5332 Nucleic Acids Research, 2009, Vol. 37, No. 16 inhibitory Ly49 receptors that are expressed on T and NKT cells as well as NK cells, the stimulatory Ly49 recep- tors are only expressed on NK cells (18). Lack of a well- deﬁned upstream promoter-1 (Pro-1) region, which likely acts as a stochastic switch for inhibitory receptor genes in immature NK cells (19,20) and reports of the higher co-expression of Ly49D and H and deviation from the product rule (21), suggest that the activating Ly49 genes, despite being surrounded by inhibitory genes in the Ly49 gene cluster, are subject to distinct regulatory mechanisms. DNA methylation of the 50-region of the inhibitory Ly49 genes correlates with their expression. In the case of Ly49a and Ly49c, where stochastic mono-allelic expression has been demonstrated, we have previously shown that DNA methylation and mono-allelic receptor expression correlate (22). The existence of stochastic mono-allelic expression and/or correlation with DNA methylation is unknown for the activating receptors. Here, we have investigated the link between DNA methy- lation of the 50-region of activating receptors and the maintenance of their expression. Furthermore, we show evidence for a mode of transcriptional regulation for the activating Ly49 genes that diﬀers from that of the inhibitory genes. MATERIALS AND METHODS Mice All C57BL/6 mice were bred and maintained in the animal facility of the British Columbia Cancer Research Centre (Vancouver, British Columbia, Canada). 129SvEvTac and 129SvEvTac/C57BL6 F1 hybrids were ordered from Taconic farms. NOD/ShiLtJ and C57BL6/NOD-ShiLtJ F1 hybrids were ordered from the Jackson Laboratory (Bar Harbor, Maine, USA). All mice used in this study were more than 6-weeks old. The B6.BXD8/B6 (23) and age-matched B6 mice were 6.5-weeks old. All experiments were according to a protocol approved by the Committee on Animal Care of the University of British Columbia. Antibodies, cell separation and flow cytometry The monoclonal antibody (mAb) anti-FcRg (2.4G2) (24,25) and 3D10-FITC (21) have been described before. Anti- CD3e-PerCP-Cy5.5, anti- NK1.1-PE, anti- NK1.1-APC, anti-DX5-PE (alpha-2 integrin, CD49b), 1F8-FITC (anti-Ly49C/I/H), 5E6-biotin (anti-Ly49C/I), 4E5-FITC (anti-Ly49D), A1-biotin (anti-Ly49AB6) and ﬂuorochrome-conjugated streptavidin were purchased from BD Biosciences (Mississauga, Ontario, Canada). The anti-mouse NKp46-PE antibody was purchased from eBioscience (San Diego, CA, USA). The 12A8 pur- iﬁed antibody was generously provided by Dr Stephen Anderson (NCI, Frederick, MD, USA) and was conju- gated to FITC via Thermo Scientiﬁc Pierce EZ-label ﬂuo- rescein isothiocyanate protein labelling kit (Rockford, IL, USA) per manufacturer’s protocol. Flow cytometry for cell sorting was performed on Cytopeia Inﬂux cell sorter and Becton Dickinson FACSAria Cell Sorting System (Mississauga, Ontario, Canada). All sorted samples were >95% pure or resorted for high purity. For the single-cell sorting, NK cells were sorted into wells containing JEG-3 (human choriocarcinoma) as carrier cells. Doublet discrimination was applied to avoid more than one cell per well. Primary cell and tissue genomic DNA extraction Genomic DNA (gDNA) was obtained from FACS sorted cells as described before (22). gDNA was extracted from fresh B6 mouse liver using DNAzol reagent (Invitrogen) per manufacturer’s instructions. Further proteinase K digestion and phenol–chloroform extraction was performed. Sodium bisulfite conversion and PCR Bisulﬁte conversion was performed as described previ- ously (22). The conversion rate was >98%. First-round PCR ampliﬁcation of Ly49h 50 region was performed using Ly49h forward and reverse ﬂanking bisulﬁte prim- ers. Converted DNA was used as template in a 45 ml reac- tion volume, containing 30 pmol of each primer, 1 mM dNTPs, 3 mM MgCl2 and 0.5 U Taq Platinum DNA poly- merase (Qiagen). After initial denaturation for 7 min at 958C, 30–40 cycles were performed, each consisting of 90 s at 958C, 55 s at 508C and 40 s at 728C with a ﬁnal extension of 7 min at 728C. Two microliters of the ﬁrst PCR was used for nested ampliﬁcation using Ly49h forward and reverse nested bisulﬁte primers. The same ampliﬁcation conditions were chosen as for ﬁrst-round PCR with the exception that the annealing temperature was changed to 48 + 0.18C/cycle. All bisulﬁte primer sequences are shown in Table 1. For the Ly49d 50 region of the B6 strain the following primers were used to amplify two regions for COBRA analysis of two CpG dinucleotides: ﬁrst round (ﬂanking) PCR was performed with Ly49d forward and reverse ﬂanking bisulﬁte primers. After initial denaturation for 7 min at 958C, 35 cycles were performed, each consisting of 90 s at 958C, 55 s at 518C and 50 s at 728C with a ﬁnal extension of 7 min at 728C. Two separate nested PCRs were performed on 3 ml of the product of the ﬂanking PCR to amplify two regions containing an upstream and a downstream (in relation to transcription start site) CpG dinucleotide. For the upstream CpG-containing fragment the Ly49d forward ﬂanking and reverse nested bisulﬁte primers were used. After initial denaturation for 7 min at 958C, 35 cycles were performed, each consisting of 90 s at 958C, 55 s at 518C and 17 s at 728C with a ﬁnal extension of 7 min at 728C. The downstream CpG-containing frag- ment was ampliﬁed via the Ly49d forward nested and reverse ﬂanking bisulﬁte primers. After initial denatura- tion for 7 min at 958C, 35 cycles were performed, each consisting of 90 s at 958C, 55 s at 538C and 40 s at 728C with a ﬁnal extension of 7 min at 728C. The resulting pro- ducts were subjected to combined bisulﬁte and restriction enzyme analysis (COBRA). For Ly49d and Ly49r 50 region ampliﬁcation from the 129SvEvTac/C57BL6 F1 hybrid cells, ﬁrst round PCR was performed with the same ﬂanking primers as Ly49d as their sequence is identical to Ly49r with the same PCR conditions as that used for Ly49d of B6. Nested PCR was