甲基转移酶Dot1L通过调控启动子甲基化水平促进骨骼肌成肌分化

李程程; 周永; 李浩可; 邓忠良

doi:10.16016/j.2097-0927.202111134

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甲基转移酶Dot1L通过调控启动子甲基化水平促进骨骼肌成肌分化

基础医学 | 更新时间：2026-01-09

- 甲基转移酶Dot1L通过调控启动子甲基化水平促进骨骼肌成肌分化
- Methyltransferase Dot1L promotes myogenic differentiation of skeletal muscle by regulating promoter methylation level
- 陆军军医大学学报 2022年44卷第11期页码：1104-1111
- 作者机构：
  
  400010 重庆，重庆医科大学附属第二医院骨科
- 作者简介：
  
  [邓忠良，E-mail: zhongliang.deng@qq.comDENG Zhongliang, E-mail: zhongliang.deng@qq.com
- 基金信息：
  
  国家自然科学基金面上项目(31870768)
- DOI：10.16016/j.2097-0927.202111134
  中图分类号： R322.74;R345;R394.1
- 收稿：2021-11-18，
  
  修回：2022-01-07，
  
  纸质出版：2022-06-15
- 稿件说明：
移动端阅览
李程程, 周永, 李浩可, 等. 甲基转移酶Dot1L通过调控启动子甲基化水平促进骨骼肌成肌分化[J]. 陆军军医大学学报, 2022,44(11):1104-1111.

Chengcheng LI, Yong ZHOU, Haoke LI, et al. Methyltransferase Dot1L promotes myogenic differentiation of skeletal muscle by regulating promoter methylation level[J]. Journal of Army Medical University, 2022, 44(11): 1104-1111.
李程程, 周永, 李浩可, 等. 甲基转移酶Dot1L通过调控启动子甲基化水平促进骨骼肌成肌分化[J]. 陆军军医大学学报, 2022,44(11):1104-1111. DOI： 10.16016/j.2097-0927.202111134.

Chengcheng LI, Yong ZHOU, Haoke LI, et al. Methyltransferase Dot1L promotes myogenic differentiation of skeletal muscle by regulating promoter methylation level[J]. Journal of Army Medical University, 2022, 44(11): 1104-1111. DOI： 10.16016/j.2097-0927.202111134.

摘要

目的

明确甲基转移酶Dot1L是否参与成肌分化调控过程，并初步探索其调控成肌分化过程的分子机制。

方法

应用小鼠体外成肌分化模型C2C12细胞系，诱导C2C12细胞在0、1、3、5 d发生成肌分化，使用Dot1L特异性抑制剂抑制其甲基转移酶活性，并应用免疫荧光实验观察Dot1L对成肌分化过程的影响；检测成肌分化标志分子肌球蛋白重链(myosin heavy chain

MHC)和肌细胞调节因子(Myogenin)的蛋白质水平和转录水平及启动子区域的组蛋白甲基化水平；使用RNA-Seq基因组范围内检测抑制剂处理与否发生变化的基因阵列；并用CHIP-qPCR实验验证H3K79me2结合位点。

结果

Dot1L组蛋白底物H3K79的甲基化水平随成肌分化进程逐渐升高(

0.01)；抑制Dot1L酶活性后，成肌分化过程明显受阻；免疫荧光实验显示C2C12细胞多核肌管形成显著下降(

0.01)，MHC和Myogenin的蛋白质水平及转录水平均显著降低(

0.01)；启动子区域组蛋白H3K4甲基化水平降低(

0.01)；RNA-Seq显示受Dot1L调控的基因阵列主要集中在骨骼肌的成肌分化和肌肉的发育；经CHIP-qPCR验证参与成肌分化的部分基因存在H3K79me2结合位点。

结论

甲基转移酶Dot1L通过直接调控一群成肌分化相关基因的转录水平参与调控骨骼肌的成肌分化过程。

Abstract

Objective

To confirm whether methyltransferase Dot1L is involved in the regulation of myogenic differentiation

and explore its molecular mechanism in the regulation process.

Methods

Myoblast differentiation model of mouse C2C12 cell line was used to induce myogenic differentiation for 0

3 and 5 d. The specific inhibitor of Dot1L was used to inhibit the activity of methyltransferase

and the impacts of Dot1L on the process of myoblast differentiation was observed by immunofluorescence assay. The protein and transcription levels of myosin heavy chain (MHC) and myogenin

markers of myogenic differentiation

were subsequently detected

so was the histone methylation level of the promoter region. Moreover

RNA-Seq analysis was adopted to determine the gene arrays affected by inhibitor treatment in the genome

and chromatin immunoprecipitation coupled with quantitative PCR (CHIP-qPCR) was performed to verify the binding site of H3K79me2.

Results

The methylation level of H3K79

Dot1L histone substrate

was gradually increased with the process of myogenic differentiation (

0.01). With the inhibition of Dot1L activity

the process of myogenic differentiation was obviously suppressed

the formation of multinucleated myotubes in C2C12 cells was significantly decreased (

0.01)

the protein and transcription levels of MHC and Myogenin were remarkably reduced (

0.01)

and the histone H3K4 methylation level in the promoter region was also declined (

0.01). RNA-Seq analysis indicated that the gene arrays regulated by Dot1L mainly focused on the myogenic differentiation and muscle development of skeletal muscle. Finally

CHIP-qPCR verified that some genes involved in myogenic differentiation have H3K79me2 binding sites.

Conclusion

Methyltransferase Dot1L participates in the regulation of the myogenic differentiation process of skeletal muscle by directly affecting the transcription level of myogenic differentiation related genes.

关键词

Keywords

references

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