Next to the significant evidence that CpG methylation of

Next to the significant evidence that CpG methylation of the HLA-G promoter is important for the transcriptional activity of the gene (Moreau et al., 2003; Mouillot et al., 2005), HLA-G order phenylephrine hydrochloride is also post-transcriptionally regulated by the 3′UTR. Several polymorphic sites in the 3′UTR have been described to modulate the expression of the HLA-G transcripts and proteins (Rousseau et al., 2003) and alternative splicing (Moreau et al., 2003; Mouillot et al., 2005; Auboeuf et al., 2002). First, there is a 14bp insertion/deletion (INDEL) containing an AUUUG sequence. It has been reported to influence the mRNA stability and splicing pattern (Rousseau et al., 2003) and has been associated with a lower level of HLA-G mRNA (Hviid et al., 2003). Next, the single nucleotide polymorphism (SNP) +3187A/G appears to mediate mRNA stability and degradation (Yie et al., 2008). The presence of an A(denine) at the +3187 position has been shown to lead to a decreased HLA-G mRNA expression (Yie et al., 2008). Furthermore, there is the +3142C/G SNP, which influences the binding affinity for specific microRNAs (miRNAs). The presence of G(uanine) at position +3142 increases the affinity for miR-148a, -148b and -152, downregulating HLA-G expression by mRNA degradation (Tan et al., 2007; Castelli et al., 2009). In hESC, despite the variation of HLA-G expression observed by the different studies, only one study reported on the promoter CpG methylation status in a mRNA negative line, the Sheffield-1 (Shef-1) line. This line displayed a partially methylated CpG island around its transcriptional start site with exception of four fully methylated CpG sites (region -211 to -272) suggesting that this methylation pattern was responsible for the lack of HLA-G mRNA expression (Suarez-Alvarez et al., 2010). However, no promoter methylation data are available for HLA-G positive lines, and the role of the polymorphisms in the 3′UTR has not been previously explored in hESC. The aim of this study was to identify the control mechanisms of HLA-G gene expression in hESC. For this, we evaluated how HLA-G expression is linked to the patterns of promoter CpG methylation and explored the role of the 3′UTR polymorphic sites and their binding miRNAs on the post-transcriptional regulation of HLA-G.
Material and methods
Results
Discussion In this work we investigated the transcriptional (promoter CpG methylation) and post-transcriptional (3′UTR SNPs and miRNAs) regulation of HLA-G expression in hESC. Our results suggest that, while the gross expression of HLA-G is controlled by CpG methylation, its levels are significantly modulated at the post-transcriptional level based on the genotypes of the lines and their affinity for miRNA binding. As for the vast majority of human genes, the promoter CpG methylation status of HLA-G is key for the transcriptional activity of the gene (Guillaudeux et al., 1995). We found three different methylation patterns and three types of epi-alleles in hESC. The epi-allele with a low methylation grade in region -121 to -188 was specific for hESC and not detected in the control lines. Treatment with the demethylating agent 5-aza-dc resulted in a 2.7 to 8.1-fold increase in mRNA levels confirming the impact of CpG methylation on gross levels of HLA-G in hESC. This is in line with previous studies reporting that hypomethylation correlates to HLA-G expression in glioma, choriocarcinoma, B-lymphoma, melanoma and ovarian cancer cell lines as well as in the Shef-1 stem cell line after 5-aza-dc treatment (Suarez-Alvarez et al., 2010; Moreau et al., 2003; Mouillot et al., 2005; Menendez et al., 2008). Suarez-Alvarez et al. suggested that four fully methylated CpG sites (region –211 to -272) were responsible for the lack of HLA-G mRNA expression in Shef-1 (Suarez-Alvarez et al., 2010). In most of our lines we found a lower methylation grade (<50%) for CpG -211 and -241. Based on these results, CpG -211 and -241 could have been an explanation for the difference in mRNA expression. However, VUB01 and VUB14 showed an almost identical methylation grade as Shef-1 with ≥50% methylation for CpG -211 and -241. This suggests that the methylation grade of this -272 to -211 region alone is not responsible for the transcriptional repression of the gene in Shef-1. This is in accordance with the study of Menendez et al. who found HLA-G mRNA expression in malignant and benign ovarian tumor samples containing a completely (=100%) methylated -272 to -211 region (Menendez et al., 2008).