Development of a new strategy to control gene expression by chromatin conversion

DNA甲基化有助于基因组调控，包括基因转录和染色质结构的控制。通常，甲基化发生在CpG二核苷酸内胞嘧啶的5位，这意味着转录失活的染色质的形成。在细胞核中，不仅DNA被甲基化，而且甲基化的DNA还必须被甲基-CpG结合结构域蛋白（MBD蛋白）解释。哺乳动物中至少有5种MBD蛋白：MeCP 2、MBD 1、MBD 2、MBD 3和MBD 4（也称为MED 1）。此外，我们已经确定了MBD的结构从MBD 1与甲基化DNA的复合物的多维heteronudear NMR光谱。其中功能上重要的残基Arg 22、Arg 30、Asp 32、Tyr 34、Arg 44、Ser 45和Tyr 52改变为丙氨酸的突变型MBD丧失了结合甲基化DNA的能力。虽然MeCP 2、MBD 2和MBD 3嵌入组蛋白脱乙酰酶复合物中，但在已知的组蛋白脱乙酰酶复合物中尚未发现MBD 1，这表明MBD 1可能形成新的阻遏物复合物或染色质。为了抑制体内基因表达，我们已经开发了一些新设计的分子，其中含有某些转录因子的DNA结合结构域和MBD 1或MeCP 2的TRD。由于MBD突变体可以逆转甲基化依赖的基因沉默，这些分子似乎对重新激活被抑制的基因有用。基于我们的研究结果，我们提供了一个甲基化依赖的方式基因沉默的机制基础和一个新的工具来控制基因表达。

DNA methylation contributes to genome regulation including control of gene transcription and chromatin structure. Normally, methylation occurs at position 5 of cytosine within CpG dinudeotides, which implicates the formation of transcriptionally inactive chromatin. In the nucleus, not only is the DNA methylated, but the methylated DNA must also be interpreted by methyl-CpG binding domain proteins (MBD proteins). There are at least five mammalian MBD proteins: MeCP2, MBD1, MBD2, MBD3, and MBD4 (also known as MED1).Recently, we have presented evidence that MBD1 acts as a transcriptional regulator through the cooperation of MBD, cysteine-rich CXXC domains, and a C-terminal transcriptional repression domain (TRD). Further, we have determined the structure of the MBD from MBD1 in complex with methylated DNA by multi-dimensional heteronudear NMR spectroscopy. Mutant-types MBD in which the functionally important residues Arg22, Arg30, Asp32, Tyr34, Arg44, Ser45 and Tyr52 were changed to alanine lost the ability to bind methylated DNA. Although MeCP2, MBD2, and MBD3 are embedded in the histone deacetylase complexes, MBD1 has not been found in known histone deacetylase complexes, suggesting that MBD1 may form a novel represser complex or chromatin. To inhibit gene expression in vivo, we have developed some newly designed molecules containing a DNA binding domain of certain transcription factor and the TRD of MBD1 or MeCP2. Because the MBD mutants can reverse methylation-dependet gene silencing, these molecules seem to be useful for reactivating the repressed genes. Based on our findings we provide a mechanistic basis for gene silencing in methylation-dependent manners and a new tool to control gene expression.