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. 2012 Nov 10;41(1):182–195. doi: 10.1093/nar/gks1051

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© The Author(s) 2012. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com.

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Figure 2. — Hypermethylated CpG island associated with the Phox2a locus. (A) Visualization of the NimbleGen array data for immortalized (in red) and primary MEFs (in green). Three independently established immortalized murine cell lines are shown (Set 1, Set 2 and Set 3). At this level of resolution, each peak corresponds to a CpG island. The methylation signal, obtained by the NimbleScan software, is plotted along the chromosome as a P-value on the y-axis (starting from 0 when P-value is 1). The P-value is derived from the Kolmogorov–Smirnov test comparing the log2 enrichment ratios between MIRA and input within a 750 bp window. (B) Snapshot of the UCSC Genome Browser indicating the location of the Phox2a gene. CpG islands are shown as green boxes. The locations of the H3K4me3, CTCF and Pol2-binding sites, are shown for low-passage MEFs, based on ENCODE/LICR published databases (http://genome.ucsc.edu/ENCODE/downloadsMouse.html). H3K4me3 and Pol2 signals are strong indicators of active promoters, whereas CTCF-binding sites are considered as a mark for potential insulator elements. (C) Genomic DNA from primary (1bc, 2bc, 3bc), senescent (2c, 3c) and immortalized (1ac, 2ac, 3ac) MEFs was treated with sodium bisulfite, and the CpG island within the Phox2a gene was amplified with gene-specific primers, and subjected to COBRA. Digested fragments on the gel are indicative of methylated BstUI restriction sites (5′-CGCG) within the CpG island. Mouse genomic DNA was methylated in vitro with the SssI methyltransferase, and served as positive control (Pos). (+) and (−) represent the presence and absence, respectively, of the BstUI restriction enzyme in the reaction mix. M = 100 bp ladder DNA marker. Given the limited amount of DNA available from sample 1c, bisulfite sequencing was performed only. (D) The extent of CpG methylation within the Phox2a CpG island was determined by sodium bisulfite sequencing in primary (1bc, 2bc, 3bc; left panel), senescent (1c, 2c, 3c; middle panel) and immortalized (1ac, 2ac, 3ac; right panel) MEFs. The sequencing results of up to 10 independent clones, and the respective percentage of methylation per sample are shown. Data were analysed using the web-based software, QUantification tool for Microarray Analysis (QUMA) (http://quma.cdb.riken.jp/). Open and closed circles represent unmethylated and mCG dinucleotides, respectively.