# DNA Methylation of Igf2r Promoter CpG Island 2 Governs Cis-Acting Inheritance and Gene Dosage in Equine Hybrids

## TL;DR

This study explores how DNA methylation in horses and their hybrids affects gene expression, revealing new insights into epigenetic inheritance.

## Contribution

The study identifies CpG island 2 in the Igf2r promoter as a novel site of cis-acting DNA methylation inheritance in equine hybrids.

## Key findings

- Parent-specific expression of Igf2r is lost in equine brain and mule liver tissues.
- DNA methylation in CpG island 2 correlates negatively with Igf2r expression in multiple tissues.
- Equine hybrids show cis-acting inheritance of DNA methylation patterns in the Igf2r promoter.

## Abstract

The Igf2r expression pattern in offspring is influenced by the parents and is important for organism growth. In this study, we examined the Igf2r gene in horses, donkeys, and their hybrids. We found that the parent-specific expression of Igf2r was lost in equine brain and mule liver tissues, whereas it was retained in most other tissues. Surprisingly, this was not related to canonical differentially methylated regions (DMRs). However, DNA methylation patterns in CpG island 2 (CpGI2) in the promoter of Igf2r could be transmitted from parents to offspring, thereby determining its expression activity, even in hybrids. This helps us to improve hybrid breeding strategies and address livestock growth issues.

Genomic imprinting is critical for mammalian development, but its regulation varies across species. The insulin-like growth factor 2 receptor (IGF2R), which is a maternally expressed imprinted gene critical for cell proliferation and differentiation, as well as embryonic and placental development, is classically regulated by differentially methylated regions (DMRs) and lncRNA-Airn in mice. However, studies on this in equus are scarce, especially in terms of mechanistic studies. In the present study, heart, liver, spleen, lung, kidney, brain, and muscle samples were obtained from horses, donkeys, and hybrids, and gene expression and imprinting state were tested to investigate the imprinting regulation of Igf2r in these animals. Bisulfite sequencing combined with an allele-specific expression analysis revealed a tissue-specific loss of imprinting in the mule liver and hybrid brain tissues. Strikingly, we found that the maternal-specific expression of equine Igf2r did not rely on the canonical DMRs or lncRNA-Airn. Surprisingly, DNA methylation of a specific region called CpG island 2 (CpGI2) in the Igf2r promoter showed cis-acting inheritance, meaning that the DNA methylation patterns of the parental alleles are retained in hybrid tissues. Notably, the DNA methylation of CpGI2 correlated negatively with Igf2r expression in the spleen (R2 = 0.8797, p = 6.46 × 10−6), lung (R2 = 0.8569, p = 1.57 × 10−5), and kidney (R2 = 0.8650, p = 3.85 × 10−6). Our findings suggest that imprinting may work differently in other species. This study provides a framework for understanding imprinting diversity in hybrids and shows that equine hybrids can be used to study how epigenetic inheritance works.