Active tissue-specific DNA demethylation conferred by somatic cell nuclei in stable heterokaryons -- Zhang et al. 104 (11): 4395 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Zhang et al. 10.1073/pnas.0700181104.

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SI Figure 5
SI Figure 6
SI Figure 7

Fig. 5. Schematic representation of combined bisulfite restriction analysis (COBRA) of DNA methylation. DNA was treated with bisulfite, which converts all cytosines to uracils, except cytosines protected by methylation (i.e., A^mCGT). The bisulfite-specific PCR amplification was carried out to amplify only the bisulfite-converted DNA samples. The primers do not contain CpG dinucleotides so that the amplification step will not discriminate templates with different methylation states. PCR products were digested with the restriction enzyme HpyCH4IV, which recognizes and cuts at ACGT. Cleavage will occur only if the CpG sequence is retained during the bisulfite conversion, i.e., if that cytosine is methylated. The unmethylated ACGT, which is converted to ATGT after PCR amplification, will not be cleaved by HpyCH4IV. Digested PCR products were separated on an agarose gel. The band pattern and intensity are analyzed by image software. The relative amounts of methylated and unmethylated DNA can be assessed by comparing the intensity of the cleaved with the uncleaved bands.

Fig. 6. Optimization of the amount of human DNA in heterokaryons for COBRA methylation analysis. (A) The standard curve used to calculate the amount of human DNA in heterokayons by quantitative PCR. Various amounts of DNA from keratinocytes (0.04, 0.2, 0.8, 2, 4, 8, 10, and 15 ng) were mixed with mouse DNA such that total DNA is 20 ng. Quantitative PCR was carried out using human-specific GAPDH primers. The correlation coefficient (R) of our standard curve is 0.99850, indicating a good fit. The table below shows various given concentrations of human DNA (ng/reaction), calculated concentrations of human DNA (ng/reaction) based on the standard curve, and the percentage of variations between these two values, indicating the level of accuracy of this method. (B) The table shows the DNA samples, including the PrePEG treatment (Pre), and heterokaryons cultured for 1, 3, 5, and 7 days (1D, 3D, 5D, and 7D), calculated concentrations of human DNA (ng/reaction) based on the standard curve, the percentages of human DNA in heterokaryons (all PCR reactions contained 20 ng of heterokaryon DNA as template), the concentration of heterokaryon DNA samples (measured by NanoDrop). The last column shows the amounts of heterokaryon DNA required to get 40 ng of human DNA, which were used in the methylation analysis. The final amount of DNA for bisulfite treatment was adjusted to 1.5 mg for each sample by adding mouse DNA to minimize the variation caused by difference in starting DNA. A wide range of ratios of human DNA to mouse DNA, were tested for the accuracy and reproducibility of the methylation assay. Twenty-five to 100 ng of human DNA gave consistent results. Therefore, 40 ng of human DNA was used in all of our COBRA methylation analysis for heterokaryons.

Fig. 7. Quantification of methylation analysis from three independent experiments. Three genes were analyzed: the MyoD core enhancer, the keratin-14 promoter, and the b-globin HS2 locus. Samples analyzed were from keratinocytes and heterokaryons during nuclear reprogramming: PrePEG, and heterokaryon DNA at days 1, 3, 5, and 7.