Epigenetic and post-transcriptional regulation of CD16 expression during human natural killer cell development1

The surface receptor FcγRIIIA (CD16a) is encoded by the FCGR3A gene and is acquired by human natural killer (NK) cells during maturation. NK cells bind the Fc portion of IgG via CD16a, and execute antibody-dependent cellular cytotoxicity, which is critical for the effectiveness of several anti-tumor monoclonal antibody therapies. The role of epigenetic regulatory mechanisms controlling transcriptional and post-transcriptional CD16 expression in NK cells is unknown. Here we compared specific patterns of DNA methylation and expression of FCGR3A to FCGR3B , which differ in cell type-specific expression despite displaying nearly identical genomic sequences. We identified a sequence within the FCGR3A promoter that selectively exhibits reduced methylation in CD16a+ NK cells versus CD16a- NK cells and neutrophils. This region contained the transcriptional start site of the most highly expressed CD16a isoform in NK cells. Luciferase assays revealed remarkable cell-type specificity and methylation-dependent activity of FCGR3A- versus FCGR3B -derived sequences. Genomic differences between FCGR3A and FCGR3B are enriched at CpG dinucleotides and mutation of variant CpGs reversed cell-type specificity. We further identified miR-218 as a post-transcriptional negative regulator of CD16a in NK cells. Forced over-expression of miR-218 in NK cells knocked down CD16a mRNA and protein expression. Moreover, miR-218 was highly expressed in CD16a- NK cells compared to CD16a+ NK cells. Taken together, we propose a system of FCGR3A regulation in human NK cells in which CpG dinucleotide sequences and concurrent DNA methylation confer developmental and cell type-specific transcriptional regulation, while miR-218 provides an additional layer of post-transcriptional regulation during the maturation process.