Murine natural killer cells selectively express members of the Ly49 family of class I MHC receptors; however, the molecular mechanism controlling probabilistic expression of Ly49 proteins has not been defined. A pair of overlapping, divergent promoters discovered in the Ly49g gene functions as a molecular switch that can produce a forward transcript containing the coding region of the gene (on position) or a noncoding transcript in the opposite direction (off position), and this element maintains transcription in the chosen direction. Competition of C/EBP and TBP transcription factors for overlapping binding sites determines the relative strength of the competing promoters and the probability of transcription in a given direction. Similar elements precede all Ly49 family members, and the relative strength of the forward promoter in each inhibitory Ly49 gene correlates with the percentage of natural killer cells that express a given receptor, supporting a promoter competition model of selective gene activation.

The Ly49 family of NK cell receptors and its MHC-binding characteristics have only been well characterized in C57BL/6 (B6) mice. Previous studies have shown that 129/J mice express unique Ly49 genes that are not found in the B6 strain. Screening of a 129/J cDNA library led to the discovery of 10 distinct full-length Ly49-related coding sequences (Ly49e, g, i, o, p, r, s, t, u, and v). Although 129/J mice share identical class I MHC (Kb and Db) transcripts with B6 mice, only one Ly49 is identical in the two strains (Ly49E). In addition to the previously characterized Ly49P, two new activating Ly49 proteins were discovered, Ly49R and U. The MHC specificity of the total 129/J Ly49 repertoire was evaluated with soluble class I MHC tetramers and found to be distinct compared with the B6 Ly49 repertoire. Ly49V bound to many types of class I MHC, suggesting that Ly49V+ NK cells may monitor host cells for a global down-regulation in MHC levels. An activating receptor, Ly49R, was shown to bind soluble class I molecules to a moderate degree, a result not previously observed for other activating Ly49 proteins. Furthermore, tetramer-binding results were confirmed functionally with cytotoxicity assays using sorted 129/J NK cells. This study shows that the Ly49 repertoire and its MHC-binding characteristics can be very different among inbred mouse strains. Ly49 divergence should be considered when using 129-derived embryonic stem cells for the production of gene-targeted mice, especially when an immune or NK-derived phenotype is under scrutiny.

The analysis of several Ly49 genes has identified a tissue-specific promoter adjacent to the previously defined first exon. The current study reveals the presence of an additional Ly49 promoter (Pro-1) and two noncoding exons upstream of the previously defined promoter (Pro-2). DNA sequences homologous to Pro-1 are present 4–10 kb upstream of Pro-2 in all Ly49 genes examined, and Pro-1 transcripts were detected from the Ly49a, e, g, o, and v genes. Pro-1 activity can be detected in bone marrow, embryonic thymus, freshly isolated liver NK cells, and the murine LNK cell line, but it does not function in adult thymus, sorted NK-T cells, spleen NK cells, or the EL-4 T cell line, even though these cells express Ly49 proteins. Luciferase reporter assays identified a Pro-1 core promoter region that functions in the LNK cell line but not EL-4 cells. The novel promoter is not active in mature NK cells, suggesting that Pro-1 represents an early Ly49 promoter.