Establishment Of Genomic Imprints Research Articles

Role of transcription in imprint establishment in the male and female germ lines.

The authors highlight an area of research that focuses on the establishment of genomic imprints: how the female and male germlines set up opposite instructions for imprinted genes in the maternally and paternally inherited chromosomes. Mouse genetics studies have solidified the role of transcription across the germline differentially methylated regions in the establishment of maternal genomic imprinting. One work now reveals that such transcription is also important in paternal imprinting establishment. This allows the authors to propose a unifying mechanism, in the form of transcription across germline differentially methylated regions, that specifies DNA methylation imprint establishment. Differences in the timing, genomic location and nature of such transcription events in the male versus female germlines in turn explain the difference between paternal and maternal imprints.

The pivotal roles of the NOD-like receptors with a PYD domain, NLRPs, in oocytes and early embryo development†.

Nucleotide-binding oligomerization domain (NOD)-like receptors with a pyrin domain (PYD), NLRPs, are pattern recognition receptors, well recognized for their important roles in innate immunity and apoptosis. However, several NLRPs have received attention for their new, specialized roles as maternally contributed genes important in reproduction and embryo development. Several NLRPs have been shown to be specifically expressed in oocytes and preimplantation embryos. Interestingly, and in line with divergent functions, NLRP genes reveal a complex evolutionary divergence. The most pronounced difference is the human-specific NLRP7 gene, not identified in rodents. However, mouse models have been extensively used to study maternally contributed NLRPs. The NLRP2 and NLRP5 proteins are components of the subcortical maternal complex (SCMC), which was recently identified as essential for mouse preimplantation development. The SCMC integrates multiple proteins, including KHDC3L, NLRP5, TLE6, OOEP, NLRP2, and PADI6. The NLRP5 (also known as MATER) has been extensively studied. In humans, inactivating variants in specific NLRP genes in the mother are associated with distinct phenotypes in the offspring, such as biparental hydatidiform moles (BiHMs) and preterm birth. Maternal-effect recessive mutations in KHDC3L and NLRP5 (and NLRP7) are associated with reduced reproductive outcomes, BiHM, and broad multilocus imprinting perturbations. The precise mechanisms of NLRPs are unknown, but research strongly indicates their pivotal roles in the establishment of genomic imprints and post-zygotic methylation maintenance, among other processes. Challenges for the future include translations of findings from the mouse model into human contexts and implementation in therapies and clinical fertility management.

NLRPs, the subcortical maternal complex and genomic imprinting

Before activation of the embryonic genome, the oocyte provides many of the RNAs and proteins required for the epigenetic reprogramming and the transition to a totipotent state. Targeted disruption of a subset of oocyte-derived transcripts in mice results in early embryonic lethality and cleavage-stage embryonic arrest as highlighted by the members of the subcortical maternal complex (SCMC). Maternal-effect recessive mutations of NLRP7, KHDC3L and NLRP5 in humans are associated with variable reproductive outcomes, biparental hydatidiform moles (BiHM) and widespread multi-locus imprinting disturbances. The precise mechanism of action of these genes is unknown, but the maternal-effect phenomenon suggests a function during early pre-implantation development, while biochemical and genetic studies implement them as SCMC members or interacting partners. In this review article, we discuss the role of the NLRP family members and the SCMC proteins in the establishment of genomic imprints and post-zygotic methylation maintenance, the recent advances made in the understanding of the biology involved in BiHM formation and the wider roles of the SCMC in mammalian reproduction.

Establishment of paternal genomic imprinting in mouse prospermatogonia analyzed by nuclear transfer.

In mice, the establishment of paternal genomic imprinting in male germ cells starts at midgestation, as suggested by DNA methylation analyses of differentially methylated regions (DMRs). However, this information is based on averages from mixed populations of germ cells, and the DNA methylation pattern might not always provide a full representation of imprinting status. To obtain more detailed information on the establishment of paternal imprinting, single prospermatogonia at Embryonic Days 15.5 (E15.5), E16.5, and E17.5 and at Day 0.5 after birth were cloned using nuclear transfer; previous reports suggested that cloned embryos reflected the donor's genomic imprinting status. Then, the resultant fetuses (E9.5) were analyzed for the DNA methylation pattern of three paternal DMRs (IG-DMR, H19 DMR, and Rasgrf1 DMR) and the expression pattern of imprinted genes therein. The overall data indicated that establishment of genomic imprinting in all paternally imprinted regions was completed by E17.5, following a short intermediate period at E16.5. Furthermore, comparison between the methylation status of DMRs and the expression profiles of imprinted genes suggested that methylation of the IG-DMR, but not the H19 DMR, solely governed the control of its imprinted gene cluster. The Rasgrf1 DMR seemed to be imprinted later than the other two genes. We also found that the methylation status of the Gtl2 DMR, the secondary DMR that acquires DNA methylation after fertilization, was likely to follow the methylation status of the upstream IG-DMR. Thus, the systematic analyses of prospermatogonium-derived embryos provided additional important information on the establishment of paternal imprinting.

Abnormal DLK1/MEG3 imprinting correlates with decreased HERV-K methylation after assisted reproduction and preimplantation genetic diagnosis

Retrotransposons participate in cellular responses elicited by stress, and DNA methylation plays an important role in retrotransposon silencing and genomic imprinting during mammalian development. Assisted reproduction technologies (ARTs) may be associated with increased stress and risk of epigenetic changes in the conceptus. There are similarities in the nature and regulation of LTR retrotransposons and imprinted genes. Here, we investigated whether the methylation status of Human Endogenous Retroviruses (HERV)-K LTR retrotransposons and the imprinting signatures of the DLK1/MEG3. p57KIP2 and IGF2/H19 gene loci are linked during early human embryogenesis by examining trophoblast samples from ART pregnancies and preimplantation genetic diagnosis (PGD) cases and matched naturally conceived controls. Methylation analysis revealed that HERV-Ks were totally methylated in the majority of controls while, in contrast, an altered pattern was detected in ART-PGD samples that were characterized by a hemi-methylated status. Importantly, DLK1/MEG3 demonstrated disturbed methylation in ART-PGD samples compared to controls and this was associated with altered HERV-K methylation. No differences were detected in p57KIP2 and IGF2/H19 methylation patterns between ART-PGD and naturally conceived controls. Using bioinformatics, we found that while the genome surrounding the p57KIP2 and IGF2/H19 genes differentially methylated regions had low coverage in transposable element (TE) sequences, the respective one of DLK1/MEG3 was characterized by an almost 2-fold higher coverage. Moreover, our analyses revealed the presence of KAP1-binding sites residing within retrotransposon sequences only in the DLK1/MEG3 locus. Our results demonstrate that altered HERV-K methylation in the ART-PGD conceptuses is correlated with abnormal imprinting of the DLK1/MEG3 locus and suggest that TEs may be affecting the establishment of genomic imprinting under stress conditions.

DNA methyltransferase 1(Dnmt1) mutation affectsSnrpnimprinting in the mouse male germ line

DNA methylation and DNA methyltransferases are essential for spermatogenesis. Mutations in the DNA methyltransferase Dnmt1 gene exert a paternal effect on epigenetic states and phenotypes of offspring, suggesting that DNMT1 is important for the epigenetic remodeling of the genome that takes place during spermatogenesis. However, the specific role of DNMT1 in spermatogenesis and the establishment of genomic imprints in the male germ line remains elusive. To further characterize the effect of DNMT1 deficiency on the resetting of methylation imprints during spermatogenesis, we analyzed the methylation profiles of imprinted regions in the spermatozoa of mice that were heterozygous for a Dnmt1 loss-of-function mutation. The mutation did not affect the H19 or IG differentially methylated regions (DMRs) that are usually highly methylated but led to a partial hypermethylation of the Snrpn DMR, a region that should normally be unmethylated in mature spermatozoa. This defect does not appear in mouse models with mutations in Dnmt3a and Mthfr genes and, therefore, it is specific for the Dnmt1 gene and is suggestive of a role of DNMT1 in imprint resetting or maintenance in the male germ line.

Partners in Imprinting: Noncoding RNA and Polycomb Group Proteins

Establishment of genomic imprints during early development involves concerted epigenetic mechanisms. Two recent studies by Terranova et al. (in this issue of Developmental Cell) and Pandey et al. (in a recent issue of Molecular Cell) have demonstrated that Polycomb group proteins (PcG) and the Kcnq1ot1 regulatory RNA, respectively, are indispensable for gene- and lineage-specific chromatin modification and compaction of the paternally imprinted Kcnq1 cluster.

Patterns of Hybrid Loss of Imprinting Reveal Tissue- and Cluster-Specific Regulation

BackgroundCrosses between natural populations of two species of deer mice, Peromyscus maniculatus (BW), and P. polionotus (PO), produce parent-of-origin effects on growth and development. BW females mated to PO males (bw×po) produce growth-retarded but otherwise healthy offspring. In contrast, PO females mated to BW males (PO×BW) produce overgrown and severely defective offspring. The hybrid phenotypes are pronounced in the placenta and include PO×BW conceptuses which lack embryonic structures. Evidence to date links variation in control of genomic imprinting with the hybrid defects, particularly in the PO×BW offspring. Establishment of genomic imprinting is typically mediated by gametic DNA methylation at sites known as gDMRs. However, imprinted gene clusters vary in their regulation by gDMR sequences.Methodology/Principal FindingsHere we further assess imprinted gene expression and DNA methylation at different cluster types in order to discern patterns. These data reveal PO×BW misexpression at the Kcnq1ot1 and Peg3 clusters, both of which lose ICR methylation in placental tissues. In contrast, some embryonic transcripts (Peg10, Kcnq1ot1) reactivated the silenced allele with little or no loss of DNA methylation. Hybrid brains also display different patterns of imprinting perturbations. Several cluster pairs thought to use analogous regulatory mechanisms are differentially affected in the hybrids.Conclusions/SignificanceThese data reinforce the hypothesis that placental and somatic gene regulation differs significantly, as does that between imprinted gene clusters and between species. That such epigenetic regulatory variation exists in recently diverged species suggests a role in reproductive isolation, and that this variation is likely to be adaptive.

DNA methylation status of H19 and Xist genes in lungs of somatic cell nuclear transfer bovines

In somatic cell nuclear transfer (SCNT) technologies, the donor cell’s nuclei need to be epigenetically reprogrammed for embryonic development. The incomplete reprogramming of donor cell nuclei has been implicated as a primary reason for the low efficiency of SCNT. DNA methylation is a major epigenetic modification of the genome that regulates crucial aspects of genome function, including establishment of genomic imprinting. In order to make sure whether the DNA methylation reprogramming is efficient in SCNT animals, we analyzed the DNA methylation status of two imprinting genes, H19 and Xist, in lungs of deceased SCNT bovines that died within 48 h of birth using bisulfite sequencing analysis. Our findings demonstrated that cloned bovines showed significantly lower DNA methylation of H19 than controls (P<0.05), and three tested CpGs sites (1, 2, 3) exhibited unmethylation in one cloned bovine (9C3); however, Xist showed similar DNA methylation levels between clones and controls, and both showed hypermethylation (96.11% and 86.67%).