5340 Nucleic Acids Research, 2009, Vol. 37, No. 16 to those located upstream in Ly49H-negative cells. This ﬁnd again raises the possibility of the existence of a prox- imal downstream regulatory element for the Ly49 genes whose function might be aﬀected by DNA methylation. Indeed for the KIR genes, a region spanning the trans- criptional start site and the 50-UTR provides the core promoter activity in T-cells but not NK cells (45). This region also binds diﬀerent protein complexes in NK and T-cells as shown with gel shift experiments (45). The multiple alignment of the 50-region of inhibitory and acti- vating Ly49 genes revealed a region of high homology in exon 1 among the activating genes that is not present in the inhibitory Ly49 sequences (Figure 6A). Further inves- tigation of this region, for the activating Ly49d and h versus the corresponding region for the inhibitory Ly49a and c, in gel shift experiments, showed diﬀerent patterns of protein complex binding between inhibitory and acti- vating gene regions (Figure 6B). YY1 was also conﬁrmed as a candidate transcription factor binding to this region. YY1 is known to have both transcriptional activating and inhibiting properties (46). In addition, the tested region showed a diﬀerent protein-binding pattern with nuclear extract from NK and T-cells (Figure 6B). These results combined with the lack of detectable activating Ly49 tran- scripts in T-cells support a varied mode of transcriptional regulation for the two types of Ly49 genes. Further ana- lysis of this region is needed to conﬁrm the extent of its role in the transcriptional regulation of the activating Ly49 genes. In contrast to the inhibitory receptors, the DNA methylation pattern of the 50-region in the Ly49D and H expressing NK populations does not follow the bimodal (half-and-half) methylation pattern. The half-and-half pattern of DNA methylation correlates with the mono- allelic expression of Ly49A, C and NKG2A as was sub- sequently shown using F1 hybrids for Ly49a and Nkg2a (22,44). If DNA methylation does not correlate with stochastic mono-allelic expression; either stochastic gene expression is maintained at the level of histone modiﬁca- tions only or this pattern is possibly an indication of high, if not complete, bi-allelic expression and even the lack of stochastic expression of the activating receptors. We have previously shown that the transcription of some Ly49 genes and Nkg2a correlates with histone acet- ylation levels (22,44,47). Ly49g transcription in the EL4 cell line is activated mostly in response to histone deace- tylase inhibitors but is mostly unaﬀected by DNA methyl- transferase inhibitors. Histone acetylation levels of the Pro-2 region as assayed via chromatin immunoprecipita- tion (ChIP) also correlate with state of expression in EL4-derived subclones (47). It is possible that the stochas- tic mono-alleleic expression of Ly49G (42) is controlled at the level of histones only. Based on these results, it is also possible that the maintenance of the activating receptor expression patterns is through diﬀerential histone modiﬁcations. However, an alternative hypothesis to explain the lack of a half-and-half DNA methylation pattern could be that the expression of the activating receptors is pre- dominantly bi-allelic. Based on this hypothesis, in the Ly49D/R expressing NK cells of the 129/B6 F1 hybrid, the 50-regions of both Ly49d and r should always be hypomethylated to reﬂect absolute bi-allelic expression. Our results show that most Ly49d and r clones are hypo-methylated (16/19) in the receptor-expressing NK cells indicating a strong deviation away from the mono- allelic DNA methylation pattern observed for the inhibi- tory genes (Figure 3B). If Ly49d and r are not alleles but are independent loci, based on the product rule, assuming no bias for co-expression given 50% surface expression of each receptor by NK cells, we would expect 25% of all NK cells to be co-expressing the two receptors. Since the total of NK cells expressing one or both of Ly49D and R would be 75% (assuming independent loci), we would expect that 1/3 of all clones sequenced for each gene (when sorting for D and/or R expressing NK cells) to show hyper-methylation patterns similar to the negative population. Our data indicates a much lower methylation frequency than would be expected with stochastic mono- allelic expression and hence we can conclude that neither Ly49d nor r show bimodal DNA methylation patterns. Our experiment does not prove or disprove the allelic relationship of Ly49d and r. The bias towards bi-allelic expression of the activating Ly49 receptors is further supported by the results of the single-cell Ly49d allele-speciﬁc RT–PCR performed on NK cells of the B6/NOD F1 hybrid (Table 2) as well as the higher than expected expression of Ly49H from a single genomic allele (Figure 5). We detected bi-allelic transcription of Ly49d in 55% (22/40) of Ly49d-transcrib- ing NK cells. If Ly49D is expressed on 50% of NK cells, the percentage of NK cells expressing this receptor bi-allelically would be 28% (Figure 5D). The single-cell RT–PCR method tends to be somewhat ineﬃcient in detecting bi-allelic gene expression. The transcription factor, Pax-5, was statistically deemed to be bi-allelically expressed by this method even though only close to 65% of the cells analysed showed bi-allelic expression (39). Transcripts for our control gene Nkg2d, that is expressed by all NK cells (40), went undetected in 9% (7/80) of NK cells (Table 2), indicating a likely underestimation of the bi-allelic expression percentage calculated for Ly49d. However, based on the few hyper-methylated clones detected from the Ly49D/R-expressing NK cells as well as the detection of only one Ly49d allele in a few single cells (from the B6/NOD F1 hybrid) even after ﬁve sets of single-cell RT–PCR, we believe that a real albeit very small population of NK cells express Ly49D mono- allelically. It is not inconceivable that the activating receptors are controlled and expressed diﬀerently from the inhibitory receptors. The lack of a well-deﬁned Pro-1 element and its transcripts (19,20) in addition to deviation of co-expression percentages from the product rule (21) sug- gests a diﬀerent mode of transcriptional regulation from that of the inhibitory genes. Also, in the Ly49h genomic transgenic mouse, Ly49H expression was restricted to NK cells as is the case with the endogenous receptor (48). This might be due to the lack of the adapter protein DAP12 in T and B cells. However, in mice carrying only an Ly49d cDNA transgene (driven by H-2Kb promoter and IgH enhancer) (32) some surface expression of