Sperlazza M Jeannette, Bilinovich Stephanie M, Sinanan Leander M, Javier Fatima R, Williams David C
Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.
Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.
J Mol Biol. 2017 May 19;429(10):1581-1594. doi: 10.1016/j.jmb.2017.04.009. Epub 2017 Apr 24.
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 dinucleotides and that this alternative binding selectivity modifies gene expression in the developing mammalian brain. The structural determinants of binding to methylated CpA (mCA) and hydroxymethylated DNA have not been previously investigated. Here, we employ isothermal titration calorimetry 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 the 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 that MeCP2 recognition of methylated/hydroxymethylated CpA dinucleotides functions as an epigenetic switch redistributing MeCP2 among mCG and mCA loci.
与瑞特综合征相关的甲基化CpG结合蛋白2（MeCP2）在成熟神经元发育过程中选择性结合甲基化DNA以调控转录。与甲基化CpG结合结构域（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，碱基水平分辨率表观遗传图谱技术的进展表明，MeCP2可以结合不对称甲基化和羟甲基化的CpA二核苷酸，并且这种替代的结合选择性会改变发育中的哺乳动物大脑中的基因表达。此前尚未研究过与甲基化CpA（mCA）和羟甲基化DNA结合的结构决定因素。在这里，我们采用等温滴定量热法和核磁共振光谱法来表征MeCP2与甲基化和羟甲基化的mCG和mCA DNA的结合，研究与瑞特综合征相关的错义突变的影响，并与相关且进化上最古老的蛋白质MBD2进行比较。这些分析表明，MeCP2以链特异性和方向依赖性方式高亲和力结合mCA。相比之下，MBD2对mCA没有显示出高亲和力或甲基特异性结合。与瑞特相关的错义突变（T158M、R106W和P101S）使MeCP2 MBD不稳定，并同样破坏对mCG和mCA的识别。最后，高亲和力mCA位点的羟甲基化不会改变结合特性，而mCG位点中同等胞嘧啶的半羟甲基化会降低亲和力和特异性。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。
