Mechanism of transcriptional silencing mediated by the methyl-CpG binding protein MBD4 and RET finger protein

肿瘤中通过许多癌症相关基因启动子中CpG岛甲基化的异常基因沉默几乎影响肿瘤进展的每一步。在这一过程中，甲基化CpG结合域（MBD）蛋白在将表观遗传信息传递给下游调控蛋白中起着重要作用，在目前已鉴定的5种MBD蛋白中，MBD4是唯一的例外，它一直被认为是一种DNA修复蛋白。在这项研究中，我们证明了MBD4也具有通过甲基CpG序列抑制转录的能力。MBD4参与HDAC依赖性阻遏，并直接结合HDAC 1和辅阻遏物SIN 3A。此外，MBD4特异性结合CDKN2A和MLH1基因的高度甲基化启动子。为了进一步研究MBD4在基于甲基化的转录抑制中的作用，进行了酵母双杂交筛选，发现RET指蛋白（RFP）是与MBD4中的转录抑制结构域相互作用的主要蛋白质之一。甲基化CDKIV 2A和MLH 1启动子中MBD4介导的转录抑制作用通过RFP的过表达而极大地增强。由于RFP已在多种肿瘤细胞系以及睾丸和胚胎中检测到高水平，RFP可能在肿瘤发生、精子发生和胚胎发生中通过MBD蛋白增强转录抑制中起重要作用。

Aberrant gene silencing in tumors via methylation of CpG islands in the promoter of many cancer-related genes affects virtually every step in tumor progression. In this process, the methyl-CpG binding domain (MBD) proteins play an essential role in transmitting epigenetic information to downstream regulatory proteins.Among the five MBD proteins identified so far, MBD4 has been the only exception; it has long been thought to be a DNA repair protein. In this study, we demonstrated that MBD4 also has the ability to repress transcription through methyl-CpG sequences. MBD4 is involved in HDAC-dependent repression and directly binds to HDAC1 and corepressor SIN3A. Furthermore MBD4 specifically binds to highly methylated promoters of CDKN2A and MLH1 genes. In order to further investigate the role of MBD4 in methylation-based transcriptional repression, a yeast two-hybrid screening was performed, and the RET finger protein (RFP) was found to be one of the major proteins that interact with the transcriptional repression domain in MBD4. The effect of the MBD4-mediated transcriptional repression in methylated CDKIV2A and MLH1 promoters was extremely enhanced by the overexpression of RFP. Because RFP has been detected at high levels in a variety of tumor cell lines as well as testis, and embryos, RFP may have an important role in the enhancement of transcriptional repression through MBD proteins in tumorigenesis, spermatogenesis, and embryogenesis.