Elsevier

Genomics

Volume 84, Issue 6, December 2004, Pages 952-960
Genomics

Timing of establishment of paternal methylation imprints in the mouse

https://doi.org/10.1016/j.ygeno.2004.08.012Get rights and content

Abstract

Imprinted genes are characterized by predominant expression from one parental allele and differential DNA methylation. Few imprinted genes have been found to acquire a methylation mark in the male germ line, however, and only one of these, H19, has been studied in detail. We examined methylation of the Rasgrf1 and Gtl2 differentially methylated regions (DMR) to determine whether methylation is erased in male germ cells at e12.5 and when the paternal allele acquires methylation. We also compared their methylation dynamics with those of H19 and the maternally methylated gene Snrpn. Our results show that methylation is erased on Rasgrf1, H19, and Snrpn at e12.5, but that Gtl2 retains substantial methylation at this stage. Erasure of methylation marks on Gtl2 appears to occur later in female germ cells to give the unmethylated profile seen in mature MII oocytes. In the male germ line, de novo methylation of Rasgrf1, Gtl2, and H19 occurs in parallel between e12.5 and e17.5, but the DMR are not completely methylated until the mature sperm stage, suggesting a methylation dynamic different from that of IAP, L1, and minor satellite sequences, which have been shown to become fully methylated by e17.5 in male germ cells. This study also indicates important differences between different imprinted DMR in timing and extent of methylation in the germ cells.

Section snippets

Methylation of H19 and Snrpn in the male germ line

We isolated germ cell populations of greater than 90% purity from e10.5 [21], e12.5, and e17.5 gonads [20] or from cauda epididymis [22] as previously described. DNA was prepared as described under Materials and methods and samples were treated with bisulfite before PCR was carried out on the target genes. The structure of the DMR and their locations relative to the transcriptional start sites of the genes are depicted in Fig. 1. The CpG’s assayed in each DMR are indicated below the cognate DMR

Discussion

The Rasgrf1 DMR is located 30 kb upstream of the body of the gene, immediately 5′ to a cluster of unique 40-bp repeats [8]. In contrast to H19, methylation of the Rasgrf1 DMR is required for expression of the paternal allele rather than its repression. Deletion of the adjacent repeats leads to a variable loss of DMR methylation and silencing of the gene to different extents [12]. These results suggested that the DMR and associated repeats may form part of the germ-line imprint for this gene [12]

Mice

Hsd:ICR(CD-1), C57BL/6, and CBA mice were purchased from Harlan UK Ltd. (Oxon, England). Natural matings were used to produce embryos for the isolation of PGCs and prospermatogonia. The day we observed a plug was taken as e0.5.

Isolation of germ cell DNA

For germ cells from embryonic stages, embryos were harvested at e10.5, e12.5, and e17.5. Germ cells from e10.5 embryos were harvested using the MiniMacs system and TG-1 antibody as described previously [21]. From e12.5 onward, the male gonads can be distinguished by the

Acknowledgments

We thank J. Trasler for advice on harvesting oocyte DNA and R. Black and F. Liu for technical support. This work was supported by grants from NSF China and the Max-Planck Society to G.L.X. and grants from the BBSRC (G12997), the Northern Ireland HPSS R&D Office (RRG 6.7), and the Royal Society (RSRG 20735) to C.P.W.

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