexpression vector was constructed,and the MyoD1 shRNA interference vector p GPH1/RFP/Neo-MyoD1-cattle-263 was synthesized and screened.After transfection of overexpression vector and interference vector into Guanling cattle primary myoblast cells,BSP detected that the methylation probability of MyoD1 promoter 1 CpG island increased from 42.86% to 50.95% in the overexpression group compared with the control group,respectively.The methylation probability of CpG-1-6 and CpG-1-7 increased from 30.00% and 26.67% to 53.33%and 50.00%,respectively.The methylation probability of the first CpG island of the MyoD1 promoter in the interference group decreased from 42.38% to 33.33%,respectively,and the methylation probability of the unit point of CpG-1-6 and CpG-1-7 decreased from 30.00% and 33.33% to 6.67% and 13.33%,respectively.The results showed that both high and low MyoD1 gene expression levels would promote the change of promoter methylation level.4 Study on the regulation of the mutual expression of MyoD1 and MSTN.MSTN overexpression vector and promoter-reporter vector pEGFP-N3-MSTN and pGL3-MSTN-Pro were constructed.The pEGFP-N3-MyoD1 and pEGFP-N3-MSTN were transfected into Guanling cattle primary myoblast cells,respectively.After the overexpression of MyoD1,the relative expression of MyoD1 in the treatment group was increased from 1.018±0.222 to 8283.987±2337.534(P=0.004)compared to the control group.Meanwhile,the relative expression of the MSTN gene was increased from1.005±0.132 to 7.849±1.089(P=0.0004).Compared with the control group,the relative expression of MSTN in the overexpression group was increased from 1.003±0.092 to121131.802±12474.046(P=0.004).Simultaneously,the relative expression of the MyoD1 gene was decreased from 1.003±0.099 to 0.103±0.016(P=0.0001).In further experiments,the relative luciferase activity in the treatment group was increased from1.131±0.051 to 3.030±0.212(P=0.0001)after overexpression of MyoD1 compared with the control group.The relative luciferase activity in the overexpressed MSTN group was decreased from 1.797±0.136 to 0.543±0.092(P=0.0002)compared with the control group.These results indicated that MyoD1 could positively regulate the transcription of MSTN,while MSTN overexpression could negatively regulate the transcription of MyoD1.