Probing the Dynamic Distribution of Bound States for Methylcytosine-binding Domains on DNA
Probing the Dynamic Distribution of Bound States for Methylcytosine-binding Domains on DNA
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DOI：
10.1074/jbc.m113.512236
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发表时间：
2014-01-17
影响因子：
4.8
通讯作者：
Williams, David C., Jr.
中科院分区：
文献类型：
--
作者：
Cramer, Jason M.;Scarsdale, J. Neel;Williams, David C., Jr.
背景：虽然MBD 3与MBD 2高度同源，但其功能仍存在疑问。结果：MBD 3优先定位于甲基化的CpG二核苷酸，在较小程度上，未甲基化的CpG二核苷酸。结论：MBD 3基因甲基化位点和非甲基化位点的动态分布改变了MBD 3的基因组定位。重要性：MBD蛋白质在DNA上的动态分布的变化决定了MBD蛋白质之间的功能差异，尽管MBD 3与其他甲基胞嘧啶结合结构域（MBD）蛋白质高度同源，但MBD 3不选择性地结合甲基化的DNA，因此MBD 3的功能作用仍然存在疑问。为了探索其结合特性和潜在功能的结构基础，我们表征了MBD 3 MBD在羟甲基化、甲基化和未甲基化DNA上的溶液结构和结合分布。该结构域的整体折叠与其他MBD非常相似，但参与DNA结合的关键环比以前观察到的更加无序。甲基化DNA的特异性识别限制了该环的结构，并导致NMR光谱中的大化学位移变化。基于这些光谱的变化，我们表明，MBD 3优先定位于甲基化，并在较小程度上，未甲基化的胞嘧啶-鸟苷二核苷酸（CpG），但不区分羟甲基化和未甲基化的网站。测量不同结合态的残余偶极耦合清楚地表明，MBD 3结构在甲基化特异性和非特异性结合模式之间不发生变化。此外，测量的MBD 3结合到甲基化DNA的残余偶极耦合可以通过甲基化和非特异性结合模式的线性组合来描述，确认优先定位到甲基化位点。高度同源的MBD 2蛋白显示类似但更强的定位于甲基化以及未甲基化的CpG。总之，这些数据建立了MBD 2和MBD 3在基因组DNA上的相对分布以及它们在活性和非活性富含CpG的启动子处观察到的占据的结构基础。
Background: Although highly homologous to MBD2, the functional role of MBD3 remains in question. Results: MBD3 preferentially localizes to methylated and, to a lesser degree, unmethylated CpG dinucleotides. Conclusion: Dynamic distribution between methylated and unmethylated sites modifies the genomic localization of MBD3. Significance: Changes in the dynamic distribution on DNA dictate functional differences between MBD proteins.Although highly homologous to other methylcytosine-binding domain (MBD) proteins, MBD3 does not selectively bind methylated DNA, and thus the functional role of MBD3 remains in question. To explore the structural basis of its binding properties and potential function, we characterized the solution structure and binding distribution of the MBD3 MBD on hydroxymethylated, methylated, and unmethylated DNA. The overall fold of this domain is very similar to other MBDs, yet a key loop involved in DNA binding is more disordered than previously observed. Specific recognition of methylated DNA constrains the structure of this loop and results in large chemical shift changes in NMR spectra. Based on these spectral changes, we show that MBD3 preferentially localizes to methylated and, to a lesser degree, unmethylated cytosine-guanosine dinucleotides (CpGs), yet does not distinguish between hydroxymethylated and unmethylated sites. Measuring residual dipolar couplings for the different bound states clearly shows that the MBD3 structure does not change between methylation-specific and nonspecific binding modes. Furthermore, residual dipolar couplings measured for MBD3 bound to methylated DNA can be described by a linear combination of those for the methylation and nonspecific binding modes, confirming the preferential localization to methylated sites. The highly homologous MBD2 protein shows similar but much stronger localization to methylated as well as unmethylated CpGs. Together, these data establish the structural basis for the relative distribution of MBD2 and MBD3 on genomic DNA and their observed occupancy at active and inactive CpG-rich promoters.