Humanization of a tandem repeat in IG-DMR causes stochastic restoration of paternal imprinting at mouseDlk1-Dio3domain

Abstract

The Dlk1-Dio3 imprinted domain, regulated by an intergenic differentially methylated region (IG-DMR), is important for mammalian embryonic development. Although previous studies have reported that DNA methylation of a tandem repeated array sequence in paternal IG-DMR (IG-DMR-Rep) plays an essential role in the maintenance of DNA methylation in mice, the function of a tandem repeated array sequence in human IG-DMR (hRep) is unknown. Here, we generated mice with a human tandem repeated sequence, which replaced the mouse IG-DMR-Rep. Mice that transmitted the humanized allele paternally exhibited variable methylation status at the IG-DMR and were stochastically rescued from the lethality of IG-DMR-Rep deficiency, suggesting that hRep plays a role in human IG-DMR for the regulation of imprinted expression. Moreover, ChIP analysis showed that TRIM28 was enriched in hypermethylated paternal hRep without ZFP57. Our results suggest that hRep contributes to the maintenance of human IG-DMR methylation imprints via the recruitment of TRIM28.

Genome-wide DNA methylation, imprinting, and gene expression in human placentas derived from Assisted Reproductive Technology

“…In the current study, we observed prominent ART-associated downregulation of paternally expressed DLK1, which locates in a paternally imprinted DLK1-DIO3 locus. Since ZFP57 binds to and stabilizes paternally methylated ICR in DLK1-DIO3 locus 58 and TRIM28 is a cofactor of ZFP57 52,53 , we examined ICR DNAm at this locus of ART (IVF: n = four, ICSI: n = four), SF ( n = four), and control ( n = six) placentas by traditional bisulphite sequencing. Interestingly, although we did not see highly methylated paternal and demethylated maternal allele in this specific ICR sequence partially overlapping with the ZFP57 binding site, we observed significantly decreased DNAm in the ICR of SF placentas compared to control (CpG1: P = 0.003, CpG2: P = 0.007) and ART placentas (CpG1: P = 0.002, CpG7: P = 0.047) (Fig.…”

“…In the current study, we observed ART-associated hypomethylation in ZFP57 as well as prominent downregulation of paternally expressed DLK1, which locates in a paternally imprinted DLK1-DIO3 locus. Since ZFP57 binds to and stabilizes paternally methylated ICR in DLK1-DIO3 locus in mouse 58 and TRIM28 is a cofactor of ZFP57 52,53 , we examined ICR DNAm at this locus of ART (IVF: n = four, ICSI: n = four), SF ( n = four), and control ( n = six) placentas by traditional bisulphite sequencing (Fig. 4a).…”

“…4a). The analyzed ICR sequence is next to the repeated sequence motifs, which contribute to the DNAm of the ICR 58 . Maternal and paternal alleles were distinguished by two single nucleotide polymorphisms (SNPs) (rs1884539 and rs75998174) when possible.…”

Identification of the sequences responsible for maternalH19-ICR hypermethylation with Beckwith-Wiedemann syndrome-like overgrowth in mice

“…For DNA methylation analysis of blastocysts, embryos at 3.5 dpc were produced via in vitro fertilization using oocytes from mutant mice and PWK sperm, as previously described (Joh et al 2018). Genomic DNA was extracted from oocytes, and blastocysts were extracted as previously described (Hara et al 2021). Next, DNA samples (70-80 oocytes and 7-10 blastocysts per sample) were subjected to bisulfite conversion through 2 µg carrier RNA (QIAGEN).…”