Methyl-CpG/MBD2 Interaction Requires Minimum Separation and Exhibits Minimal Sequence Specificity.
Methyl-CpG/MBD2 Interaction Requires Minimum Separation and Exhibits Minimal Sequence Specificity.
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DOI：
10.1016/j.bpj.2016.11.014
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发表时间：
2016-12
影响因子：
3.4
通讯作者：
Blythe Moreland;Kenji M. Oman;John Curfman;P. Yan;R. Bundschuh
中科院分区：
文献类型：
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作者：
Blythe Moreland;Kenji M. Oman;John Curfman;P. Yan;R. Bundschuh
确定细胞中CpG二核苷酸的甲基化模式仍然是表观遗传分析的重要组成部分。甲基化、基因表达和伴随疾病之间的相关性才刚刚开始探索。许多用于检测甲基化的实验使用相对便宜的高通量方法，其具有优先结合甲基化CpG的甲基结合结构域（MBD）蛋白。在这里，我们的特点MBD 2-DNA结合的协同性和序列特异性在下拉实验揭示了三个潜在的偏见，在这样的实验。第一种是由两个MBD 2蛋白在mCpG之间的空间冲突引起的，mCpG之间相隔2 bp或更少，这表明在这些位点的同时结合受到抑制。通过比较M. SssI处理的样品上的输入与下拉高通量测序数据证实了这一点，从中我们还发现，对于具有四个或更多mCpG的DNA片段，下拉效率急剧增加。再次采用这两个数据集的分析来研究MBD 2围绕甲基化CpG（mCpG）的序列偏好。在比较mCpG位置上的碱基分布时，发现对某些碱基的统计学显着偏好，尽管下拉效率的相应偏差均<5%。虽然这表明mCpG序列背景在MBD 2结合中大多可以被忽略，但我们的高通量方法所表现出的统计确定性预示着未来的应用。
Determining the pattern of methylation at CpG dinucleotides in a cell remains an essential component of epigenetic profiling. The correlations among methylation, gene expression, and accompanying disease have just begun to be explored. Many experiments for sensing methylation use a relatively inexpensive, high-throughput approach with a methyl-binding domain (MBD) protein that preferentially binds to methylated CpGs. Here, we characterize the cooperativity and sequence specificity of MBD2-DNA binding in a pulldown experiment revealing three potential biases in such experiments. The first is caused by steric clashes between two MBD2 proteins at mCpGs separated by 2 bp or less, which suggests that simultaneous binding at these sites is inhibited. This is confirmed by comparing input versus pulldown high-throughput sequencing data on M.SssI-treated samples, from which we also find that pulldown efficiency sharply increases for DNA fragments with four or more mCpGs. Analysis of these two data sets was again employed to investigate MBD2's sequence preferences surrounding a methylated CpG (mCpG). In comparing the distributions of bases at positions with respect to an mCpG, statistically significant preferences for certain bases were found, although the corresponding biases in pulldown efficiency were all <5%. While this suggests that mCpG sequence context can mostly be ignored in MBD2 binding, the statistical certainty exhibited by our high-throughput approach bodes well for future applications.