Epigenetic silencing of potentially functional KIR2DL5 alleles: Implications for the acquisition of KIR repertoires by NK cells

Abstract

NK cells detect altered patterns of HLA expression in infections and tumors using a variegated repertoire of killer cell Ig-like receptors (KIR). Each clone surveys different HLA molecules by expressing a limited subset of the KIR encoded in its genome, which is maintained throughout cell divisions by epigenetic mechanisms (methylation of the nonexpressed genes). How KIR repertoires are acquired remains, however, unexplained. Human KIR2DL5 is a useful model for studying KIR expression because it has alleles with similar coding regions, but drastically divergent expression -whilst some are transcribed in a typically clonal manner, others, with distinctive promoter polymorphisms, are nonexpressed. Here we investigate the relationship between the sequence diversity of KIR2DL5, including three novel alleles, and its variable transcription. The promoters of the transcribed alleles recruit the transcriptional regulator RUNX3, whilst a mutation shared by all silent alleles precludes this binding. However, all promoters are functional in vitro, and pharmacological DNA demethylation of NK cells rescues the transcription of silent alleles, indicating that only epigenetic mechanisms prevent their inclusion in a normal KIR repertoire. Our results are consistent with a model in which RUNX factors could function as switch elements in the acquisition of KIR repertoires by NK cell precursors.

Duplication, mutation and recombination of the human orphan gene KIR2DS3 contribute to the diversity of KIR haplotypes

“…19,24 Moreover, the intergenic region is indistinguishable in the two KIR2DL5-KIR2DS3 clusters (our own unpublished observation), which therefore constitute two continuous segments of nearly identical sequence, each spanning more than 20 kb. These regions are possibly prone to chromatid misalignment during meiosis, and to further asymmetrical recombination between the centromeric and the telomeric regions of the KIR gene complex.…”

“…24,34 In summary, the intergenic region of interest, along with the preceding and consecutive exons of the flanking KIR genes, was amplified by combining one gene-specific and one KIRgeneric primers in a long-range PCR in the following cycling conditions: 95 1C 2 min, 10 cycles of 10 s at 95 1C, 10-20 min at 72 1C and 20 cycles of 10 s at 95 1C, 10-20 min at 68-70 1C. The same technique was used in cell C43 (sibling 1 in Figure 2) to demonstrate the juxtaposition of KIR2DL5B*002 and KIR2DS3*002.…”

Molecular characterisation of KIR2DS2*005, a fusion gene associated with a shortened KIR haplotype

“…Generic KIR typing, allelic KIR2DL5 and KIR2DS3 typing, and long-range PCR for initial KIR2DS2*005 identification were done as described. 11,19,20 A new PCR-SSP method for specific detection of KIR2DS2*005 was designed: genomic DNA was amplified using 0.2 U of BioTaq DNA polymerase (Bioline, London, UK), and primers Fi6a þ 3019b (forward, The KIR genes flanking KIR2DS2*005 in donor C180F2 genomic DNA were determined using KIR-gene walking protocols similar to those we have reported previously DNA sequencing. All PCR products were submitted to direct nucleotide sequencing with no cloning step.…”

Lineage-Specific Transition of Histone Signatures in the Killer Cell Ig-Like Receptor Locus from Hematopoietic Progenitor to NK Cells

“…Suitable candidates would be members of the family of runt-related proteins (RUNX), transcription factors that have a conserved binding site in the KIR promoter (46). A mutation of this site, that abolishes binding of RUNX to the KIR promoter is selectively found in KIR variants that are transcriptionally silent (47,48). As RUNX1 is known to interact with various histone acetylases (49,50), the inability to recruit these epigenetic modifiers via RUNX1 could inhibit the establishment of active histone acetylation marks in these silent KIR variants.…”