Promoter Region Architecture and Transcriptional Regulation of the Genes for the MHC Class I-Related Chain A and B Ligands of NKG2D 1 Gopalakrishnan M. Venkataraman,* Dominic Suciu,* Veronika Groh,* Jeremy M. Boss, † and Thomas Spies 2 * Ligands of the NKG2D receptor, which activates NK cells and costimulates effector T cells, are inducibly expressed under harmful conditions, such as malignancies and microbial infections. Moreover, aberrant expression in autoimmune disease lesions may contribute to disease progression. Among these ligands are the closely related human MHC class I-related chains (MIC) A and B, which appear to be regulated by cellular stress. Analyses of MIC gene 5-end flanking regions in epithelial tumor cells defined minimal core promoters that directed near maximum heat shock- or oxidative stress-induced transcriptional activation. Consid- erably larger fully functional promoters were required for maximum proliferation-associated activation. These activities were dependent on core promoter sequences that included heat shock elements, which inducibly bound heat shock factor 1, TATA-like elements, and constitutively occupied Sp1 and inverted CCAAT box factor sites. By contrast, MIC gene activation by CMV infection was largely independent of these and upstream promoter sequences, and expression of viral immediate early gene (IE1 or IE2) products was sufficient for induction of transcription and surface protein expression. Altogether, these results reveal distinct modes of activation of the genes for the MIC ligands of NKG2D and provide a molecular framework for analyses of gene regulation under different cellular insult conditions. The Journal of Immunology, 2007, 178: 961–969. D iverse MHC class I-like molecules that have no role in Ag presentation and limited tissue distributions serve as ligands for the NKG2D-DAP10 receptor complex, which activates NK cells and costimulates effector T cell subsets (1, 2). In humans, these ligands include the closely related MHC class I-related chains (MIC) 3 A and B (MICA and MICB) trans- membrane glycoproteins, which are encoded near HLA-B in the MHC and are represented by orthologous sequences in most mam- mals, except rodents (3). Expression of MIC is mostly restricted to intestinal mucosa, but can be induced by CMV infection in fibro- blasts and endothelial cells, and by mycobacterial infection in den- dritic and epithelial cells (4 – 6). Moreover, MIC are frequently associated with epithelial tumors of diverse tissue origins and are aberrantly expressed in rheumatoid arthritis synoviocytes and ce- liac disease intestinal epithelial cells (7–9). Thus, NKG2D trigger- ing by MIC delivers immunostimulatory signals that can be beneficial under adverse conditions, such as infections and malig- nancies, but may exacerbate autoimmune disease progression. Despite the immunological significance of MIC, molecular mechanisms controlling gene regulation are poorly defined, and it is unknown whether and how different cellular and environmental stimuli converge to induce gene expression. Recent evidence has indicated that activation of DNA damage control pathways results in induction of NKG2D ligands, including UL16-binding protein family members and possibly MICA (10). Moreover, the 5'-end flanking regions of MICA and MICB contain putative heat shock elements (HSE), which are prototypic transcription inducer sites in heat shock protein 70 (HSP70) genes that bind activated trimeric heat shock factor 1 (HSF1) (4, 11–13). With cell lines, MIC mRNA and protein expression are mostly limited to proliferating epithelial cells. Quiescent epithelial cells grown for extended time at high confluence display relatively small amounts of MIC mRNA and surface proteins that are sharply increased upon exposure to heat shock (14). Oxidative stress has also been found to induce MIC gene expression in colon carcinoma cells, although increased cell surface expression was not observed (15). The present study has used molecular and functional approaches to define the archi- tecture of MIC gene promoter regions and the significance of tran- scriptional control elements for cell stress-induced, proliferation- associated, and CMV-mediated transcriptional activation. Materials and Methods Cell culture, heat shock, and flow cytometry Cell lines were from the American Type Culture Collection. Primary hu- man fibroblasts (passages 4 – 6) and Hela S3 cells were grown in Way- mouth’s and MEM-Joklik medium (Invitrogen Life Technologies) supple- mented with 10% FBS (HyClone), glutamine, and antibiotics. Surface expression of MICA, or of MICA and MICB, was monitored by flow cytometry using mAbs 2C10 and 6G6, respectively (4, 14). For heat shock, culture plates with adherent HCT116 cells grown for 7 days at high con- fluence were sealed with parafilm and floated for 1 h on a 42.5°C water *Fred Hutchinson Cancer Research Center, Seattle, WA 98109; and † Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322 Received for publication November 22, 2005. Accepted for publication October 27, 2006. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Grants GM47310 (to J.M.B.) and AI30581 and AI52319 (to T.S.) from the National Institutes of Health. 2 Address correspondence and reprint requests to Dr. Thomas Spies, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, D1-100, Seattle, WA 98109. E-mail address: tspies@fhcrc.org 3 Abbreviations used in this paper: MIC, MHC class I-related chain; CBF, CCAAT box factor; ChIP, chromatin immunoprecipitation; HSE, heat shock element; HSF1, heat shock factor 1; HSP, heat shock protein; ICE, inverted CCAAT box-like element; IE, immediate early; Inr, initiator. Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 The Journal of Immunology www.jimmunol.org