The Journal of Immunology Modulation of Gene Expression by the MHC Class II Transactivator1 Uma M. Nagarajan, Alyssa Bushey, and Jeremy M. Boss2 The class II transactivator (CIITA) is a master regulator of MHC class II expression. CIITA also modulates the expression of MHC class I genes, suggesting that it may have a more global role in gene expression. To determine whether CIITA regulates genes other than the MHC class II and I family, DNA microarray analysis was used to compare the expression profiles of the CIITA expressing B cell line Raji and its CIITA-negative counterpart RJ2.2.5. The comparison identified a wide variety of genes whose expression was modulated by CIITA. Real time RT-PCR from Raji, RJ2.2.5, an RJ2.2.5 cell line complemented with CIITA, was performed to confirm the results and to further identify CIITA-regulated genes. CIITA-regulated genes were found to have diverse functions, which could impact Ag processing, signaling, and proliferation. Of note was the identification of a set of genes localized to chromosome 1p34-35. The global modulation of genes in a local region suggests that this region may share some regulatory control with the MHC. The Journal of Immunology, 2002, 169: 5078–5088. y mediating both constitutive and IFN-␥ inducible ex- (1, 15–17). The RFX complex, CREB (16, 18), and NFY (19) bind pression, the class II transactivator (CIITA)3 functions as to the X1, X2, and Y box motifs, respectively. CIITA’s role in B a master regulator of MHC class II genes (1, 2). CIITA activating gene expression appears to be through the recruitment of acts through a compact series of conserved cis-acting elements components of the basal transcription machinery (20–23), as well termed the W/Z, X1, X2, and Y boxes found 100–200 bp upstream as histone acetyltransferases that acetylate the local nucleosomes of all MHC class II isotypes (reviewed in Ref. 3). These cis-acting (15). CIITA has also been reported to possess intrinsic acetyltrans- elements are also found upstream of the invariant chain, HLA-DM, ferase activity (24). Besides its role in class II regulation, CIITA and HLA-DOA genes, which are coordinately regulated with the has also been shown to down-regulate IL-4 (25), Fas ligand (26), classical MHC class II genes (4, 5). Several years ago, it was and collagen ␣2 expression (27). This down-modulatory activity ␤ discovered that MHC class I and 2-microglobulin genes, which has been suggested to be due to CIITA’s ability to titrate CREB- are expressed constitutively, also contained WXY sequences, and binding protein (CBP) from the promoter/enhancers of these that CIITA could directly augment their expression through these genes. Thus, CIITA has the ability to regulate genes by at least two elements (6, 7). We recently found that the expression of HLA- mechanisms: the MHC-class II regulatory pathway and by titration DOB is decreased in B cells lacking CIITA (8), implying that its of limiting transcription factors/coactivators. expression is modulated by CIITA as well. Thus, the expression of The fact that CIITA can positively and negatively regulate genes a large number of genes involved in Ag processing and presenta- suggests that the presence of CIITA in cells may have broad con- tion is controlled either completely or in part by CIITA and the sequences on global gene activity. To determine whether CIITA class II regulatory pathway. regulates genes other than the MHC class II and I family, and to Genetic deficiencies in CIITA and members of the RFX com- plex RFX-B/ANK, RFX5, and RFXAP (9–12) are the basis of the identify novel CIITA regulated genes, DNA microarray analysis bare lymphocyte syndrome, a series of rare autosomal diseases that was used. The global expression profiles of the CIITA-expressing manifest in a severe combined immunodeficiency (reviewed in B cell line Raji (28) and its CIITA-negative counterpart RJ2.2.5 Refs. 13 and 14). Cell lines established from bare lymphocyte (29) were compared. RJ2.2.5 cells were derived directly from Raji syndrome patients have provided key reagents for the elucidation cells by gamma irradiation and selection for the loss of MHC class of the molecular mechanism of CIITA function and MHC class II II surface protein expression (29), and are mutant for both CIITA regulation. CIITA does not directly bind to MHC class II promoter alleles (1, 30). The results of this comparison revealed a set of DNA, but appears to exert its function by interacting with MHC genes that appeared to be up-regulated and down-modulated by class II DNA-bound transcription factors RFX, CREB, and NFY CIITA. Quantitative RT-PCR analysis using RNA from Raji, RJ2.2.5, and RJ2.2.5 stably transfected with CIITA demonstrated that many of these genes were in fact regulated by CIITA. A subset Department of Microbiology and Immunology, Emory University School of Medi- of the CIITA-regulated genes was tested and found to be induced cine, Atlanta, GA 30322 in fibroblasts following IFN-␥ treatment, suggesting that CIITA, Received for publication May 22, 2002. Accepted for publication August 28, 2002. which is induced by IFN-␥, can modulate their activity in other cell 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 types. Bioinformatics analysis was used to organize the genes by with 18 U.S.C. Section 1734 solely to indicate this fact. function and determine their chromosome location. The genes 1 This work was supported by National Institutes of Health Grants AI34000 and a modulated by CIITA were found to have diverse functions, sug- supplement to GM47310. gesting that CIITA may play a general role in the physiology of 2 Address correspondence and reprint requests to Dr. Jeremy M. Boss, Department of cells that are focused on presenting and processing Ags. A set of Microbiology and Immunology, Emory University School of Medicine, Room 3131, 1510 Clifton Road, Atlanta, GA 30322. E-mail address: [email protected] CIITA-modulated genes was found to be clustered on chromosome 3 Abbreviations used in this paper: CIITA, class II transactivator; HSP, heat shock 1p34-35, suggesting a novel mechanism of CIITA-mediated gene protein; CBP, CREB-binding protein; EST, expressed sequence tag. control. Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 Downloaded by guest on October 1, 2021 The Journal of Immunology 5079 Materials and Methods using primers for the GAPDH transcripts were conducted in each plate to Cell lines provide a normalization reference. The primer sets used are listed in Table I. The HLA-DRA and GAPDH primers sets have been previously described Raji, a Burkitt’s lymphoma derived cell line, is wild type for CIITA and (8, 15). The threshold cycle values for all genes were normalized to the positive for MHC class II gene expression (28). The human B cell line threshold cycle of GAPDH and converted to linear scale. All real-time RJ2.2.5 was derived by mutagenesis from Raji cells and selected for loss RT-PCR were conducted at least three times from independent RNA of HLA class II Ag expression (29). RJ2.2.5 is null for CIITA (1, 30). Raji preparations. and RJ2.2.5 were grown in RPMI supplemented with 5% FBS, 5% bovine Genomic DNA was isolated from Raji and RJ2.2.5 as described in Aus- calf serum, 2 mM glutamine, penicillin (5 U/ml), and streptomycin sulfate ubel et al. (32). Quantitative real-time PCR assays were conducted using (5 ␮g/ml). A431, a vulvar epithelial carcinoma cell line, was grown in various concentrations (25, 50, 100, and 200 ng) of genomic DNA for DMEM with 10% FBS and above supplements. A431 cells express both importin ␣ 7(KPNA6), tyrosyl tRNA synthetase (YARS), TGF-␤ receptor CIITA and MHC class II genes following IFN-␥ treatment (15). interacting protein (EIF3S2), and RbAp48 (RBBP4) genes. A three-step PCR with denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and cRNA preparation and DNA microarray hybridization extension at 68°C for 30 s was conducted for 40 cycles. The PCR primers Ј Ј Total RNA was isolated from cells by the Nonidet P-40-lysis method (31) used were: KPNA6,5-gacttgctggcagatgcttgctg, and 5 -gaccactcacatcag ␮ cagctc; YARS,5Ј-gtagggtatcacatctgcactgag, and 5Ј-ccagaacctcctattgtg or the RNeasy method (Qiagen, Valencia, CA). Total RNA (20 g) was Ј Ј used directly for cDNA synthesis using “The Super Choice” system for gaagc; and EIF3S2,5-gcgaagatggttacgtccgtatcc and 5 -gggattacaggcat cDNA synthesis (Life Technologies, Grand Island, NY) with an oligo(dT) gagccacc. For RBBP4, the primers used for RT-PCR (Table I) were used primer containing a T7 phage promoter sequence. cRNA was prepared for genomic PCR as they primed a single exon. from cDNA as described earlier (8). Bioinformatics Test DNA microarray chips from Affymetrix (Santa Clara, CA) were used to check the cRNA for equal hybridization to 5Ј and 3Ј oligonucle- Public and corporate databases were searched for information about the otides of housekeeping genes before each experiment. Three independent genes identified during microarray analysis. GenBank accession numbers experiments were conducted using the Affymetrix human U95A chips. Pre- used by Affymetrix were used to confirm gene identities in searches against hybridization and hybridizations were conducted as previously described GenBank (www.ncbi.nlm.nih.gov/entrez) and LocusLink (www.ncbi.nlm. (8). Arrays were scanned on a Hewlett-Packard gene array scanner nih.gov/LocusLink). The genes were grouped based on known or inferred (Hewlett-Packard, Palo Alto, CA).