Affinity-based enrichment strategies to assay methyl-CpG binding activity and DNA methylation in early Xenopus embryos.
Affinity-based enrichment strategies to assay methyl-CpG binding activity and DNA methylation in early Xenopus embryos.
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DOI：
10.1186/1756-0500-4-300
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发表时间：
2011-08-18
影响因子：
1.8
通讯作者：
Veenstra GJ
中科院分区：
文献类型：
--
作者：
Bogdanović O;Veenstra GJ
DNA甲基化是脊椎动物基因组中广泛存在的表观遗传修饰。DNA甲基化的基因组位点可以被甲基-CpG结合域蛋白（MBD）和特异性锌指蛋白结合，其可以募集共阻遏物复合物以沉默靶位点上的转录。与甲基化DNA的结合可以通过翻译后MBD修饰来调节。实施甲基化DNA亲和沉淀法以测定蛋白质与甲基化DNA的结合。从非洲爪蟾卵母细胞提取物中沉淀内源性MeCP 2和MBD 3，并优化甲基化特异性结合的条件。对于反向实验，通过MBD亲和捕获评估早期爪蟾胚胎中的DNA甲基化。甲基化DNA亲和树脂可用于探测提取物中的MBD活性。该测定具有广泛的应用潜力，因为它可以与下游程序如蛋白质印迹、荧光HDAC测定和定量质谱联用。甲基-CpG结合蛋白的甲基化DNA亲和捕获产生高度富集甲基化DNA的组分，适合与下一代测序技术偶联。这两种富集策略可以探测早期脊椎动物卵母细胞和胚胎中甲基-CpG蛋白的相互作用。
DNA methylation is a widespread epigenetic modification in vertebrate genomes. Genomic sites of DNA methylation can be bound by methyl-CpG-binding domain proteins (MBDs) and specific zinc finger proteins, which can recruit co-repressor complexes to silence transcription on targeted loci. The binding to methylated DNA may be regulated by post-translational MBD modifications. A methylated DNA affinity precipitation method was implemented to assay binding of proteins to methylated DNA. Endogenous MeCP2 and MBD3 were precipitated from Xenopus oocyte extracts and conditions for methylation-specific binding were optimized. For a reverse experiment, DNA methylation in early Xenopus embryos was assessed by MBD affinity capture. A methylated DNA affinity resin can be applied to probe for MBD activity in extracts. This assay has a broad application potential as it can be coupled to downstream procedures such as western blotting, fluorimetric HDAC assays and quantitative mass spectrometry. Methylated DNA affinity capture by methyl-CpG binding proteins produces fractions highly enriched for methylated DNA, suitable for coupling to next generation sequencing technologies. The two enrichment strategies allow probing of methyl-CpG protein interactions in early vertebrate oocytes and embryos.