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Updated: May 30, 2026
Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos
Published on: August 18, 2022
Affinity-based enrichment strategies to assay methyl-CpG binding activity and DNA methylation in early Xenopus
Ozren Bogdanović1, Gert Jan C Veenstra
1Department of Molecular Biology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen, Faculty of Science, (Geert Grooteplein 28), Nijmegen, (6525 GA), The Netherlands. g.veenstra@ncmls.ru.nl.
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.
