The Role of High-Mobility Group I(Y) Proteins in Expression of IL-2 and T Cell Proliferation 1 S. Roy Himes, 2 * Raymond Reeves, ² Joanne Attema, ‡ Mark Nissen, ² Ying Li, ² and M. Frances Shannon 3‡ The high-mobility group I(Y) (HMGI(Y)) family of proteins plays an important architectural role in chromatin and have been implicated in the control of inducible gene expression. We have previously shown that expression of HMGI antisense RNA in Jurkat T cells inhibits the activity of the IL-2 promoter. Here we have investigated the role of HMGI(Y) in controlling IL-2 promoter-reporter constructs as well as the endogenous IL-2 gene in both Jurkat T cells and human PBL. We found that the IL-2 promoter has numerous binding sites for HMGI(Y), which overlap or are adjacent to the known transcription factor binding sites. HMGI(Y) modulates binding to the IL-2 promoter of at least three transcription factor families, AP-1, NF-AT and NF-B. By using a mutant HMGI that cannot bind to DNA but can still interact with the transcription factors, we found that DNA binding by HMGI was not essential for the promotion of transcription factor binding. However, the non-DNA binding mutant acts as a dominant negative protein in transfection assays, suggesting that the formation of functional HMGI(Y)-containing complexes requires DNA binding as well as protein:protein interactions. The alteration of HMGI(Y) levels affects IL-2 promoter activity not only in Jurkat T cells but also in PBL. Importantly, we also show here that expression of the endogenous IL-2 gene as well as proliferation of PBL are affected by changes in HMGI(Y) levels. These results demonstrate a major role for HMGI(Y) in IL-2 expression and hence T cell proliferation. The Journal of Immunology, 2000, 164: 3157–3168. A ctivation of Th cells requires the specific binding of the TCR to an Ag-MHC class II complex on the surface of APCs as well as costimulation through the interaction of cell-surface molecule pairs such as CD28 on T cells and B7 on APCs (1, 2). Signal transduction through the TCR and CD28 in- duces the activation and nuclear expression of a number of tran- scription factors, including NF-B/Rel, NF-AT and AP-1 family proteins (reviewed in Refs. 3 and 4). This cascade of events results in the coordinate expression of a variety of genes including the cytokine IL-2, which functions as a major growth factor for T cells, and expression of IL-2R, which allows the formation of a high-affinity receptor for IL-2. The resulting autocrine loop can act as a strong growth stimulus allowing a specific clonal expansion of the T cell. The requirements for activation of IL-2 gene expression in re- sponse to T cell activation have been studied in detail. Initially, two major TCR responsive regions were identified in the IL-2 promoter (reviewed in Refs. 3 and 5). These were termed Ag re- sponse elements (ARRE), 4 i.e., ARRE-1 (or NF-IL-2A) and ARRE-2 (or NF-IL-2E). The ARRE-2 region was the first to be identified as a composite binding site for NF-AT and AP-1 pro- teins, whereas ARRE-1 was thought to function via the binding of AP-1 and Oct transcription factors (6 –10). Recently, the ARRE-1 region has been found to bind a complex of NF-AT and AP-1 as well as Oct (11). Several other regions of the promoter have also recently been identified as binding sites for NF-AT alone or as complexes with AP-1 (11). The mechanism of costimulation through the CD28 receptor involves enhanced nuclear expression of NF-B and c-Rel transcription factors (12–14), as well as phos- phorylation of c-Jun by JNK kinase, resulting in increased trans- activation competence for AP-1 (15, 16). The region of the IL-2 gene that responds to CD28 (the CD28RR) is composed of a vari- ant NF-B site (referred to as the CD28RE), which has a high affinity for c-Rel-containing complexes, as well as an adjacent AP-1 site. These elements cooperate to lead to increased IL-2 pro- moter activity (17–20). The highly inducible nature of the IL-2 promoter seems to be the result of coordinate binding of many transcription factors to their recognition sequences on the promoter leading to the assembly of a functional unit (3, 4, 11, 14, 21–24). Such a unit has been termed an enhanceosome on the IFN- promoter (25). The family of nu- clear proteins known as high-mobility group I(Y) (HMGI(Y)) are known to play a major role in the assembly of the IFN- enhan- ceosome (26 –28). We have previously shown that HMGI(Y) is essential for the selective binding of c-Rel to the IL-2 CD28RE and that modulating HMGI(Y) levels affects not only the activity of the CD28RR but of the entire promoter (14). HMGI(Y) proteins are small nonhistone nuclear proteins that bind the narrow minor groove of A:T sequence-rich B form DNA *Hanson Center for Cancer Research, Adelaide, South Australia; ² Department of Biochemistry and Biophysics, Washington State University, Pullman, WA 99164; and ‡ Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra, Australia Received for publication September 22, 1999. Accepted for publication January 4, 2000. 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, in part, by National Institutes of Health Grant RO1- GM46352 (to R.R) and a National Health and Medical Research Council (Australia) Program Grant (to M.F.S.). 2 Current address: Department of Microbiology, University of Queensland, Brisbane, Australia. 3 Address correspondence and reprint requests to Dr. M. Frances Shannon, Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra ACT 2601, Australia. E-mail address: frances.shannon@anu.edu.au 4 Abbreviations used in this paper: ARRE, Ag response element; HMGI(Y), high- mobility group protein I or Y; CD28RR, CD28 response region; CD28RE, CD28 response element; P/I, PMA and calcium ionophore; LTR, long terminal repeat; GFP, green fluorescence protein. Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00