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

Abstract

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.

Keywords: DNA methylation; Igf2r; cis-acting inheritance; epigenetic regulation; equus; genomic imprinting.

Figure 1

DNAMAN alignment of Igf2r (A) and H19 (B) at horse and donkey mRNA SNV sites.

Figure 2

Allele-specific expression analysis of equine Igf2r and H19 in hybrids. (A) SNV validation of Igf2r. (B) Tissue-specific ASE ratios of Igf2r. (C) SNV validation of H19. (D) Tissue-specific ASE ratios of H19.

Figure 3

DNA methylation analysis of Igf2r promoter regions. (A) Schematic of equine Igf2r locus and SNV sites (black triangle), with 12 CpGs in CpGI1, 10 CpGs in CpGI2, and 16 CpGs in CpGI3 (white box). SNVs between horse and donkey are as follows: CpGI1: A (horse)/G (donkey); CpGI2: G (horse)/C (donkey); and CpGI3: A (horse)/G (donkey). (B) Percent methylation of CpGI1 in the Igf2r promoter. (C) Percent methylation of CpGI3 in the Igf2r promoter. (D) Percent methylation of CpGI2 in the Igf2r promoter. Effective readings ≥ 10 (****: p < 5.48 × 10⁻⁵). (E) Bisulfite sequencing of CpGI2 in the Igf2r promoter with donkey tissues (liver and kidney). Effective readings = 8.

Figure 4

DNA methylation analysis of Igf2r promoter and intronic regions. (A) Schematic of equine Igf2r locus, including transcriptional direction (arrow), transcription unit (black box), DMR, and CpG islands (gray box). Bisulfite mutagenesis and sequencing are indicated below, including 1 SNV site (black triangle) and 40 CpGs (white box). SNVs between horse and donkey are as follows: G (horse)/A (donkey). (B) Maternal allele-specific methylation in intron 2 of Igf2r in equine hearts; × represents the deletion of a 14 bp repetitive sequence fragment (AGGGCGCTGTCCGC). Each row of circles represents an individual strand sequenced. Black circles represent methylated cytosines, white circles represent unmethylated cytosine. (C) Comparative results of bisulfite sequencing (BS-Seq) of the horse2 intron 2 DMR region: 14 bp deletions are shown in black boxes, and 12 bp repeat sequences are shown in red boxes. (D) Conservation of the intronic DMR in germ cells (sperm and oocytes). (E) Maternal allele-specific methylation in intron 2 of Igf2r in the liver. (F) Maternal allele-specific methylation in intron 2 of Igf2r in the brain. Effective readings ≥ 6.

Figure 5

DNA methylation cis-acting inheritance of Igf2r in promoter CpGI2 in equines. (A) DNA methylation rates of seven equine tissues (heart, liver, spleen, lung, kidney, brain, and muscle) in CpGI1. (B) DNA methylation rates in CpGI1, and DNA methylation in hybrid tissue traced back to parents using SNV information. (C) Bar chart: DNA methylation rates in CpGI1. (D) DNA methylation rates in CpGI2, tending towards the result of horse > hybrids > donkey. (E) DNA methylation rates in CpGI2. (F) Bar chart: DNA methylation rates in CpGI2, (****: p < 6.46 × 10⁻⁷). (G) DNA methylation rates of horse-derived alleles of Igf2r in CpGI2 in hybrid tissues. (H) DNA methylation rates of donkey-derived alleles of Igf2r in CpGI2 in hybrid tissues. n = 3.

Figure 6

Tissue-dependent correlation between CpGI2 DNA methylation and Igf2r expression. (A) Igf2r expression levels in mules and hinnies mirror their respective maternal species. (B) CpGI2 DNA methylation states align with parental origin (***: p = 1.82 × 10⁻⁴). (C) Linear regression between DNA methylation and expression of Igf2r in spleen, lung, and kidney. (D) Relationship between DNA methylation and Igf2r gene expression. Data transformation methods: copy number values were subjected to double natural log transformation (LN(LN)), while DNA methylation levels were normalized to a range of 0–1, followed by × −1 adjustment.