甲基CpG结合蛋白2(MeCP2)遏制HEK293细胞甲基化修饰的IL-6启动子活性及其分子机制

Methyl CpG-binding protein 2(MeCP2)inhibits the activity of methylated IL-6 promoter of HEK293 cells and its molecular mechanism

目的 在HEK293细胞甲基化修饰白细胞介素6(IL-6)启动子序列并过表达甲基CpG结合蛋白2(MeCP2)以及转录因子P300,探讨MeCP2抑制IL-6转录活性的分子机制。方法 通过电泳迁移率实验(EMSA)对P300 IL-6启动子区域的P300结合位点进行验证,IL-6启动子序列连接于荧光素酶报告质粒并转染HEK293细胞,同时利用规律间隔的成簇短回文重复序列-去活性Cas9核酸酶-DNA甲基转移酶3A(CRISPR-dCas9-DNMT3A)转染介导启动子的甲基化修饰,再转染MeCP2以及P300过表达质粒。亚硫酸氢盐测序PCR分析各组IL-6启动子区域胞嘧啶甲基化修饰情况;Western blot法检测胞内MeCP2、 P300蛋白水平;通过化学发光检测仪测定HEK293荧光素酶活性,并结合染色质免疫沉淀-测序技术(ChIP-seq)分析各组P300以及MeCP2在IL-6启动子区域的结合水平。结果 EMSA证实小鼠IL-6启动子部位存在P300结合位点;CRISPR-dCas9-DNMT3A转染HEK293细胞能够成功甲基化小鼠IL-6启动子;MeCP2以及P300过表达质粒能够稳定合成目标蛋白,且不受其他转染影响。相比于未被修饰的启动子,甲基化修饰可降低启动子转录活性,且于P300过表达情况下,较之单纯甲基化修饰,过表达MeCP2还将进一步遏制启动子转录活性。此外,甲基化修饰的启动子与MeCP2结合后,过表达P300未提升启动子转录活性;而在其他转染条件下,过表达P300均能促进转录活性。Chip-seq分析进一步表明,启动子的甲基化修饰并不干扰P300结合;然而甲基化修饰的启动子与MeCP2结合后,P300与启动子的结合则受到抑制。结论 MeCP2与甲基化修饰的IL-6启动子相结合,可阻遏转录因子与启动子结合,从而进一步抑制启动子的转录活性。

Objective To investigate the underlying molecular mechanism of methyl-CpG-binding protein 2(MeCP2) inhibiting interleukin 6(IL-6) transcriptional activity by observing the sequence of methylated IL-6 promoter, overexpression of MeCP2, and transcription factor P300 in HEK293 cells. Methods The binding site of P300 in the IL-6 promoter region was confirmed by electrophoretic mobility shift assay(EMSA);the IL-6 promoter sequence was ligated into luciferase reporter plasmid and transfected into HEK293 cells. The methylation of the promoter was mediated by clustered regularly interspaced short palindromic repeats-deactivated Cas9(CRISPR-dCas9)-mediated DNA methyltransferase 3A(DNMT3A) transfection, and then MeCP2 and P300 overexpression plasmids were transfected. The bisulfate sequencing PCR(BSP)was used to analyze the cytosine methylation in the IL-6 promoter region of each group. The contents of intracellular MeCP2 and P300 were detected by the Western blot. A chemiluminescence detector was used to determine the luciferase activity of HEK293 cells. The binding level of P300 and MeCP2 in the IL-6 promoter region was analyzed by chromatin immunoprecipitationfollowed by sequencing(ChIP-seq). Results EMSA confirmed the presence of P300 binding sites in the IL-6 promoter of mice. CRISPR-dCas9-DNMT3A transfection into HEK293 cells successfully methylated the IL-6 promoter. MeCP2 and P300 overexpression plasmid steadfastly synthesized the target protein and was not affected by other transfection. Compared with the unmodified promoter, methylation could reduce the transcriptional activity of the promoter. When P300 was overexpressed, MeCP2 could further inhibit the transcriptional activity of the promoter, when compared with methylation alone. Also, overexpression of P300 could not promote the transcriptional activity of IL-6 promoter after the methylation modified promoter combined with MeCP2, while the overexpression of P300 enhanced the transcriptional activity when the promoter was not methylated or MeCP2 was not overexpressed. ChIP-seq analysis revealed that the methylated IL-6 promoter showed no difference in binding to P300;however, when combined with MeCP2, the binding capacity would be repressed. Conclusion The combination of MeCP2 with methylated IL-6 promoter can inhibit the binding of the transcription factor to the promoter, thereby impeding the transcriptional activity of the promoter.