CpG methylation patterns in the 5' part of the nonclassical HLA-G gene in peripheral blood CD34+ cells and CD2+ lymphocytes

Abstract

A dominant goal of research focused on the nonclassical human leukocyte antigen G (HLA-G) gene is to understand the molecular mechanism involved in its limited expression. In the present report, we examined DNA methylation as a potential regulatory mechanism of HLA-G transcription in two cell types of the adult lymphomyeloid lineage: CD2+ lymphocytes express several mRNA isoforms while transcripts are undetectable in CD34+ hematopoietic cells. The methylation status of 63 CpG sites in the promoter and in the 5' CpG island was established using bisulfite-treated genomic DNA sequencing. Methylation was first analyzed by the direct sequencing of bisulfite-treated and amplified products. The general patterns of CpG methylation in the 5' part of the gene were found to be similar for CD34+ cells and CD2+ lymphocytes: the distribution of methylation was not uniform across the 63 CpG sites. In the promoter region, both CpG dinucleotides were partially or fully methylated whereas in the CpG island, several CpG sites were totally demethylated. Unexpectedly, in HLA-G positive CD2+ lymphocytes, a great number of CpG dinucleotides displayed a higher frequency of methylation relative to that found in CD34+ cells. However, the sequence analysis of cloned products revealed that the molecules have different methylation patterns which suggests that the HLA-G gene is differentially expressed in CD2+ cells. Our results suggest that methylation is not the sole mechanism that achieves the repression of HLA-G transcription in immature CD34+ cells.