Antisense Transcripts Negatively Regulate Transcription of Multiple Variegated Killer Immunoglobulin-Like Receptor (KIR) Genes

Natural killer cells are CD3 negative large granular lymphocytes that lyse virally infected and malignantly transformed targets. NK cell functions are regulated by an array of inhibitory and activating receptors, including members of the killer immunoglobulin-like receptor (KIR) family. Human KIR genes are expressed in a variegated and clonally restricted manner on the surface of mature NK cells. While it is well established that individual KIR gene expression is strongly correlated with the DNA methylation status of CpG dinucleotides within the promoter region proximal to the transcriptional start site, the mechanisms that regulate variegated KIR expression are largely unknown. Our goal is to uncover the genetic mechanisms governing KIR transcriptional regulation. We have recently identified an active distal promoter element approximately 1 Kb upstream of the transcriptional start site. This region contains c-MYC binding sites, and KIR expression is increased when MYC is overexpressed in developing NK cells or when c-MYC is physiologically increased by IL-15 activation. Bi-directional promoter activity is found within the previously characterized proximal promoter of all KIR genes. Therefore, double-stranded RNA with homology to the KIR promoter can be generated within this non-coding region. The generation of double-stranded RNA has recently been shown to contribute to promoter DNA methylation of the p15 gene in mammalian cells through a Dicer-independent mechanism. Using quantitative real-time PCR, we found that purified peripheral blood CD56⁺/KIR3DL1⁻ NK cells express 5-fold more KIR3DL1 antisense transcript than purified CD56⁺/KIR3DL1⁺ cells. Therefore, the transcriptional activation of the KIR3DL1 gene correlates with a significant decrease in KIR3DL1 antisense expression. To explore the possibility that KIR antisense transcripts can silence KIR expression, we over-expressed the KIR3DL1 antisense transcript in CD34⁺ hematopoietic precursor cells and differentiated these cells into NK cells in vitro. After 21 days in culture, we assayed KIR3DL1 mRNA levels using quantitative real-time PCR. KIR3DL1 expression was reduced 4-fold compared to eGFP control cultures, further supporting the hypothesis that antisense transcripts negatively regulate KIR expression. Importantly, expression of the KIR2DL4 gene, which does not contain a promoter with significant homology to KIR3DL1 promoter, was not affected by KIR3DL1 antisense overexpression. This provides the first evidence for gene silencing through double-stranded RNA in the immune system and may be the key mechanism for the establishment of DNA promoter methylation within the KIR locus. Understanding the mechanisms of KIR expression, a requisite for NK cell education/licensing, may allow us to understand the acquisition of effector function and manipulate it for therapeutic benefit.