Bisulfite sequencing in preimplantation embryos: DNA methylation profile of the upstream region of the mouse imprinted H19 gene.
In this study we describe a modification of the bisulfite genomic sequencing protocol that enables detection of methylation from as few as five diploid cells from preimplantation mouse embryos. We have used bisulfite genomic sequencing to study the methylation profile of the putative imprinting element upstream of the mouse H19 gene at several stages of embryonic development, including fertilized oocytes and two-cell embryos. The methylation of the H19 imprinting element has recently been described extensively for midgestation embryos, but remains poorly characterized for the preimplantation stages of development, despite widespread changes in genomic DNA methylation occurring at this time. We studied the methylation profile of 35 CpG sites spanning two regions within the H19 imprinting element and found that an overall pattern of allele-specific methylation was maintained at all developmental stages examined, including fertilized oocytes and two-cell embryos. However, allele-specific methylation was not maintained in an absolute fashion subsequent to the first cell division, with a clear flux between partial de novo methylation of the maternal allele and partial demethylation of the paternal allele. Our findings highlight the dynamics of methylation in the early embryo and suggest that it is the overall level of methylation that is responsible for maintenance of the imprinting element and not the methylation of individual CpG sites.
Kanematsu Laboratories, Royal Prince Alfred Hospital, Camperdown, NSW, Australia.
P M
PM
Warnecke
J R
JR
Mann
M
M
Frommer
S J
SJ
Clark
D000483:Alleles; D000818:Animals; D001483:Base Sequence; D001755:Blastocyst; D003596:Cytosine / Q000737:chemistry; D019175:DNA Methylation; D018392:Genomic Imprinting / Q000235:genetics*; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites
C094335:H19 long non-coding RNA; D009124:Muscle Proteins; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D013447:Sulfites; C005200:sodium metabisulfite; D003596:Cytosine