Structural Basis of MeCP2 Distribution on Non-CpG Methylated and Hydroxymethylated DNA.
Structural Basis of MeCP2 Distribution on Non-CpG Methylated and Hydroxymethylated DNA.
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DOI：
10.1016/j.jmb.2017.04.009
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发表时间：
2017-05-19
影响因子：
5.6
通讯作者：
Williams DC Jr
中科院分区：
文献类型：
--
作者：
Sperlazza MJ;Bilinovich SM;Sinanan LM;Javier FR;Williams DC Jr
Rett综合征相关甲基- cpg结合蛋白2 （MeCP2）选择性结合甲基化DNA来调节成熟神经元发育过程中的转录。与甲基-CpG结合域（MBD）家族的其他成员一样，MeCP2通过识别CpG （mCG）二核苷酸中的对称5-甲基胞嘧啶发挥作用。然而，基础水平分辨率表观遗传定位技术的进展表明，MeCP2可以结合不对称甲基化和羟甲基化的CpA （h/mCA）二核苷酸，这种选择性结合改变了发育中的哺乳动物大脑中的基因表达。与mCA和羟甲基化DNA结合的结构决定因素以前没有研究过。在这里，我们使用ITC和NMR光谱来表征MeCP2与甲基化和羟甲基化的mCG和mCA DNA的结合；检查Rett综合征相关错义突变的影响；并与相关的、进化上最古老的蛋白质MBD2进行比较。这些分析表明，MeCP2以一种链特异性和定向依赖的方式与mCA具有高亲和力。相反，MBD2对mCA没有高亲和力或甲基特异性结合。ret相关的错义突变（T158M、R106W和P101S）破坏MeCP2 MBD的稳定性，同时破坏mCG和mCA的识别。最后，高亲和力mCA位点的羟甲基化不会改变其结合特性；然而，在mCG位点上的等效胞嘧啶的半羟基化降低了亲和力和特异性。基于这些发现，我们认为MeCP2对甲基化/羟甲基化CpA二核苷酸的识别作为一种表观遗传开关，在mCG和mCA位点之间重新分配MeCP2。
The Rett syndrome-associated methyl-CpG binding protein 2 (MeCP2) selectively binds methylated DNA to regulate transcription during the development of mature neurons. Like other members of the methyl-CpG binding domain (MBD) family, MeCP2 functions through the recognition of symmetrical 5-methylcytosines in CpG (mCG) dinucleotides. Advances in base level resolution epigenetic mapping techniques have revealed, however, that MeCP2 can bind asymmetrically methylated and hydroxymethylated CpA (h/mCA) dinucleotides and this alternative binding selectivity modifies gene expression in the developing mammalian brain. The structural determinants of binding to mCA and hydroxymethylated DNA have not been previously investigated. Here, we employ ITC and NMR spectroscopy to characterize MeCP2 binding to methylated and hydroxymethylated mCG and mCA DNA; examine the effects of Rett syndrome-associated missense mutations; and make comparisons to the related and evolutionarily most ancient protein, MBD2. These analyses reveal that MeCP2 binds mCA with high affinity in a strand-specific and orientation-dependent manner. In contrast, MBD2 does not show high affinity or methyl-specific binding to mCA. The Rett-associated missense mutations (T158M, R106W, and P101S) destabilize the MeCP2 MBD and disrupt recognition of mCG and mCA equally. Finally, hydroxymethylation of a high-affinity mCA site does not alter the binding properties; whereas, hemi-hydroxylation of the equivalent cytosine in an mCG site decreases affinity and specificity. Based on these findings, we suggest MeCP2 recognition of methylated/hydroxymethylated CpA dinucleotides functions as an epigenetic switch redistributing MeCP2 among mCG and mCA loci.