Figure 5.

The Rhox and other LSH-dependent loci are hypomethylated in Ehmt2^−/− ES cells. (A) Western blots show G9a, LSH, and HDAC1 in nuclear extracts from wild-type and mutant ES cells and MEFs. (B) Western blots for H3K9me1, H3K9me2, and H3K9me3 in wild-type and mutant ES cells and MEFs. “Ehmt2^−/− Tg” indicates Ehmt2^−/− (G9a-null) cells expressing catalytically inactive Ehmt2 (G9a) transgene. (C) ELISA assays with anti-5meC antibody on genomic DNA purified from wild-type and mutant ES cells and MEFs. Note than Hells^−/− MEFs and Ehmt2^−/− ES cells have comparable amounts of 5-meC. Catalytically inactive Ehmt2 transgene partially rescues DNA methylation defects in Ehmt2^−/− ES cells. (D,E) Methylation-sensitive PCR (D) and bisulfite sequencing (E) detect loss of DNA methylation at Rhox and other LSH-dependent promoters in Ehmt2^−/− ES cells. DNA methylation is partly restored in Ehmt2^−/− cells expressing catalytically inactive Ehmt2 transgene (Tg). MspI and HpaII digested genomic DNA is indicated by “M” and “H,” respectively. “Mr” is DNA size marker. In F, methylated CpGs are shown as black circles. The percentage of methylated CpGs relative to the total number of CpGs investigated by bisulfite sequencing for any given sequence is shown in parentheses. (F) A Venn diagram showing the overlap between hypomethylated (relative to ES cells and MEFs, group I) promoters in Hells^−/− MEFs and hypomethylated loci in Ehmt2^−/− ES cells.