Dynamics of the Equine Placental DNA Methylome and Transcriptome from Mid- to Late Gestation

Abstract

The placenta is a temporary organ that is essential for the survival of the fetus, with a lifelong effect on the health of both the offspring and the dam. The functions of the placenta are controlled by its dynamic gene expression during gestation. In this study, we aimed to investigate the equine placental DNA methylome as one of the fundamental mechanisms that controls the gene expression dynamic. Chorioallantois samples from four (4M), six (6M), and ten (10M) months of gestation were used to map the methylation pattern of the placenta. Globally, methylation levels increased toward the end of gestation. We identified 921 differentially methylated regions (DMRs) between 4M and 6M, 1225 DMRs between 4M and 10M, and 1026 DMRs between 6M and 10M. A total of 817 genes carried DMRs comparing 4M and 6M, 978 comparing 4M and 10M, and 804 comparing 6M and 10M. We compared the transcriptomes between the samples and found 1381 differentially expressed genes (DEGs) when comparing 4M and 6M, 1428 DEGs between 4M and 10M, and 741 DEGs between 6M and 10M. Finally, we overlapped the DEGs and genes carrying DMRs (DMRs-DEGs). Genes exhibiting (a) higher expression, low methylation and (b) low expression, high methylation at different time points were identified. The majority of these DMRs-DEGs were located in introns (48.4%), promoters (25.8%), and exons (17.7%) and were involved in changes in the extracellular matrix; regulation of epithelial cell migration; vascularization; and regulation of minerals, glucose, and metabolites, among other factors. Overall, this is the first report highlighting the dynamics in the equine placenta methylome during normal pregnancy. The findings presented serve as a foundation for future studies on the impact of abnormal methylation on the outcomes of equine pregnancies.

Keywords: chorioallantois; differentially methylated regions; horse; methylation; placental methylome; reduced representation bisulfate sequencing.

Figure 1

Descriptive representation of mCs in the equine placenta at 4M, 6M, and 10M in the CG context. (A) Correlation of the detected mCs among the samples. (B) Distribution of mCs shows a similar pattern across chromosomes. (C) Description of the methylation levels in the genomic functional regions. All true methylated cytosine sites were annotated to the reference equine transcriptome. NS: no significant difference (p > 0.05).

Figure 2

Differentially methylated regions (DMRs) across gestational times. (A) DMR distribution across chromosomes. (B) Distribution of DMRs according to the contexts and genomic features. The majority of DMRs were identified in the CG context and landed in introns (~40%) and exons (~20%), followed by repeats (~11%) and promoters (~10%). (C) Methylation changes in DMRs across the time points and among the genomic features. An ~30% change (median) in methylation percentages on the DMRs was noted across all genomic features, represented by the pink dashed lines in the figure.

Figure 3

Differentially methylated genes and their gene ontologies among different gestational ages. (A) A total of 452 genes were differentially methylated in at least two of the comparisons (4M vs. 6M, 6M vs. 10M, 4M vs. 10M). (B) The GO analysis for these groups of genes revealed their enrichment in Wnt signaling, the developmental process, cell communication, and the extracellular matrix.

Figure 4

Differentially expressed genes among different gestational time points. (A) Heatmap of gene expression in chorioallantois from 4M, 6M, and 10M of gestation. (B) Differentially expressed genes were demonstrated using volcano plots.

Figure 5

Association between gene expression and methylation. Based on the assumption that methylation influences gene expression, we identified the genes with reduced methylation that had an increased expression and genes with increased methylation that had a decreased expression ((A): 4M vs. 6M, (B): 4M vs. 6M, and (C): 6M vs. 10M; red squares indicate the genes which followed the expected patterns). (D) Gene ontology analysis of the 38 genes displaying accordance between methylation of DMRs and gene expression.