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In mammals, several genes influenced by the phenomenon of genomic imprinting are critical during development. Recently, Kono et al. (2004 Nature 428, 860?864) reported the production of intact female mouse individuals that had only two haploid sets of maternal genome. They obtained these mice by combining a normal haploid maternal genome and a mutant haploid maternal genome with a 13 k base deletion in the H19 gene and its 5?-upstream region. This genomic combination resulted in the appropriate expression of the Igf2, H19, and other imprinted genes. In the mouse genome, there are four CTCF-binding sites in the 5?-upstream region of the H19 gene. The binding of CTCF to these binding sites regulates the expression of the Igf2 and H19 genes. The binding of CTCF to its binding sites is regulated by methylation of CpG sites in binding sites. In this study, as the first step to elucidate the role of the paternal genomic imprinting during development, we investigated the methylation of CpG sites in the 5?-upstream region of the H19 gene in mouse somatic cells, gametes, and two types of ES cells. Genomic DNA was isolated from BDF1 (C57BL/6N _ DBA/2N) mouse's tail (male and female somatic tissue, mST and fST, respectively), spermatozoa (S), oocytes (O), and wild type and androgenetic embryonic stem cells (wtES and agES, respectively). The methylation of CpG sites was evaluated by using the bisulfite sequencing assay. There were 13 CpG sites and a CTCF-binding site in the region from ?4413 to ?3976 in the H19 gene relative to the transcription start site. The percentages of CpG sites in this region that were methylated were 88% (160/182), 79% (27/130), 93% (230/247), 8% (10/130), 77% (10/13) and 89% (314/351) for mST, fST, S, O, wtES, and agES, respectively. In the CTCF-binding site core motif (CCGCGTGGTGGCAG), the percentages of methylated CpG sites were 93% (26/28), 80% (16/20), 95% (36/38), 0% (0/20), 50% (1/2) and 96% (52/54) for mST, fST, S, O, wtES, and agES, respectively. The CpG sites in the sequence of agES were highly methylated similar to the finding in spermatozoa. However, an aberrant methylation pattern was observed in some clones of agES. From these results, it was concluded that the methylation of CpG sites in the genomic sequence of agES was well conserved and, therefore, agES is useful to elucidate the role of the paternal genomic imprinting during development. This work was supported by Wakayama Prefecture Collaboration of Regional Entities for the Advanced of Technological Excellence, Japan, and by a Grant-in-Aid for the 21st Century COE Program of the Japan MEXT. |
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