Methylation analysis of the intergenic differentially methylated region of DLK1-GTL2 in human

Abstract

Imprinting is a non-Mendelian form of inheritance where epigenetic modifications control mono-allelic expression depending on the parental origin. Methylation of CpG-dinucleotides at differentially methylated regions (DMRs) is one of the best-studied mechanisms directing expression to one specific parental allele. We studied the methylation patterns of the intergenic (IG)-DMR of DLK1 and GTL2. The methylation marks of the IG-DMR were analysed in human gametes, preimplantation embryos, amniocytes and blood of babies born after intracytoplasmic sperm injection (ICSI) and blood from adults using a bisulphite sequencing technique. In oocytes, the IG-DMR was mainly unmethylated while in sperm cells a generally methylated pattern was detected. This germ-line specific methylation mark was maintained in the preimplantation embryos until the second cleavage stage. Afterwards in the preimplantation embryos, intermediate methylation patterns (26 -74% methylation) occurred, which may point to relaxation of the imprints. Intermediate patterns were also present in amniocytes, blood from ICSI babies and adults. We hypothesise that in the early cleavage stage embryo a strict differential methylation pattern is needed for the correct imprint establishment of surrounding imprinted genes. Once correct imprinting of the involved gene(s) is acquired, a more relaxed state of the IG-region is allowed.

Epigenetic profiling at mouse imprinted gene clusters reveals novel epigenetic and genetic features at differentially methylated regions

“…3; see Methods). As expected, we detected sDMRs at each known imprinted DMR (Arima et al 2006;Geuns et al 2007), which provided a positive control for the specificity of the MeDIP method in our analysis. At these regions, there was an absence of methylation at each ICR in sperm, except for the H19 ICR and Dlk1-Meg3 intergenic (IG) DMR, which displayed a higher level of enrichment for methylated DNA relative to the somatic tissue samples (Fig.…”

Aberrant DNA methylation at imprinted genes in testicular sperm retrieved from men with obstructive azoospermia and undergoing vasectomy reversal

“…Differences in DNA methylation at the IG-GTL2 and MEST DMRs were not significant between the groups studied. The H19 and IG-GTL2 DMR are paternally methylated DMRs (Kerjean et al 2000, Geuns et al 2007); however, our results suggest the H19 DMR is more prone to the loss of methylation compared with the IG-GTL2 DMR. The propensity of the H19 DMR to disturbances of DNA methylation may be related to the molecular structure of the DMR or of the surrounding sequences.…”

“…Previously published primer sequences were used for the amplification of the selected sequences (Kerjean et al 2000, Geuns et al 2007. Amplification was carried out as outlined by Kerjean et al (2000).…”

“…While most imprinted genes are methylated in the oocyte, including MEST (Lucifero et al 2002), two imprinted genes, H19 and the intragenic region (IG) of GTL2, show sperm-specific DNA methylation (Kerjean et al 2000, Geuns et al 2007. The H19/IGF2, GTL2/ DLK1 regions and MEST are important regulators of prenatal growth (DeChiara et al 1990, Kaneko-Ishino et al 1995, Georgiades et al 2000.…”

Genomic imprints as a model for the analysis of epigenetic stability during assisted reproductive technologies

“…KCNQ1OT1 LOM was also observed in 5/23 and 2/37 DNA strands from IVM MII oocyte pools (Khoueiry et al 2008. This contrasts with short-term IVM MII oocytes that had normal SNRPN (three single oocytes), KCNQ1OT1 (four single oocytes), and MEG3 (GTL2) (three pools of two oocytes) methylation (Geuns et al 2003(Geuns et al , 2007a(Geuns et al , 2007b. In the above studies, where zona pellucidae were not removed, there is the possibility of cumulus cell contamination.…”