Ly49D was observed on T cells (49) indicating possible pairing of Ly49D with other adaptor proteins in these cells. The ability of T-cells to express some Ly49D on their surface in the absence of DAP12 in this transgenic mouse (49) supports the notion that the endogenous Ly49d promoter might be inactive in T-cells due to lack of necessary transcription factors and/or the presence of repressive epigenetic marks at regulatory sequences. Our results support both possibilities. The Ly49h 50 region is hypermethylated in T-cells (Figure 2B) indicating a possi- ble epigenetic hindrance to transcription of this gene in these cells. The lack of detectable Ly49d and h transcripts in T-cells (Figure 1A) also supports this notion. Furthermore, the 50-UTRs of both Ly49d and h show dif- ferent protein complex binding pattern compared to that of the inhibitory Ly49 genes (Figure 6B). In contrast to the endogenous receptor, in the Ly49a genomic transgenic mouse, expression of Ly49A was seen on the majority of splenic B-cells for all transgenic lines regardless of copy number of the transgene (50). The 30Kb Ly49a genomic transgene contained the Pro-1 region and it was subsequently shown that the deletion of this region in the same transgene abrogated the expres- sion of Ly49A in NK, T and B cells (50). Mice carrying a 79kb Ly49h genomic transgene spanning the complete length of the gene displayed Ly49H expression patterns similar to the B6 Ly49h gene indicating that the transcrip- tional regulatory sequences necessary for the ‘wild-type’ expression of this gene are contained within the 79 kb region (48). Ly49h transcript and percentage of NK cells expressing Ly49H correlated in the transgenic mice and increased according to the transgene copy number, but only up to a certain threshold (48). This phenomenon indicates a possible control mechanism for an upper limit of expression likely at the level of transcription and/or at the level of NK selection. The activating Ly49 genes evolve faster than their inhib- itory family members (51). This is evident from the large variation in the number and sequence of the activating Ly49 genes and the high number of activating psuedo- genes in nearly all the sequenced mouse strains (7,11). It is also possible that the regulatory sequences of the activating receptors evolved diﬀerently from that of the inhibitory receptors in order to allow a tighter control in NK cells and as a side eﬀect of this evolution, mouse T-cells lost the ability to express these receptors. Deviation from stochastic expression might also allow more control on the number of activating receptors on the surface of NK cells because mono-allelic expression might lead to a lower number of receptors on the cell surface compared to bi-alleleic expression (41). Hence, in a non-stochastic system, the expression level of these receptors is homoge- neous among diﬀerent NK cells. In conclusion, our results suggest that distinct modes of transcriptional regulation govern the expression of the activating and inhibitory Ly49 genes. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. Nucleic Acids Research, 2009, Vol. 37, No. 16 5341 ACKNOWLEDGEMENTS We sincerely thank Liane Gagnier, Hyun-Jung Goo, Vivian Lam and Dr Maura Gasparetto for technical assis- tance; Dr Florian Kuchenbauer, Dr Sally Rogers and Dr Nooshin Tabatabaei for sharing expertise; Dr Claudia Luther, Evette Haddad and Tim Halim for sharing reagents. We are very grateful to the staﬀ of the TFL cell sorting facility and the BCCRC animal facility. We are thankful to Dr Stephen K. Anderson for the gift of 12A8 antibody. FUNDING Canadian Institutes of Health Research with core support from the British Columbia Cancer Agency. 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