Cutting Edge: KIR Antisense Transcripts Are Processed into a 28-Base PIWI-Like RNA in Human NK Cells

Signaling through MHC class I-specific inhibitory receptors, such as killer Ig-like receptors (KIRs) is important not only to dampen activation signals, but also for the acquisition of NK cell function and the establishment of self-tolerance during development. Despite significant promoter homologies within the KIR locus, individual KIR genes are regulated independently and probabilistically (1–4). The question of how KIR transcriptional patterns are established in NK cells is largely unresolved. A thorough analysis of the complete 2-kb intergenic region upstream of the KIR3DL1 gene revealed that the promoter proximal to the translational start site has bidirectional activity, and a unidirectional distal promoter exists upstream of the conventional proximal promoter (5, 6). Polyadenylated antisense transcripts and full-length distal transcripts were previously cloned from several KIR genes, leading to the hypothesis that transcription across proximal KIR promoters may be involved in establishing epigenetic marks that influence gene expression (7). In support of this hypothesis, we show that a novel 28-base PIWI-like RNA processed from KIR3DL1 antisense transcripts is essential for transcriptional silencing.

The use of all human tissue was approved by the Committee on the Use of Human Subjects in Research at the University of Minnesota (Minneapolis, MN), and informed consent was secured in accordance with the Declaration of Helsinki. CD56⁺ NK cells were column-isolated from peripheral blood using magnetic beads (Miltenyi Biotec, Auburn, CA). KIR3DL1 subsets were isolated by staining CD56⁺ NK cells with PE-labeled DX9 Ab (BD Biosciences, San Jose, CA) and separating PE-positive and -negative populations using anti-PE magnetic beads (Miltenyi Biotec). CD34⁺ hematopoietic progenitor cells (HPCs) were isolated from umbilical cord blood by double-column positive selection using anti-CD34 microbeads (Miltenyi Biotec).

For the quantification of the KIR3DL1 antisense transcripts, cDNA synthesis was carried out at 55°C using a KIR3DL1/S1-specific reverse transcription primer. A TaqMan primer and probe set was used for KIR3DL1 antisense transcript amplification. Primer probe sets for KIR3DL1 distal transcripts and KIR coding transcripts have been published previously (8, 9). Additional primers used in this study are shown in Supplemental Table I. Bisulfite sequencing analysis of the KIR3DL1 promoter was performed as previously described (2).

The small RNA fraction from 2 × 10⁶ NK cells was isolated using the PureLink miRNA Isolation Kit (Invitrogen, Carlsbad, CA). For periodate oxidation/β-elimination, we followed published methods (10).

Lentiviral vectors were created by MluI/BamHI restriction digest and oligoannealing using pELNS with an H1 PolIII promoter. Oligo sequences are shown in Supplemental Table I. All murine stem cell virus (MSCV)-enhanced GFP (eGFP) vector constructs were created using the Gateway cloning system (Invitrogen). Primer sequences are shown in Supplemental Table I.

CD34⁺ cells isolated from umbilical cord blood were transduced with retrovirus or lentivirus and cultured for 21 d on the EL08-1D2 stromal line (11). The culture medium and cytokines for NK cell differentiation are published (8).

Cell sorting was performed on the FACSAria (BD Biosciences), and phenotypic analysis was performed on the FACSCalibur (BD Biosciences) using CellQuest Pro Software (BD Biosciences). The Abs used in this study were allophycocyanin-conjugated NCAM16.2 (CD56) and CD34 and PE-conjugated DX9 (anti-CD158e), EB6 (anti-CD158a/h), and GL183 (CD158b/j) (BD Biosciences).

To determine how expression of KIR3DL1 gene transcripts (Supplemental Fig. 1) relates to receptor expression, we isolated CD56⁺ NK cells from the peripheral blood of healthy donors and separated KIR3DL1⁻ and KIR3DL1⁺ NK cell populations. As expected, the expression of KIR3DL1 coding transcripts strongly correlated with surface receptor expression (Fig. 1A). In contrast, KIR3DL1 antisense transcript expression was inversely correlated with surface receptor expression. Virtually all of the KIR3DL1 antisense transcripts detected in mature CD56⁺ NK cells were confined to the KIR3DL1⁻ subset (Fig. 1B). KIR3DL1 distal transcripts were detected at high levels within the KIR3DL1⁺ NK cell population, which is consistent with a previous analysis of the distal promoter (8). Interestingly, low levels of KIR3DL1 distal transcript were present in the KIR3DL1⁻ NK cell population (Fig. 1C). Therefore, the potential for creation of dsRNA is restricted to cells lacking receptor expression.

We performed an S1 nuclease protection assay using RNA purified from CD56⁺ peripheral blood NK cells to determine whether KIR3DL1 distal and antisense transcripts form dsRNA. Sequencing analysis of cloned products led to the identification of a 228-bp dsRNA that extends from position −18 to −306 bp relative to the KIR3DL1 mRNA transcriptional start site. The 5′ boundary of the cloned dsRNA region (−306) coincides precisely with the transcriptional start site of the KIR3DL1 distal transcript, consistent with a direct role of the distal promoter in the generation of dsRNA (Supplemental Fig. 2).

To test whether small RNAs are processed within the dsRNA region, expression vectors producing sense and antisense KIR3DL1 transcripts spanning the promoter region were transfected into HEK293 cells to generate dsRNA. Twenty-four hours posttransfection, total RNA was isolated, and the small RNA fraction was enriched. A cDNA library was generated and screened with a probe containing the entire KIR3DL1 dsRNA region. A single 28-base antisense small RNA was identified in each of three independent small RNA libraries generated. The specific generation of this small RNA from dsRNA was confirmed in additional experiments in which KIR3DL1 sense- or antisense-expressing vectors were transfected separately into HEK293 cells, and the small RNA was not detected (Supplemental Fig. 2). We also identified a 28-base RNA processed from the KIR2DL1 antisense transcript (Supplemental Fig. 3). To confirm that the 28-base RNA is not generated as a cell line artifact, we harvested CD56⁺ NK cell small RNAs from multiple donors and were able to isolate the KIR3DL1 28-base RNA using a PCR-based cloning strategy.

The only known species of small RNAs in the 25–30 nt range are PIWI-interacting RNAs (piRNAs), which are implicated in the germline silencing of transposons (12). Mammalian piRNAs are 2′-0-methylated at their 3′ terminal ribose, whereas small interfering RNAs and microRNAs have terminal hydroxyl groups at both the 2′ and 3′ positions (13). To determine whether the KIR3DL1 28-base small RNA has a 3′ terminal modification, a periodate oxidation/β-elimination reaction was performed on total RNA from CD56⁺ peripheral blood NK cells mixed with synthetic control RNA lacking any 3′ terminal modifications. Only RNAs containing both 2′ and 3′ hydroxyl groups react with NaIO₄, and β-elimination shortens NaIO₄-reacted RNA by 1 nt, leaving a 3′-monophosphate terminus. Both the KIR3DL1 28-base RNA and control RNA were cloned out of the total reacted RNA and sequenced. As expected, the majority (8 out of 10) of control RNA sequences were shortened by 1 nt. In contrast, all 10 sequences for the KIR3DL1 28-base RNA were full-length (Table I). Therefore, the KIR3DL1 28-base RNA contains a protective group at either the 2′ or 3′ ribose position of its 3′ terminus that renders it resistant to periodate oxidation/β-elimination.

We transduced primary CD34⁺ hematopoietic progenitor cells with a retroviral vector expressing the full-length KIR3DL1 antisense transcript and differentiated these cells into mature CD56⁺ NK cells. Overexpression of KIR3DL1 antisense transcripts led to an ∼70% reduction in KIR expression. This effect was dependent upon transcript orientation, as overexpression of sense transcripts homologous to the KIR3DL1 promoter did not lead to any reduction in KIR expression. Importantly, overexpression of KIR3DL1 antisense transcripts with the 28-base RNA sequence removed did not result in any reduction in KIR expression, implying that the processed KIR3DL1 28-base PIWI-like RNA is necessary for silencing (Fig. 2A). We also transduced HPCs with a lentiviral vector expressing the 28-base PIWI-like RNA. Compared with eGFP and scramble controls, overexpression of the 28-base PIWI-like RNA led to an ∼90% reduction in KIR expression (Fig. 3A).

The mRNA expression levels for each gene were reduced 4- to 5-fold in cells overexpressing KIR3DL1 antisense transcripts. Interestingly, expression of the KIR2DL4 gene, which shares only 67% sequence identity with the KIR3DL1 promoter and is constitutively expressed by mature CD56⁺ NK cells (1), was not affected by overexpression of the KIR3DL1 antisense transcript (Fig. 2B). The silencing effect of the KIR3DL1 antisense on other KIR genes with significant promoter homologies is likely due to the presence of high levels of antisense transcript produced by the constitutive MSCV promoter throughout development.

Because the correlation between DNA methylation of KIR promoters and transcriptional silencing is well established (2–4), we analyzed the methylation status of the endogenous KIR3DL1 promoter region in cells that were transduced with either the eGFP control construct or the 28-base PIWI-like RNA and then differentiated along the NK lineage. CD56⁺ KIR3DL1⁺ and KIR3DL1⁻ cells were sorted from eGFP cultures, and CD56⁺ KIR3DL1⁻ cells were sorted from 28-base PIWI-like RNA cultures. Genomic DNA was extracted, and bisulfite sequencing was performed using primers specific for the KIR3DL1 promoter region (2). Consistent with previous studies, we observed very little methylation of the KIR3DL1 promoter in KIR3DL1⁺ cells and dense methylation in the KIR3DL1⁻ cells from the eGFP control culture. There were too few KIR3DL1⁺ cells from 28-base PIWI-like RNA cultures for analysis. However, when we analyzed the KIR3DL1 promoter in sorted KIR3DL1⁻ cells from 28-base PIWI-like RNA overexpressing cultures, we observed dense methylation (Fig. 3B). These results show that the 28-base PIWI-like RNA participates in KIR promoter methylation during human NK cell development.

Three facts support the conclusion that the KIR small RNAs described in this study belong to the piRNA family: 1) the 28-base size of the RNA; 2) the presence of a protective group at the 3′ terminus of the RNA; and 3) the finding that the KIR small RNAs are processed exclusively from antisense transcripts. Although we have not yet identified the protein(s) that interact with the 28-base PIWI-like RNA, we have confirmed the expression of PIWIL4 in human NK cells (Supplemental Fig. 4).

During human NK cell development, CpG sites within KIR promoters are demethylated by a mechanism that remains to be elucidated. We propose that at a late stage in human NK cell development, transcription is initiated in a probabilistic fashion from the bidirectional promoters of clonally restricted KIR genes. If the promoter initiates in the forward direction, coding transcripts are generated, and the open epigenetic state of the promoter becomes locked in. If the promoter initiates in the reverse direction, antisense transcripts are generated and quickly processed into the 28-base PIWI-like RNA, which participates in cis in the maintenance or establishment of DNA methylation and stable, clonal silencing of the promoter (Supplemental Fig. 5).

It may appear contradictory that distal transcript expression is higher in KIR⁺ cells (Fig. 1C), yet it is required for formation of dsRNA and processing of the 28-base PIWI-like RNA. This paradox may be explained by the observation that distal transcripts appear to have dual functions. If antisense transcripts from the bidirectional proximal promoter are present, distal transcripts are involved in formation of dsRNA for subsequent processing of the 28-base PIWI-like RNA. However, if antisense transcripts are not present, transcription from the distal promoter appears to promote transcription of KIR genes in response to IL-15 stimulation (8).

Although there is significant homology in the 28-base PIWI-like RNA sequence among the clonally restricted KIR genes, we envision the shutdown of single genes to occur though the local action of chromatin-modifying enzymes that interact with the 28-base PIWI-like RNA at the promoter. We suggest that antisense transcripts are rapidly and efficiently processed into the 28-base PIWI-like RNA, which mediates epigenetic silencing without diffusion or transport away from the promoter.

Disclosures The authors have no financial conflicts of interest.