Published online 15 July 2009 Nucleic Acids Research, 2009, Vol. 37, No. 16 5331–5342 doi:10.1093/nar/gkp592 Evidence for high bi-allelic expression of activating Ly49 receptors Arefeh Rouhi1, C. Benjamin Lai1,2, Tammy P. Cheng 3 , Fumio Takei1,4, Wayne M. Yokoyama3 and Dixie L. Mager1,2,* 1The Terry Fox laboratory, British Columbia Cancer Agency, 2Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada, 3Howard Hughes Medical Institute, Rheumatology Division, Washington University School of Medicine, St Louis, MO 63110, USA and 4Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada Received April 4, 2009; Revised May 28, 2009; Accepted June 27, 2009 ABSTRACT Stochastic expression is a hallmark of the Ly49 family that encode the main MHC class-I-recogniz- ing receptors of mouse natural killer (NK) cells. This highly polygenic and polymorphic family includes both activating and inhibitory receptor genes and is one of genome’s fastest evolving loci. The inhibitory Ly49 genes are expressed in a sto- chastic mono-allelic manner, possibly under the control of an upstream bi-directional early promoter and show mono-allelic DNA methylation patterns. To date, no studies have directly addressed the transcriptional regulation of the activating Ly49 receptors. Our study shows differences in DNA methylation pattern between activating and inhibi- tory genes in C57BL/6 and F1 hybrid mouse strains. We also show a bias towards bi-allelic expression of the activating receptors based on allele-specific single-cell RT–PCR in F1 hybrid NK cells for Ly49d and Ly49H expression in Ly49h+/ mice. Further- more, we have identified a region of high sequence identity with possible transcriptional regulatory capacity for the activating Ly49 genes. Our results also point to a likely difference between NK and T-cells in their ability to transcribe the activating Ly49 genes. These studies highlight the complex regulation of this rapidly evolving gene family of central importance in mouse NK cell function. INTRODUCTION Natural killer (NK) cells constitute an important part of the body’s defence both as centurions of innate immunity and as communicators and collaborators of the adaptive immune system. They exert their function via the interpretation of signals received from their surface receptors. Normal cells display major histocompatibility complex (MHC) class I molecules that are recognized by a number of inhibitory receptors on the surface of NK cells. The inhibitory receptors prevent activation of NK cells and the destruction of normal, MHC class I-possessing cells (1). NK cells also possess stimulatory (activating) receptors that recognize other molecules on the surface of potential target cells which are mostly pathogen- encoded or stress-induced self proteins (2–4). NK activation and the killing of target cells therefore depend on the balance between stimulatory and inhibitory signals received from these surface receptors. In primates and some other mammals, the killer cell immunoglobulin- like (KIR) family of genes code for the main MHC class-I recognizing receptors (5). However, in rodents, the struc- turally unrelated Ly49 family provides this function. Both KIR and Ly49 families are polygenic and polymorphic among diﬀerent individuals and mouse strains (6,7). The KIR gene cluster of human is 150 kb containing approximately 14 genes and pseudogenes with very high coding and regulatory sequence similarity (8) indicating that they arose through recent gene duplication events (9). There is great diversity in both gene number and sequence polymorphisms for the KIRs among diﬀerent people (6). The Ly49 gene cluster has also arisen from recent duplications and gene conversions of ancestral genes (10–12). Hence, there is high sequence similarity both in the coding and non-coding regions among the majority of the genes. The Ly49 cluster includes 16 genes and pseudogenes spanning over 600 kb in the C57BL/6 (B6) mouse strain and is located in the natural killer gene complex (NKC) region of chromosome 6 (8,13). There are two functional Ly49 activating receptors, Ly49D and H, coded by the B6 genome. Ly49D binds to the MHC-class-I allele H2-Dd (14,15) and Ly49H binds to the m157 protein of mouse cytomegalovirus (MCMV) conferring resistance towards virus infection in the strains of mice that express Ly49H (2,3,16,17). Unlike the *To whom correspondence should be addressed. Tel: +604 675 8139; Fax: +604 877 0712; Email: dmager@bccrc.ca 2009 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.