原创力文档affinity-based enrichment strategies to assay methyl-cpg binding activity and dna methylation in early xenopus embryosaffinity-based浓缩策略分析methyl-cpg绑定活动和dna甲基化在非洲爪蟾蜍早期胚胎.pdfVIP
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affinity-based enrichment strategies to assay methyl-cpg binding activity and dna methylation in early xenopus embryosaffinity-based浓缩策略分析methyl-cpg绑定活动和dna甲基化在非洲爪蟾蜍早期胚胎
Bogdanović and Veenstra BMC Research Notes 2011, 4:300
/1756-0500/4/300
TECHNICAL NOTE Open Access
Affinity-based enrichment strategies to assay
methyl-CpG binding activity and DNA
methylation in early Xenopus embryos
*
Ozren Bogdanović and Gert Jan C Veenstra
Abstract
Background: 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.
Findings: 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.
Conclusions: 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.
Background [8]. In addition, several structurally unrelated methyl-
DNA methylation is an epigenetic modification mostly
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