Embryos generated with the use of assisted reproductive technologies (ART) can develop overgrowth syndromes. In ruminants, the condition is referred to as large offspring syndrome (LOS) and exhibits variable phenotypic abnormalities including overgrowth, enlarged tongue, and abdominal wall defects. These characteristics recapitulate those observed in the human loss-of-imprinting (LOI) overgrowth syndrome Beckwith-Wiedemann (BWS). We have recently shown LOI at the KCNQ1 locus in LOS, the most common epimutation in BWS. Although the first case of ARTinduced LOS was reported in 1995, studies have not yet determined the extent of LOI in this condition. Here, we determined allele-specific expression of imprinted genes previously identified in human and/or mouse in day ∼105 Bos taurus indicus × Bos taurus taurus F1 hybrid control and LOS fetuses using RNAseq. Our analysis allowed us to determine the monoallelic expression of 20 genes in tissues of control fetuses. LOS fetuses displayed variable LOI compared with controls. Biallelic expression of imprinted genes in LOS was associated with tissue-specific hypomethylation of the normally methylated parental allele. In addition, a positive correlation was observed between body weight and the number of biallelically expressed imprinted genes in LOS fetuses. Furthermore, not only was there loss of allele-specific expression of imprinted genes in LOS, but also differential transcript amounts of these genes between control and overgrown fetuses. In summary, we characterized previously unidentified imprinted genes in bovines and identified misregulation of imprinting at multiple loci in LOS. We concluded that LOS is a multilocus LOI syndrome, as is BWS.genomic imprinting | assisted reproductive technologies | large offspring syndrome | Beckwith-Wiedemann syndrome | loss of imprinting

Genomic imprinting is essential for mammalian development. Recent studies have revealed that maternal histone H3 Lys27 trimethylation (H3K27me3) can mediate DNA methylation-independent genomic imprinting. However, the regulatory mechanisms and functions of this new imprinting mechanism are largely unknown. Here we demonstrate that maternal Eed, an essential component of the Polycomb group complex 2 (PRC2), is required for establishing H3K27me3 imprinting. We found that all H3K27me3-imprinted genes, including Xist, lose their imprinted expression in Eed maternal knockout (matKO) embryos, resulting in male-biased lethality. Surprisingly, although maternal X-chromosome inactivation (XmCI) occurs in Eed matKO embryos at preimplantation due to loss of Xist imprinting, it is resolved at peri-implantation. Ultimately, both X chromosomes are reactivated in the embryonic cell lineage prior to random XCI, and only a single X chromosome undergoes random XCI in the extraembryonic cell lineage. Thus, our study not only demonstrates an essential role of Eed in H3K27me3 imprinting establishment but also reveals a unique XCI dynamic in the absence of Xist imprinting.

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DNA methylation at the 5-position of cytosine (5mC) plays vital roles in mammalian development. DNA methylation is catalyzed by DNA methyltransferases (DNMTs), and the two DNMT families, DNMT3 and DNMT1, are responsible for methylation establishment and maintenance, respectively. Since their discovery, biochemical and structural studies have revealed the key mechanisms underlying how DNMTs catalyze de novo and maintenance DNA methylation. In particular, recent development of low-input genomic and epigenomic technologies has deepened our understanding of DNA methylation regulation in germ lines and early stage embryos. In this review, we first describe the methylation machinery including the DNMTs and their essential cofactors. We then discuss how DNMTs are recruited to or excluded from certain genomic elements. Lastly, we summarize recent understanding of the regulation of DNA methylation dynamics in mammalian germ lines and early embryos with a focus on both mice and humans.