Department of Genetics and The Edison Family Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, MO 63108-8510, USA.
Sci Adv. 2017 Nov 17;3(11):eaao1799. doi: 10.1126/sciadv.aao1799. eCollection 2017 Nov.
Methylation of CpG (cytosine-phosphate-guanine) dinucleotides is a common epigenetic mark that influences gene expression. The effects of methylation on transcription factor (TF) binding are unknown for most TFs and, even when known, such knowledge is often only qualitative. In reality, methylation sensitivity is a quantitative effect, just as changes to the DNA sequence have quantitative effects on TF binding affinity. We describe Methyl-Spec-seq, an easy-to-use method that measures the effects of CpG methylation (mCPG) on binding affinity for hundreds to thousands of variants in parallel, allowing one to quantitatively assess the effects at every position in a binding site. We demonstrate its use on several important DNA binding proteins. We calibrate the accuracy of Methyl-Spec-seq using a novel two-color competitive fluorescence anisotropy method that can accurately determine the relative affinities of two sequences in solution. We also present software that extends standard methods for representing, visualizing, and searching for matches to binding site motifs to include the effects of methylation. These tools facilitate the study of the consequences for gene regulation of epigenetic marks on DNA.
CpG（胞嘧啶-磷酸-鸟嘌呤）二核苷酸的甲基化是一种常见的表观遗传标记，影响基因表达。对于大多数转录因子（TF）而言，甲基化对转录因子结合的影响是未知的，即使已知，这种知识通常也只是定性的。实际上，甲基化敏感性是一种定量效应，就像 DNA 序列的变化对转录因子结合亲和力有定量影响一样。我们描述了 Methyl-Spec-seq，这是一种易于使用的方法，可以同时测量数百到数千个变体的 CpG 甲基化（mCPG）对结合亲和力的影响，从而可以定量评估结合位点中每个位置的影响。我们在几个重要的 DNA 结合蛋白上展示了它的用途。我们使用一种新颖的双色竞争荧光各向异性方法来校准 Methyl-Spec-seq 的准确性，该方法可以准确地确定溶液中两个序列的相对亲和力。我们还提供了软件，可以扩展表示、可视化和搜索结合位点模体匹配的标准方法，以包括甲基化的影响。这些工具促进了对 DNA 上的表观遗传标记对基因调控的后果的研究。
