Abstract：
The epigenetic factor Methyl-CpG-Binding Protein 2 (MeCP2) is a nuclear protein that binds methylated DNA molecules (both 5-methylcytosine and 5-hydroxymethylcytosine) and controls gene transcription. MeCP2 is an important transcription factor that acts in a dose-dependent manner in the brain; thus, its optimal expression level in brain cells is important. As such, its deregulated expression, as well as gain- or loss-of-function mutation, lead to impaired neurodevelopment, and compromised structure and function of brain cells, particularly in neurons. Studies from others and us have characterized two well-recognized MeCP2 isoforms: MeCP2E1 and MeCP2E2. We have reported that in Daoy medulloblastoma brain cells, MeCP2E2 overexpression leads to MeCP2E1 protein degradation. Whether MeCP2 isoforms regulate the Mecp2 promoter regulatory elements remains unexplored. We previously showed that in Daoy cells, metformin (an anti-diabetic drug) induces MECP2E1 transcripts. However, possible impact of metformin on the Mecp2 promoter activity was not studied. Here, we generated stably transduced Daoy cell reporters to express EGFP driven by the Mecp2 promoter. Transduced cells were sorted into four EGFP-expressing groups (R4-to-R7) with different intensities of EGFP expression. Our results confirm that the Mecp2 promoter is active in Daoy cells, and that overexpression of either isoform inhibits the Mecp2 promoter activity, as detected by flow cytometry and luciferase reporter assays. Interestingly, metformin partially relieved the inhibitory effect of MeCP2E1 on the Mecp2 promoter, detected by flow cytometry. Taken together, our data provide important insight towards the regulation of MeCP2 isoforms at the promoter level, which might have biological relevance to the neurobiology of the brain.
摘要：
表观遗传因子甲基-CpG-结合蛋白2(MeCP2)是结合甲基化DNA分子(5-甲基胞嘧啶和5-羟甲基胞嘧啶)并控制基因转录的核蛋白。MeCP2是一种重要的转录因子，在大脑中以剂量依赖的方式起作用；因此，其在脑细胞中的最佳表达水平是重要的。因此，它的表达失调，以及功能的增益或丧失突变，导致神经发育受损，脑细胞的结构和功能受损，特别是在神经元中。其他人和我们的研究已经表征了两种公认的MeCP2同工型:MeCP2E1和MeCP2E2。我们已经报道了在Daoy髓母细胞瘤脑细胞中，MeCP2E2过表达导致MeCP2E1蛋白降解。MeCP2同种型是否调节Mecp2启动子调节元件仍未探索。我们之前证明了在Daoy细胞中，二甲双胍（一种抗糖尿病药物）诱导MECP2E1转录本。然而,未研究二甲双胍对Mecp2启动子活性的可能影响。这里,我们产生了稳定转导的Daoy细胞报告基因，以表达由Mecp2启动子驱动的EGFP。将转导的细胞分成4个EGFP表达组(R4-至-R7),它们具有不同的EGFP表达强度。我们的结果证实Mecp2启动子在Daoy细胞中具有活性，任何一种同种型的过表达都会抑制Mecp2启动子的活性，通过流式细胞术和荧光素酶报告基因检测。有趣的是,二甲双胍部分缓解了MeCP2E1对Mecp2启动子的抑制作用，流式细胞仪检测。一起来看,我们的数据为MeCP2同工型在启动子水平的调控提供了重要的见解，这可能与大脑的神经生物学具有生物学相关性。
