5338 Nucleic Acids Research, 2009, Vol. 37, No. 16 Figure 5. Expression of Ly49H in B6 Ly49h+/ NK cells and comparison to the stochastic mono-allelic expression model. (A) Expression of a theoretical Ly49 receptor expressed on 50% of NK cells based on the stochastic mono-allelic model in an inbred mouse strain. (B) The percentage of peripheral blood NK cells expressing Ly49H in B6.BXD8/B6 (Ly49h+/ ) mice and age-matched B6 (Ly49h+/+) were determined by FACS. P-value was calculated according to the two-tailed unpaired t-test. (C) Allelic expression of Ly49H in a theoretical B6 mouse based on the expression of this receptor in B6.BXD8/B6 (Ly49h+/ ) mice. The dashed circle and brackets indicated predicted expression patterns. (D) Allelic expression of Ly49D on NK cells based on the single-cell RT–PCR results (Table 2) in B6/NOD F1 hybrid. Each chromosome is shown with a line, the alleles are shown with rectangle, arrows indicate transcription and cross shows the deleted allele. shows a region in exon 1 that is present for the activating Ly49 genes but is either absent or diﬀerent for the inhib- itory ones (Figure 6A). This region contains a number of potential binding sites for the transcription factor Yin Yang-1 (YY1) in the activating but not the inhibitory genes. Since more than half of the assayed CpG dinucleotides of the 50-region of Ly49 genes fall downstream of the transcriptional start site, it is possible that diﬀerential methylation of this region might aﬀect transcription of the Ly49 genes. Furthermore, investigation of the possible diﬀerential regulatory role of this region might explain some of the diﬀerences between inhibitory and activating receptor expression. We therefore probed the possible existence of activating gene speciﬁc transcription factor binding sites by gel-shift assay with nuclear extracts from FACS sorted IL-2 cultured NK cells and T-cells (Figure 6B and Supplementary Figure 2). Fragments spanning the region of interest from Ly49a, c, d and h were used as probe. In both NK and T-cells a diﬀerential pattern of protein binding was observed between the activating and inhibitory Ly49 probes. A large band (indicated by a bracket in Figure 6B and Supplementary Figure 2), likely consisting of multiple pro- tein complexes, was most prominent for the activating genes. In addition, a protein complex only bound to the activating Ly49 probes (indicated by arrow in Figure 6B and Supplementary Figure 2) disappeared (supershifted) in the presence of YY1 antibody. DISCUSSION Here, we have analyzed the DNA methylation of the 50-region of B6 activating NK receptor genes, Ly49d and h. We have shown that DNA methylation of this region correlates with expression patterns of these receptors as with the inhibitory Ly49A, Ly49C (22) and NKG2A (44). Furthermore, as in the case of Ly49a, the CpG dinu- cleotides downstream of the transcriptional start site of Ly49h seem to have higher levels of methylation compared