Paternally expressed genes predominate in the placenta

Abstract

The discovery of genomic imprinting through studies of manipulated mouse embryos indicated that the paternal genome has a major influence on placental development. However, previous research has not demonstrated paternal bias in imprinted genes. We applied RNA sequencing to trophoblast tissue from reciprocal hybrids of horse and donkey, where genotypic differences allowed parent-of-origin identification of most expressed genes. Using this approach, we identified a core group of 15 ancient imprinted genes, of which 10 were paternally expressed. An additional 78 candidate imprinted genes identified by RNA sequencing also showed paternal bias. Pyrosequencing was used to confirm the imprinting status of six of the genes, including the insulin receptor (INSR), which may play a role in growth regulation with its reciprocally imprinted ligand, histone acetyltransferase-1 (HAT1), a gene involved in chromatin modification, and lymphocyte antigen 6 complex, locus G6C, a newly identified imprinted gene in the major histocompatibility complex. The 78 candidate imprinted genes displayed parent-of-origin expression bias in placenta but not fetus, and most showed less than 100% silencing of the imprinted allele. Some displayed variability in imprinting status among individuals. This variability results in a unique epigenetic signature for each placenta that contributes to variation in the intrauterine environment and thus presents the opportunity for natural selection to operate on parent-of-origin differential regulation. Taken together, these features highlight the plasticity of imprinting in mammals and the central importance of the placenta as a target tissue for genomic imprinting.

Keywords: chorionic girdle; hinny; interspecific hybrid equids; mule.

Fig. 1.

The mule–hinny chorionic girdle and fetal imprinting assay system. (A) Day 33 horse conceptus, showing the principal fetal and placental tissues, including the invasive trophoblast of the chorionic girdle. (B) Day 19 mule embryo. (C) Distribution of allelic expression ratio and parent-of-origin effect along equine chromosome 10 for all transcribed genes. (Left) Horse chromosome 10, color-coded with human chromosome synteny, showing two large syntenic blocks of human chromosomes 19 and 6. (Center) Plots of allelic expression bias for genes on chromosome 10 for both mule and hinny chorionic girdle samples. The x-axis shows the percentage of allelic expression from the horse allele (0–100%) in the two reciprocal F1 hybrids. The red bar depicts the percentage of horse allelic expression (p₁) in the mule (horse allele is maternal in mules), and the blue bar represents the proportion of horse allelic expression (p₂) in the hinny (paternal allele). (Right) Plot of the degree of parent-of-origin bias on chromosome 10. Red indicates statistically significant overexpression of the maternal allele, and blue indicates statistically significant overexpression of the paternal allele with a cutoff of q value < 0.01. Gray represents nonsignificant genes. The height of each bar is the degree of parent-of-origin effect, which is computed as (p₂ − p₁). Gene names of significant candidates are labeled (q-value < 0.05, Storer–Kim test).

Fig. 2.

Change of imprinting status of H19 and IGF2 under 5-aza-2′-deoxycytidine (AzadC) and trichostatin A (TSA) treatments. The effect of methylation and deacetylase inhibitor on imprinting status for the H19 and IGF2 genes in mule placenta was checked in the chorionic girdle cells from a day 34 mule conceptus cultured for 33 d. In vitro treatment with the DNA methyltransferase inhibitor AzadC abolished the H19 imprinting status, but IGF2 was unaffected. When the cells were treated with the histone deacetylase inhibitor TSA, we observed biallelic expression for IGF2, but H19 imprinting status was not affected. The results in cultured mule cells are consistent with the report in mouse fibroblast cells.

Fig. 3.

Strand-specific methylation of CpG islands of equine imprinted genes. Bisulfite sequencing of the DMRs in H19 and PEG3 shows allele-specific differential methylation corresponding to the allelic expression bias. Parental allele-specific methylation in mule and hinny is inferred from differences in single-nucleotide sequences in horse and donkey. Yellow boxes depict methylated CpGs, and blue boxes depict unmethylated CpGs. Each panel shows multiple CpG sites at the DMR for the corresponding gene. (A) H19 is 100% methylated in sperm, consistent with paternal silencing. The H19 DMR is differentially methylated in day 33–34 chorionic girdle (CG) samples from horse, donkey, mule, and hinny, with paternal methylation. Note the concordance between the SNP allelic state and the methylation state in mule and hinny. (B) PEG3 is unmethylated in sperm, consistent with paternal expression. The PEG3 DMR is differentially methylated in day 33–34 chorionic girdle samples from horse, donkey, mule, and hinny, with maternal methylation.

Fig. 4.

HAT1, a newly identified imprinted gene in the equine placenta. (A) Allele counts from RNA-seq data of HAT1 in chorionic girdle of mule and hinny showing the strong paternally biased allelic expression. (B) SNP genotyping in mule and hinny and their parents by Sanger sequencing, showing that HAT1 exonic SNP CUHSNP00046513 is informative. (C) Allele-specific pyrosequencing confirmation of the paternal allele overexpression of HAT1 in mule and hinny day 33 chorionic girdle samples. The target sequence is on the opposite strand. (D) Total expression levels expressed as RPKM in RNA-seq data indicating that total HAT1 expression tracks with the paternal parent. (E) Total expression levels expressed as log₂ probe intensity in Agilent 4 × 44K Horse Gene Expression Microarray of the same individual samples showing that total HAT1 expression tracks with the paternal parent, as is consistent with the results shown in D. (F) Allele-specific pyrosequencing verification of HAT1 imprinting in six different mule/hinny individuals in day 33 chorionic girdle and fetus samples and in adult liver and lymphocytes in one mule and one hinny, demonstrating that imprinting is limited to the placental tissue and that fetal and adult tissues are biallelically expressed. (G) The HAT1 DMR is differentially methylated in horse, donkey, mule, and hinny day 33–34 chorionic girdle samples with maternal-only methylation, consistent with paternal allelic expression. In the horse day 34 fetus and adult lymphocytes, the HAT1 DMR was 100% unmethylated, consistent with biallelic expression.