Identification of the porcine IG-DMR and abnormal imprinting of DLK1- DIO3 in cloned pigs

Junliang Li; Dawei Yu; Jing Wang; Chongyang Li; Qingwei Wang; Jing Wang; Weihua Du; Shanjiang Zhao; Yunwei Pang; Haisheng Hao; Xueming Zhao; Huabin Zhu; Shijie Li; Huiying Zou

doi:10.3389/fcell.2022.964045

Identification of the porcine IG-DMR and abnormal imprinting of DLK1- DIO3 in cloned pigs

Front Cell Dev Biol. 2022 Aug 10:10:964045. doi: 10.3389/fcell.2022.964045. eCollection 2022.

Authors

Junliang Li^{1

2}, Dawei Yu^{1

3}, Jing Wang⁴, Chongyang Li⁴, Qingwei Wang⁴, Jing Wang⁵, Weihua Du¹, Shanjiang Zhao¹, Yunwei Pang¹, Haisheng Hao¹, Xueming Zhao¹, Huabin Zhu¹, Shijie Li², Huiying Zou¹

Affiliations

¹ Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.
² College of Life Sciences, Hebei Agricultural University, Baoding, Hebei, China.
³ National Germplasm Center of Domestic Animal Resources, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.
⁴ State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
⁵ Department of Human Genetics David Geffen School of Medicine University of California Los Angeles, Los Angeles, CA, United States.

Abstract

Correct reprogramming of the DLK1-DIO3 imprinted region is critical for the development of cloned animals. However, in pigs, the imprinting and regulation of the DLK1-DIO3 region has not been systematically analyzed. The objective of this study was to investigate the imprinting status and methylation regulation of the DLK1-DIO3 region in wild-type and cloned neonatal pigs. We mapped the imprinting control region, IG-DMR, by homologous alignment and validated it in sperm, oocytes, fibroblasts, and parthenogenetic embryos. Subsequently, single nucleotide polymorphism-based sequencing and bisulfite sequencing polymerase chain reaction were conducted to analyze imprinting and methylation in different types of fibroblasts, as well as wild-type and cloned neonatal pigs. The results showed that Somatic cell nuclear transfer (SCNT) resulted in hypermethylation of the IG-DMR and aberrant gene expression in the DLK1-DIO3 region. Similar to wild-type pigs, imprinted expression and methylation were observed in the surviving cloned pigs, whereas in dead cloned pigs, the IG-DMR was hypermethylated and the expression of GTL2 was nearly undetectable. Our study reveals that abnormal imprinting of the DLK1-DIO3 region occurs in cloned pigs, which provides a theoretical basis for improving the cloning efficiency by gene editing to correct abnormal imprinting.

Keywords: DNA methylation; IG-DMR; SCNT; genomic imprinting; pig.