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Importance of Acidic, Proline/Serine/Threonine-Rich, And Proc. Natl. Acad. Sci. USA Vol. 94, pp. 2501–2506, March 1997 Immunology Importance of acidic, prolineyserineythreonine-rich, and GTP- binding regions in the major histocompatibility complex class II transactivator: Generation of transdominant- negative mutants KEH-CHUANG CHIN*†,GEORGE G.-X. LI†‡, AND JENNY P.-Y. TING†‡ *Department of Biochemistry and Biophysics, †Lineberger Comprehensive Cancer Center, and ‡Department of Microbiology–Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295 Communicated by George Stark, Cleveland Clinic Foundation, Cleveland, OH, December 31, 1996 (received for review September 3, 1996) ABSTRACT The class II transactivator (CIITA) is a (11). CIITA was cloned by its ability to complement RJ2.2.5, master transcription regulator of gene products involved in an in vitro-generated MHC class II negative cell derived from the exogenous antigen presentation pathway, including major Raji (11, 18). Several groups, including our own, have shown histocompatibility complex (MHC) class II, invariant chain, that CIITA is induced by IFN-g and that transfection of CIITA and DM. An extensive analysis of the putative functional alone into cells is sufficient to activate MHC class II (19–21), domains of CIITA is undertaken here to explore the action of invariant chain (19, 22), and HLA-DM genes (22). CIITA. Antibodies to CIITA protein were produced to verify A major issue in the field concerns the mode of action of that these mutant proteins are expressed. Both acidic and CIITA. Although CIITA is a strong transactivator, it does not prolineyserineythreonine-rich domains are essential for class bind MHC class II promoter elements, nor does it appear to II MHC promoter activation. In addition, three guanine interact with transcription factors that bind these elements. In nucleotide-binding motifs are essential for CIITA activity. Of this report, we have undertaken an extensive domain analysis these mutants, two exhibited strong transdominant-negative to delineate functional domains of CIITA. The results show functions. These two mutants provide a plausible approach to that the proline-, serine-, and threonine-rich domains are manipulate MHC class II expression and immune responses. critical for function. In addition, a GTP-binding domain consisting of phosphate-, Mg21-, and guanine-binding motifs is Major histocompatibility complex (MHC) gene products DR, crucial for CIITA function, strongly implicating a role for GTP DP, and DQ play a critical role in the presentation of processed binding. Two of the mutants are highly efficient transdomi- exogenous antigen to T cells (1–3). MHC class II molecules are nant-negative molecules that nearly abolished the normal constitutively expressed at high levels in B lymphocytes and function of CIITA. The biological implications of these ob- dendritic cells and are induced in certain cell types such as servations are discussed. macrophages, endothelial cells, astrocytes, and microglia upon treatment with interferon-g (IFN-g). The appropriate consti- MATERIALS AND METHODS tutive and inducible expression of MHC class II molecules is essential for normal immune response, whereas aberrantly Cell Cultures and Transient Transfection. 2fTGH, G3A, high and low expression have been correlated with various Raji, Namalwa, and RJ2.2.5 cells were cultured as previously autoimmune diseases (4, 5) and a type of severe combined described (18, 19, 23). COS-7, a T antigen-transformed mon- immunodeficiency disease, the bare lymphocyte syndrome key kidney cell line, was maintained in DMEM-H medium (BLS), respectively (6, 7). Patients with BLS lack MHC class containing 10% fetal calf serum (GIBCOyBRL). 2fTGH, II antigen expression on both constitutive and IFN-g-inducible G3A, and RJ2.2.5 cells were transfected as described previ- cells (8, 9). The lack of MHC class II antigen expression on ously (19, 24). cells from group A of type II BLS patients is primarily due to DNA Constructs. The mammalian expression vector, a defect in the transcription factor, class II transactivator pcDNA3.FLAG.CIITA8 (FLAG.CIITA8), contains the (CIITA), initially identified as AIR-1 (10, 11). FLAG epitope (DYKDDDDK) upstream of the first methi- The primary regulation of constitutive and IFN-g-induced onine of CIITA8. All mutant constructs were generated by a MHC class II genes is at the transcriptional level (9, 12, 13). published method (25) from the parental plasmid, FLAG.CI- The MHC class II, invariant chain, and DMAyDMB genes ITA8, with a selection primer, 59-AAATGCTTCAAT- contain three highly conserved DNA cis-acting elements: the gcTAGcgAAAAAGGAAG-39, which mutated a SspItoNheI W, X, and Y boxes (14), also known collectively as the class II restriction site, and a mutagenic primer. All mutants were box. These three elements exhibit conservation in sequence, as initially identified by the presence of an NheI site and later well as spacing constraint and regimented stereospecific align- confirmed by sequencing. ment (15, 16). Construction Of ProlineySerineyThreonine Deletion Mu- The transcription factors that directly bind to the MHC class tants. CIITA(D132–301) was constructed by creating ClaI II promoter elements are well studied (9, 12, 17), but expres- restriction sites at nucleotides 511 (amino acids 132–133) and sion of these proteins is generally ubiquitous and does not 1013 (amino acids 300–301) of CIITA8 using two mutagenic parallel MHC class II gene expression. In contrast, expression primers: 59-533-GCCCAACTTCTGCTGGCATaTCgATAC- of CIITA closely parallels that of MHC class II gene expression TCTCACCGATCAC-493-39 and 59-1036-GAGGTCAGGG- CAGGTTCAtcgATGCTGGGCAGGTCAGTG-997-39. The The publication costs of this article were defrayed in part by page charge mutagenized construct was digested with ClaI to release the payment. This article must therefore be hereby marked ‘‘advertisement’’ in prolineyserineythreonine-containing fragment, and the re- accordance with 18 U.S.C. §1734 solely to indicate this fact. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA Abbreviations: MHC, major histocompatibility complex; IFN-g, in- 0027-8424y97y942501-6$2.00y0 terferon-g; CIITA, class II transactivator; CAT, chloramphenicol PNAS is available online at http:yywww.pnas.org. acetyltransferase. 2501 2502 Immunology: Chin et al. Proc. Natl. Acad. Sci. USA 94 (1997) maining sequence was in the plasmid religated. CIITA(D132– 209) was constructed in the same way as mentioned above with mutagenic primers, 59-533-GCCCAACTTCTGCTGGCATa- TCgATACTCTCACCGATCAC-493-39 and 59-766-CAAC- GAGGAACTGGAGAAAtcgATGGGAATCTGGTCGGT- TTTC-724-39, and it contained a clean deletion of the proliney serine-rich domain. CIITA(D209–301) has deleted threonine- rich domain from amino acid residues of 209–301. This mutant construct was mutagenized with primers: 59-766-CAACGAG- GAACTGGAGAAAtcgATGGGAATCTGGTCGGTTT- TC-724-39 and 59-1036-GAGGTCAGGGCAGGTTCAtcgA- TGCTGGGCAGGTCAGTG-997-39. Construction Of Nucleotide-Binding Motif Mutants. CI- ITA-GTP1(DGK) and CIITA-GTP1(K3E) were mutated in the phosphate-binding motif. CIITA-GTP1(DGK) has a de- letion of two amino acid residues, 420 and 421, by a ‘‘loop out’’ mutagenic primer: 59-1395-CTCTTGCCCTGACCAGCCAC- AGCAATCACTCGTG-1353-39. CIITA-GTP1(K 3 E) has a point mutation at residue 427 from lysine to glutamic acid. The mutagenic primer used to generate this mutant was 59-1408- CCCAGCCCAATAGCTCTcGCCCTGACCAGCTTTGCC- 1373-39. CIITA-GTP2(DDAYG) and CIITA-GTP3(DSKAD) have deletions in the Mg21- and guanine-binding motifs, respectively. CIITA-GTP2(DDAYG) was created by a loop out primer: 59-1522-GAGCAGATCCTGCAGCCCCG- GACGGTTCAAG-1480-39. Mutagenic primer used to con- struct GTP3(DSKAD) was 59-1813-CAGCTCAAAT- AGGGCCAGGCTCTGGACCAG-1777-39. Generation of Stable Transfectants Expressing Cell Surface MHC Class II Antigen with FLAG.CIITA8 Expression Vector. Ten micrograms of FLAG.CIITA8 plasmid was transfected into G3A cell. The stable transfectants expressing cell surface MHC class II antigens were selected by the immunomagnetic selection, according to manufacturer is protocol (Dynal, Oslo). The cells underwent immunoselection three times for the next 4 weeks. Cells expressing MHC class II antigens were con- firmed by fluorescence-activated cell sorting analysis (19). Anti-CIITA (a-CIITA) Antibody Production. Anti-human CIITA antibody was raised against a peptide, 726GEIKD- KELPQYLALTPR742, crosslinked to KLH (keyhole limpet FIG. 1. The FLAG foreign epitope did not alter CIITA activity. (A) hemocyanin; Pierce). The antiserum was tested after numer- FLAG.CIITA8 activated the DRA promoter in G3A cells. G3A cells ous injections of the rabbit with the antigen. were cotransfected with the pDRA300CAT construct and an empty Western Blot. Raji, Namalwa, and RJ2.2.5 nuclear extracts vector (lane 1), CIITA expression vector (lane 2), or FLAG.CIITA8 were prepared according to Dignam et al. (26). Whole cell (lanes 3 and 4). The cells were harvested and analyzed for CAT activity extract was prepared as described (27). The samples were 48 h after transfection. (B) FLAG.CIITA8 induces HLA-DRA ex- analyzed by immunoblotting with a-FLAG (10 mgyml; IBI– pression on the MHC class II-negative G3A mutant cells. 2fTGH cells Kodak) or a-CIITA (1.5 mgyml) antibodies using standard (Ba and Bb), G3A (Bc and Bd), and G3A (Be and Bf) stably integrated techniques (27). Immunoblots were detected by enhanced with FLAG.CIITA8 were not treated (Left) or treated (Right) with 500 unitsyml of IFN-g for 72 h. The cells were
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