Genetic and epigenetic regulation of major histocompatibility complex class I gene expression in bovine trophoblast cells

Abstract

Problem: The regulatory mechanisms governing differential expression of classical major histocompatibility complex (MHC) class I (MHC-Ia) and non-classical MHC class I (MHC-Ib) genes are poorly understood.

Method of study: Quantitative reverse transcription- polymerase chain reaction (PCR) was used to compare the abundance of MHC-I transcripts and related transcription factors in peripheral blood mononuclear cells (PBMC) and placental trophoblast cells (PTC). Methylation of MHC-I CpG islands was detected by bisulfite treatment and next-generation sequencing. Demethylation of PBMC and PTC with 5'-aza-deoxycytidine was used to assess the role of methylation in gene regulation.

Results: MHC-I expression was higher in PBMC than PTC and was correlated with expression of IRF1, class II MHC transactivator (CIITA), and STAT1. The MHC-Ia genes and BoLA-NC1 were devoid of CpG methylation in PBMC and PTC. In contrast, CpG sites in the gene body of BoLA-NC2, -NC3, and -NC4 were highly methylated in PBMC but largely unmethylated in normal PTC and moderately methylated in somatic cell nuclear transfer PTC. In PBMC, demethylation resulted in upregulation of MHC-Ib by 2.8- to 6-fold, whereas MHC-Ia transcripts were elevated less than 2-fold.

Conclusion: DNA methylation regulates bovine MHC-Ib expression and is likely responsible for the different relative levels of MHC-Ib to MHC-Ia transcripts in PBMC and PTC.

Keywords: DNA methylation; bovine; non-classical MHC-I; transcription.

Figure 1

qRT‐PCR analysis of bovine major histocompatibility complex (MHC)‐I and transcription factor gene expression in peripheral blood mononuclear cells (PBMC) and placental trophoblast cells (PTC). Data were normalized to GAPDH. The heatmap shows the fold change of gene expression relative to the average for the artificial insemination (AI) PTC samples

Figure 2

Major histocompatibility complex (MHC)‐Ia and MHC‐Ib transcript percentages in placental trophoblast cells (PTC) and peripheral blood mononuclear cells (PBMC). Expression of MHC‐Ia and MHC‐Ib (BoLA‐NC1, ‐NC2, ‐NC3, ‐NC4) was detected by qRT‐PCR. Average percentages are shown

Figure 3

Methylation profile of CpG islands in major histocompatibility complex (MHC)‐Ia and MHC‐Ib genes of peripheral blood mononuclear cells (PBMC). (A) Genomic organization of bovine MHC‐I CpG islands, from ‐300 bp upstream of the transcription start site through the third exon, which is about 1500 Kb. (B‐E) Methylation status of the CpG sites of each MHC‐I allele in four PBMC samples. Each horizontal line of circles represents the methylation status of an individual allele. Different colors of circles denote variation in the methylation level. The numbers above each line of circles stand for the position in the genomic sequence relative to the first base of the start codon

Figure 4

Methylation profile of CpG islands in major histocompatibility complex (MHC)‐Ia and MHC‐Ib genes of placental trophoblast cells (PTC). (A) Genomic organization of bovine MHC‐I CpG islands. (B‐C) Methylation status of the CpG sites of each MHC‐I allele in two artificial insemination (AI) PTC samples and (D‐E) in two somatic cell nuclear transfer (SCNT) PTC samples

Figure 5

qRT‐PCR analysis of DNMT expression in placental trophoblast cells (PTC) and peripheral blood mononuclear cells (PBMC). Data are presented as the fold change (mean±SEM) of gene expression in other groups relative to that of the artificial insemination (AI) PTC group (AI PTC n = 5; somatic cell nuclear transfer (SCNT) PTC n = 5; PBMC n = 3). Statistical analyses were performed using one‐way ANOVA

Figure 6

Change in major histocompatibility complex (MHC)‐I transcription following demethylation treatment of peripheral blood mononuclear cells (PBMC) and placental trophoblast cells (PTC). Bovine PBMC and PTC were treated with 5′‐aza‐deoxycytidine for 3 days (n = 3 per group). (A) Fold change (mean ± SEM) of expression in the treated cells compared to cells cultured without 5′‐aza‐deoxycytidine. (B) Percentage of MHC‐I subtypes in the overall pool of MHC‐I transcripts