Department of Biology, Technische Universität Darmstadt, Darmstadt, Germany.
PLoS One. 2013;8(1):e53730. doi: 10.1371/journal.pone.0053730. Epub 2013 Jan 15.
Epigenetic marks like methylation of cytosines at CpG dinucleotides are essential for mammalian development and play a major role in the regulation of gene expression and chromatin architecture. The methyl-cytosine binding domain (MBD) protein family recognizes and translates this methylation mark. We have recently shown that the level of MeCP2 and MBD2, two members of the MBD family, increased during differentiation and their ectopic expression induced heterochromatin clustering in vivo. As oligomerization of these MBD proteins could constitute a factor contributing to the chromatin clustering effect, we addressed potential associations among the MBD family performing a series of different interaction assays in vitro as well as in vivo. Using recombinant purified MBDs we found that MeCP2 and MBD2 showed the stronger self and cross association as compared to the other family members. Besides demonstrating that these homo- and hetero-interactions occur in the absence of DNA, we could confirm them in mammalian cells using co-immunoprecipitation analysis. Employing a modified form of the fluorescent two-hybrid protein-protein interaction assay, we could clearly visualize these associations in single cells in vivo. Deletion analysis indicated that the region of MeCP2 comprising amino acids 163-309 as well the first 152 amino acids of MBD2 are the domains responsible for MeCP2 and MBD2 associations. Our results strengthen the possibility that MeCP2 and MBD2 direct interactions could crosslink chromatin fibers and therefore give novel insight into the molecular mechanism of MBD mediated global heterochromatin architecture.
表观遗传标记，如 CpG 二核苷酸胞嘧啶的甲基化，对哺乳动物的发育至关重要，并在基因表达和染色质结构的调控中起主要作用。甲基化胞嘧啶结合域（MBD）蛋白家族识别并翻译这种甲基化标记。我们最近表明，MBD 家族的两个成员 MeCP2 和 MBD2 的水平在分化过程中增加，并且它们的异位表达诱导体内异染色质聚集。由于这些 MBD 蛋白的寡聚化可能是导致染色质聚集效应的因素之一，我们通过体外和体内进行一系列不同的相互作用测定来解决 MBD 家族成员之间的潜在关联。使用重组纯化的 MBD，我们发现 MeCP2 和 MBD2 与其家族成员相比具有更强的自身和交叉关联。除了证明这些同源和异源相互作用在没有 DNA 的情况下发生外，我们还可以使用共免疫沉淀分析在哺乳动物细胞中证实它们。通过使用荧光双杂交蛋白-蛋白相互作用测定的改良形式，我们可以在体内单个细胞中清楚地观察到这些关联。缺失分析表明，包含氨基酸 163-309 的 MeCP2 区域以及 MBD2 的前 152 个氨基酸是负责 MeCP2 和 MBD2 关联的结构域。我们的结果加强了 MeCP2 和 MBD2 直接相互作用可能交联染色质纤维的可能性，从而为 MBD 介导的全局异染色质结构的分子机制提供了新的见解。
