Figure 3

The G9a non-cage histone H3 binding surface of the ankyrin repeat (ANK) domain is required for de novo DNA methylation of the Oct3/4 gene promoter during retinoic acid (RA)-induced embryonic stem cell (ESC) differentiation. DNA extracted from the indicated mouse embryonic stem cell (mESC) lines, grown under self-renewal conditions or treated with RA for 8 days to induce differentiation, was treated with sodium bisulfite, amplified using specific Oct3/4 promoter primers, cloned and subjected to sequence analysis. (A) Schematic diagram of CpG positions within the Oct3/4 gene promoter is shown. The methylation status of eight CpG sites (no. 1 to 8) within the promoter (-90 to -258 relative to the transcription start site) was examined. (B) Percentage of DNA methylation for the Oct3/4 promoter region encompassing eight CpG sites of each clonal cell line is indicated in the table, which summarizes data shown in (C) and (D). DNA extracted from the indicated mESC lines that were untreated (C) or treated with RA for 8 days (D) was treated with sodium bisulfite, amplified using specific Oct3/4 promoter primers, cloned and subjected to sequence analysis. Each horizontal row represents results from sequencing a separate DNA clone. The overall percentage of methylated CpG sites is indicated in parentheses beside the clone designation. The number of independent biological replicate experiments (n) from which the total number of DNA clones (c) were derived is also indicated.