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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.
Figure 1. Methylated DNA affinity precipitation. (A) A methylated, concatamerized oligo is incubated with oocyte extract to probe for proteins with methyl-CpG binding activity. After performing the necessary washing steps, the eluate contains proteins specifically binding methylated DNA. (B) The concatamerization of the oligo is monitored by agarose gel electrophoresis. The majority of concatamerized DNA consists of fragments ranging in size from 100 - 300 bp. (C) The extent of oligo methylation is measured by restriction digestion. MspI enzyme cuts both methylated and unmethylated DNA whereas its isoschizomer HpaII cuts only unmethylated DNA. (D) The binding of the biotinylated oligo to streptavidin beads is measured by agarose electrophoresis. An aliquot of the probe before incubation with streptavidin beads (before - B) is compared to a probe aliquot after 30 minutes of incubation time (after - A).
Figure 2. DNA affinity precipitation of recombinant and endogenous oocyte extracts proteins. (A) In vitro translated, S35 labeled MBD domain of MeCP2 specifically binds the methylated immobilized probe (SssI+). Conversely, the negative control (TBP2) displays only residual affinity for non-methylated DNA (SssI-). (B) Quantification of DNA methylation-specific binding using the Image-quant software. ~35% of the recombinant MBD domain is recovered after washing and elution from methylated DNA oligos. (C) Western blotting of eluates obtained after 100 mM, 200 mM and 400 mM KCl washes. MeCP2 tolerates 100 mM and 200 mM KCl concentration in the washing buffer whereas most of the MBD3 is eluted with 200 mM salt. (D) DNA affinity precipitation of MBD3, MeCP2 and Mi-2 from oocyte extracts. MeCP2 displays strong preference for methylated DNA, whereas Mi-2 and MBD3 bind both methylated and unmethylated DNA. The short form of MBD3, however, displays a preference for methylated DNA. (E) Establishment of positive (ISWI) and negative (TFIIB) controls for DNA affinity precipitation.
Figure 3. Genomic DNA isolation and sonication. (A) Xenopus tropicalis genomic DNA isolated from blastula (bl) and gastrula (ga) embryos. (B) Genomic DNA analyzed after 5 minutes or 15 minutes of sonication. Genomic DNA sonicated for 15 minutes yields fragments of 100 - 300 bp. (C) A schematic representation of methylated DNA affinity capture (MethylCap). Methylated DNA is bound by the immobilized methyl-CpG binding domain of MeCP2, washed and eluted with increasing NaCl concentrations. (D) PCR quantitation of methylated DNA recovery. DNA methylation targets (tcea, tfcp2 and gs17) mostly elute at high NaCl concentrations (500 mM, 600 mM and 700 mM) whereas the non-methylated targets (Xbra, trim33, hes4) are washed out with the first washing step (200 mM). (E) The methylation status of genomic targets tcea, tfcp2, gs17, trim33 and hes4 was validated by MeDIP (methylated DNA immunoprecipitation). The error bars represent standard deviation (SD) of two experiments.
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