Analysis of the Defect in IFN-γ Induction of MHC Class II in G1B Cells: Identification of a Novel and Functionally Critical Leucine-Rich Motif This information is current as (62-LYLYLQL-68) in the Regulatory Factor of September 28, 2021. X 5 W. June Brickey, Kenneth L. Wright, Xin-Sheng Zhu and Jenny P.-Y. Ting Downloaded from J Immunol 1999; 163:6622-6630; ; http://www.jimmunol.org/content/163/12/6622

References This article cites 62 articles, 36 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/163/12/6622.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Analysis of the Defect in IFN-␥ Induction of MHC Class II Genes in G1B Cells: Identification of a Novel and Functionally Critical Leucine-Rich Motif (62-LYLYLQL-68) in the Regulatory Factor X 5 Transcription Factor1

W. June Brickey,* Kenneth L. Wright,2* Xin-Sheng Zhu,† and Jenny P.-Y. Ting3*

MHC class II deficiency found in patients results from the absence or dysfunction of MHC class II transcriptional regulators, such as regulatory factor X (RFX) and class II transactivator (CIITA). Understanding the roles of these factors has been greatly facilitated by the study of genetic defects in cell lines of bare lymphocyte syndrome patients, as well as in cell lines that have been generated by chemical mutagenesis in vitro. The latter group includes MHC class II-deficient lines that are no longer responsive to induction by IFN-␥. Here, we show that the defect in G1B, one such cell line, is attributed to the lack Downloaded from of functional RFX5, the largest subunit of RFX. The RFX5 isolated from G1B cells contains two separate single- mutations. One alteration does not exhibit a phenotype, whereas a leucine-to-histidine mutation eliminates DNA-binding and transactivating functions. This mutation lies outside of previously defined functional domains of RFX5 but within an unusual, leucine-rich region (62-LYLYLQL-68). To further investigate the significance of the leucine-rich region, we targeted all neigh- boring leucine residues for mutagenesis. These mutants were also unable to transactivate a MHC class II reporter gene, confirming http://www.jimmunol.org/ that these leucine residues play an essential role in RFX activity and characterize a novel leucine-rich motif. The Journal of Immunology, 1999, 163: 6622–6630.

he IFN-␥ response of most genes requires a common cas- Ags. These include the invariant chain (Ii)4 (3) and the noncon- cade of activating that regulate the induction of a ventional DM molecules. The expression of HLA-D, Ii, and DM T variety of genes. Many of these molecules have been well are in general synchronously regulated, and their levels fluctuate defined by biochemical and genetic approaches. However, the coordinately upon modulation (3–5). Their presence is restricted to IFN-␥ induction of MHC class II molecules requires additional APCs, such as B cells, macrophages, thymic epithelia, and acti-

specific transcriptional activators. The strongest evidence for this vated T cells. Their level of expression is primarily regulated by by guest on September 28, 2021 requirement comes from the analyses of somatic mutant cell lines the activity of transcription factors (6–10). Therefore, understand- that are normal in their IFN-␥ responses except for the lack of ing the players and mechanisms of transcriptional regulation of induction of genes encoding MHC class II and associated mole- these genes will have significant biologic ramifications. cules (1, 2). This report focuses on the understanding of the mo- The MHC class II promoters as well as the Ii and DM promoters lecular and genetic basis for one such mutant cell line. are unique for the presence and stereochemical arrangement of The MHC class II molecules, HLA-D, play key regulatory roles three regulatory elements, the S/W, X, and Y (also known as a in T cell-mediated immune responses by presenting antigenic pep- CCAAT motif) elements (10–13). The factors that bind to X and tides on cell surfaces for recognition by class II-restricted T cells. Y boxes have been identified as the multimeric regulatory factor X Molecules other than the conventional MHC class II molecules are (RFX; Refs. 14 and 15) and trimeric nuclear factor Y (NF-Y) DNA required for optimal processing and presentation of a spectrum of binding proteins (16), respectively. An additional factor, known as X2 binding (X2BP), binds to the X2 element present in some promoters (17). Binding of factors to the X and Y elements serves novel roles, such as opening up previously closed promoters *UNC Lineberger Comprehensive Cancer Center and Department of Immunology and Microbiology and †Curriculum in Oral Biology, School of Dentistry, University in vivo and promoting accessibility to regulatory elements (18, 19). of North Carolina, Chapel Hill, NC 27599 Mutant cell lines that are defective in the expression of HLA-D Received for publication . Accepted for publication . molecules provide an important means for understanding the tran- The costs of publication of this article were defrayed in part by the payment of page scriptional regulation of MHC class II and related genes. These charges. This article must therefore be hereby marked advertisement in accordance systems include cells derived from patients afflicted with bare lym- with 18 U.S.C. Section 1734 solely to indicate this fact. phocyte syndrome (BLS), as well as cells generated in vitro by 1 This work was supported by National Institutes of Health Grants AI29564 and negative selection for the loss of HLA-D expression. The latter AI41580 to J. P.-Y. T.), and Multiple Sclerosis Society Grant RG1785 (to J. P.-Y. T.). W. J. B. was supported by National Institutes of Health Postdoctoral Training Grant includes both B cell lines that have lost constitutive MHC class II CA09156 and is the recipient of a National Kidney Foundation postdoctoral fellow- expression and IFN-␥-defective cell lines that have selectively lost ship. K. L. W. is an Arthritis Foundation Fellowship awardee. the IFN-␥ induction of HLA-D expression (1, 20–23). The mutant 2 Present address: H. Lee Moffitt Cancer Center, Department of Biochemistry and Molecular Biology, University of South Florida, 12901 Magnolia Drive, Tampa, FL 33612. 4 Abbreviations used in this paper: Ii, invariant chain; RFX, regulatory factor X; 3 Address correspondence and reprint requests to Dr. Jenny P.-Y. Ting, UNC NF-Y, nuclear factor Y; X2BP, X2 binding protein; BLS, bare lymphocyte syndrome; Lineberger Comprehensive Cancer Center, Campus Box 7295, Room 209, University CIITA, class II transactivator; DBD, DNA binding domain; MMTV, mouse mam- of North Carolina, Chapel Hill, NC 27599-7295. E-mail address: panyun@med. mary tumor virus; CAT, chloramphenicol acetyltransferase; RT, reverse transcriptase; unc.edu wt, wild type; LRR, leucine-rich repeats.

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 6623

EBV-transformed B cell lines fall into four complementation COOH. Each was conjugated to keyhole limpet hemocyanin before groups, and the genetic defects in all of these cell lines have been immunization. High titer antisera was subsequently purified by passage cataloged as defects either in the class II transactivator (CIITA) over protein A/G Sepharose columns as described by the manufacturer (Pierce, Rockford, IL). (24) or in the individual components of RFX (25, 26). To date, mutant cells defective in NF-Y have not been found, and this is EMSA, oligonucleotide competitions, and Ab supershift presumably due to the critical role NF-Y plays in the regulation of a large repertoire of CCAAT-containing promoters (19, 27, 28). Crude nuclear extracts were prepared as described (37). Gel-shift reactions were performed essentially as previously described (38), with slight mod- The focus of this study, RFX5, is defective in the complemen- ifications. Binding reactions (20 ␮l volume) were conducted in 12% glyc- tation group C of HLA-D-negative cell lines. The gene for RFX5 erol, 60 mM KCl, 12 mM HEPES (pH 7.9), 5 mM MgCl2, 0.06 mM was identified by complementation cloning using the BLS cell line EDTA, 0.5 mM DTT, and 0.5 mM PMSF with the nonspecific competitor SJO (25). RFX5 belongs to a family of novel DNA binding pro- mix of 1 ␮g poly(dI-dC) (Pharmacia, Piscataway, NJ) and 0.5 ␮g sheared teins, including among its members RFX1 to RFX4 (29). This salmon sperm DNA (Sigma, St. Louis, MO). To eliminate RFX5 family members that bind preferentially to methylated DNA, 50 ng of a high family shares an unusual and highly conserved DNA binding do- affinity methylated pBR322 oligonucleotide, 5Ј-GATMGCMGTGAM main (DBD) (30, 31). It has been shown that RFX5 is the largest GATC-3Ј (where M ϭ 5-methyl cytosine), was included in each binding component, 75 kDa (3), of the multimeric nuclear complex RFX reaction. Approximately 0.2–0.5 ng of the S/WXY DNA probe and 4–6 and that this subunit directs the binding of this complex exclusively to ␮g of nuclear extract were used. Binding reactions were incubated for 40 min on ice and then for 20 min at 30°C to promote inclusion of the X2BP. the X element in MHC class II and related genes (8, 10, 32). The samples were resolved by electrophoresis through 4% (40:1 acryl- In parallel to the study of mutant B cell lines, our earlier exam- amide:bis-acrylamide) native polyacrylamide gels with a TGE running ination of IFN-␥-defective mutant cell lines revealed that one of buffer (25 mM Tris-HCl, pH 7.9, 190 mM glycine, and 1 mM EDTA) and Downloaded from the mutant cell lines, namely G3A, is defective in the expression of electrophoresed for 2.6 h at 200 V in a 4°C room. The S/WXY probe was CIITA, whereas G1B is defective in X box binding activity, pre- generated by PCR using the p152DRA-chloramphenicol acetyltransferase (CAT) (3) plasmid (18) and the following radioactive primers: DRA152s1, sumably RFX (4). This present report constitutes the first detailed 5Ј-GAACGGAGTATCTTGTGTC-3Ј, and DRA48s2, 5Ј-CAAATCAAT analysis of the defect in G1B, which lies in two point mutations of TACTCTTTGG-3Ј. the RFX5 subunit of RFX. One of these two mutations (Leu66His) The sequences of the oligonucleotides used for competition are: Y box, 5Ј-AAATATTTTTCTGATTGGCCAAAGAGTAAT-3Ј, and S/W box, 5Ј- is responsible for IFN-␥ nonresponsiveness, and it lies outside of http://www.jimmunol.org/ CTTGTGTCCTGGACCCTTTGCAAGAACCCTTC-3Ј. The X1, X2, the previously defined DNA-binding domain. Although this mu- X1/Y, X2/Y, and mut oligonucleotide competitors are all based on a 60-bp tation does not affect the level of RFX5 expression, it eliminates HLA-DRA fragment spanning the X/Y motif (–118 to –59) with appro- the DNA binding activity and transactivation potential of RFX5. priate mutations underlined in each of the elements. The X1 competitor To ascertain the significance of this leucine-rich region, mutations sequence is 5Ј-GAACCCTTCCCCTAGCAACAGATTGTGAGTCTCAA Ј Ј in neighboring leucines were generated in vitro. Analysis of these AATATTTTTCGGAGGTTCCAAAGAG-3 . The X2 competitor is 5 - GAACCCTTAGAACTAGTCCAGATGCGTCATCTCAAAATATTTTT novel RFX5 mutants revealed that alteration of any of the leucines CGGAGGTTCCAAAGG-3Ј. The X1/Y and X2/Y competitors are the eliminated transactivation function of RFX5. These results de- same as the X1 and X2 competitors, respectively, except that the Y box is scribe an important characterization of the G1B mutant RFX5 not mutated. The mut competitor contains mutations in all three sites. (G1B-RFX5) and have important implications for suppressing the Ab supershift reactions were performed as stated above, except 2 ␮lof by guest on September 28, 2021 protein A/G affinity-purified preimmune or immune Ab were added to each function of RFX5 and therefore modulating MHC class II gene reaction for2hat4°Cbefore the addition of the oligonucleotide probe. The expression. Equally significant, this study identified an unusual, probe was then added, and the reactions were incubated as described but pivotal leucine-rich motif that is important for the function above. The protein-DNA complexes were resolved by electrophoresis as of RFX5. described.

Materials and Methods Western blot analysis Cell culture About 25 million cells were lysed in 50 mM HEPES (pH 7.9), 500 mM KCl, 10% glycerol, 0.1 mM EDTA, 0.5% Nonidet P-40, DTT, and PMSF The human fibrosarcoma cell lines that are defective in IFN-␥ induction of at 4°C for 1 h. Samples were prepared and analyzed by SDS-PAGE elec- MHC class II were generated earlier (1). G1B, G2A, G3A, trophoresis and electroblotting to nitrocellulose as described (39). Protein and G4A HLA-D-negative cell lines were generously provided by George A/G affinity-purified rabbit anti-RFX5 Ab and anti-NF-YA (18) polyclonal R. Stark and Catherine Mao (The Cleveland Research Foundation, Cleve- sera were used at a final concentration of 2 ␮g/ml to probe the blotted land, OH). The 2fTGH cell line (wild-type (wt) control) derived from the proteins, as described previously (40). To detect the primary Ab-protein human sarcoma line HT1080 (American Type Culture Collection, Manas- complexes, the blots were incubated with secondary goat anti-rabbit Abs sas, VA) only expresses high levels of MHC class II with IFN-␥ induction. conjugated with horseradish peroxidase (Bio-Rad Laboratories, Gaithers- For the experiments here, these lines were cultured in DMEM supple- burg, MD) at a dilution of 1:4000. Signals were visualized using the en- mented with 10% FBS and 2 mM L-glutamine. hanced chemiluminescence detection system (Amersham Life Science, Ar- Several human B lymphoid cell lines were utilized in this study. The lington Heights, IL) and autoradiography. SJO human B cell line is defective in RFX binding and belongs to the complementation group C of MHC class II-deficient patients with BLS (33–35). The RJ2.2.5 cell line lacking CIITA activity is a MHC class DNA constructs II-negative mutant B cell line (36). Raji is a human EBV-positive Burkitt’s Complementary DNAs were prepared from total RNA isolated from the wt lymphoma cell line that expresses high levels of MHC class II molecules. control 2fTGH and the IFN-␥-defective G1B cell lines. The cDNAs were Namalwa cells, a gift from R. Roeder (Rockefeller University, New York, generated using mouse mammary tumor virus reverse transcriptase (RT; NY), were used as a positive human B cell control. All the B cell suspen- Bio-Rad Laboratories) and random hexamers. These cDNA samples were sions were maintained in RPMI 1640 supplemented with 7.5–10% FBS and subjected to PCR amplification using the Extend Long Template PCR kit 2mML-glutamine. (Boehringer Mannheim, Indianapolis, IN) and the following primers: 5Ј- Ab generation and purification CAAGTGCCCTCATGCCGGGATGG-3Ј and 5Ј-CATGGGGGTGTTGC TTTTGGGTCTTTATGC-3Ј (13). The PCR product of the appropriate size Synthetic peptides of human RFX5 were designed and prepared by David was then subcloned into the TA cloning vector (Invitrogen, Carlsbad, CA). Klapper (University of North Carolina). Polyclonal rabbit antisera were Subsequently, a DNA fragment containing RFX5 was inserted directionally generated against these peptides by Rockland Immunochemicals (Gilberts- into the HindIII and EcoRV sites of the pcDNA3 expression vector for ville, PA). The peptide used as the immunogen for anti-RFX5 amino ter- functional testing in transient transfection studies. The point mutants 66 409 minus was NH2-CAEDEPDAKSPKTGGR-COOH and that for the anti- Leu His and Pro Arg were created by replacing either the upstream RFX5 carboxyl terminus was NH2-CNKDLKEHVLQSSLSQEHKD- HindIII (from pcDNA3 polycloning site) to AflII (897) or downstream AflII 6624 RFX5 MUTATION IN MHC CLASS II-DEFICIENT G1B CELLS

(897) to EcoRV (from pcDNA3 polycloning site) wt RFX5 DNA restric- tion fragments with the corresponding G1B mutant DNA fragments, re- spectively. The pDRCAT300 reporter plasmid containing ϳ300 bases of the HLA-DRA promoter just upstream of the transcription start site has been described (41). All plasmid DNAs were purified using Qiagen col- umns (Qiagen, Santa Clarita, CA) before transfection analyses. Plasmid DNAs were subjected to dideoxy sequencing using Sequenase, version 2.0, according to the manufacturer’s protocol (Amersham Life Science).

Transient transfection and CAT activity assay Transient transfections were performed by electroporation using 200 mV, 960 ␮F, and 3–4 ϫ 106 SJO cells with a gene pulser (Bio-Rad Laborato- ries) as described (42). After 48 h, cells were harvested and CAT enzyme activity in the transfected cell extracts was measured as described (43). The radioactivity on the TLC plates was quantitated by PhosphorImager scan- ning (Molecular Dynamics, Sunnyvale, CA). Then, percentage acetylation and fold induction, defined as the ratio of percentage acetylation of trans- fectant with RFX5 expression construct to the percentage acetylation of sample transfected with pcDNA3 vector alone, were determined.

Stable transfection and FACS analysis Downloaded from Each construct (pREP4 or pcDNA3 empty vectors and wt or G1B RFX5 vectors) carrying the G418 drug resistance marker was electroporated into SJO cells as described above. After 48 h, the cultures were subjected to selection with increasing amounts of G418 (Bio-Rad Laboratories) at doses ranging from 400 to 1200 ␮g/␮l over a 4-wk period. To prepare stably transfected G1B lines, pREP8 vector alone or carrying wt RFX5, along with the hygromycin drug resistance marker, was introduced into cells by the standard calcium phosphate transfection method. After 48 h of incu- http://www.jimmunol.org/ bation, transfected G1B cells were selected by treatment with increasing doses of hygromycin (75 to 150 ␮g/␮l) (Boehringer Mannheim). For anal- ysis of surface MHC class II expression, the cells were stained first with the mouse anti-human HLA-DR-specific Ab L243 at a dilution of 1:25 and then goat anti-mouse Ig fluorescein-conjugated secondary Ab (PharMin- gen, San Diego, CA) at a dilution of 1:300. The stained cells were exam- ined by flow cytometry (Becton Dickinson, Franklin Lakes, NJ).

Results

FIGURE 1. RFX binding activity to HLA-DRA promoter is absent in by guest on September 28, 2021 Clues to the understanding of the regulation of MHC class II mol- G1B cells. A, Gel-shift analysis of a Raji B cell nuclear extract binding to ecules lie at the dissection of the transcriptional machinery and the S/WXY domain of HLA-DRA. RFX is the factor that binds the X1 box, mechanisms responsible for directing the synthesis of these im- X2BP binds the X2 box, and NF-Y is the factor that binds the Y box. Oligonucleotide competition, indicated at the top of each lane, identifies mune regulatory molecules. To that end, we have identified the both individual complexes involving the Y and X1 boxes, as well as mul- ␥ molecular defect in a human cell line deficient in IFN- induction tiprotein complexes involving Y and X1; X1 and X2; and Y, X1, and X2. of MHC class II gene expression. The absence of X box promoter ns, nonspecific complex. The protein-DNA complexes are indicated at the binding activity and the correlative lack of transcriptional activa- left. B, G1B cells lack X1 box binding activity. Crude nuclear extracts of tion capability in G1B cells have been demonstrated previously wt 2fTGH and IFN-␥-defective cell lines (G1B, G2A, G3A, and G4A) and (4). This report describes the localization of this defect to the larg- various B cell lines (Namalwa, RFX5-deficient SJO, Raji, and CIITA- est subunit of RFX, specifically to RFX5. deficient RJ2.2.5) were prepared. A gel-shift analysis was conducted using these nuclear extracts and a radioactive oligonucleotide containing the In vitro multiprotein/DNA complexes are formed with the S/WXY S/WXY domain of HLA-DRA. A series of nonradioactive competitors for domain of HLA-DRA and contain the known MHC class II the S/W box, X2 box, RFX1 activity, and Y/CCAAT box were included in the assay. The RFX complex is indicated by the labeled bar. binding proteins Due to the complexity of gel-shift complexes formed on the S/WXY DNA, it is important to establish the pattern before de- fining the specific defect in the G1B cell line. Fig. 1 displays the protein/DNA complexes formed with a B cell nuclear extract (lane with the X1 box element, the site of RFX binding, alone or in 1) followed by specific oligonucleotide competition to identify the combination with the Y box oligo, abolishes nearly all of the upper individual components of each band (lanes 2–8). The predominant prominent bands as well as the RFX band (Fig. 1A, lanes 3 and 6). complexes formed in the absence of competition are three closely The X2 element is a weak competitor, but it eliminates the upper- spaced slow migrating bands labeled NF-Y/RFX/X2BP, RFX/ most NF-Y/RFX/X2BP band and partially diminishes the RFX/ X2BP, and NF-Y/RFX. In addition, two weaker and faster migrat- X2BP band (Fig. 1A, lane 4). This is consistent with the X2 box ing complexes are detected: RFX and NF-Y. The bands are des- protein present in these two uppermost complexes. The S motif ignated by the proteins present in the complex as defined by does not compete for any of the bands (Fig. 1A, lane 5). Compe- competition (Fig. 1A) and Ab reactivity (i.e., Fig. 2), and they tition with the X2 and Y oligos excludes all protein-shifted DR agree with an earlier report (38). Competition with the Y box el- complexes, except for the RFX-DNA complex (Fig. 1A, lane 7). ement abolishes two of the prominent bands, each containing An oligo mutated at the S/W, X1, X2, and Y sites does not block NF-Y, leaves the RFX/X2BP complex intact, and increases the the formation of specific complexes (Fig. 1A, compare lane 8 with amount migrating as RFX alone (Fig. 1A, lane 2). Competition lane 1). The Journal of Immunology 6625

FIGURE 3. RFX5 protein is detected in G1B cells. A Western blot of wt (2fTGH) and mutant (G1B or SJO) whole-cell extracts was probed with the carboxyl-terminal RFX5 Ab as described. The 75-kDa RFX5 protein is indicated by the bar on the left. Lane 10 shows the biotinylated protein marker with sizes (kd) indicated at the right. Detection of NF-YA protein in each lane of an identical blot reflected equivalent amounts of protein per volume for each extract loaded on the gel (data not shown). Downloaded from The RFX5 protein from the G1B cells is expressed at a normal level We wanted to investigate the properties of the RFX5 protein and to confirm the composition of the DNA-protein gel-shift com- plexes. So, two new Abs specific for RFX5 were generated. As

shown in Fig. 2 (left), the Ab to the carboxyl-terminal domain of http://www.jimmunol.org/ FIGURE 2. RFX5 protein in G1B cell extracts is detected with RFX5- RFX5 recognizes a single 75-kDa band in Western blot analysis of specific Abs. Left, Western blot characterization of a new Ab that specif- a G3A nuclear extract as predicted from the amino acid sequence ically reacts with the RFX5 subunit of the X1 binding protein. The Ab was and biochemical purification (25). Both amino- and carboxyl-ter- made to a carboxyl-terminal peptide of RFX5. The specific band of 75 kDa 14 minal-specific RFX5 Abs were used in an in vitro binding assay, is detected in a G3A fibrosarcoma nuclear extract (NE). M, C-labeled and both clearly shifted the RFX, the NF-Y/RFX, and RFX/X2BP size markers in lane 1. Right, The RFX5 Abs react to and supershift all of complexes (Fig. 2, right). The shifted complexes migrate very the X1-containing complexes. Binding reactions were as in Fig. 1A, lane 1, except the B cell extract was prepared from Namalwa cells. The antigenic slowly and overlap the position of the NF-Y/RFX/X2BP band, specificity of the RFX5 Ab is indicated above lanes 2 and 3. Asterisks thereby obscuring the effect of Abs on this band. Neither Ab af- indicate the supershifted bands. Neither RFX5 Ab affected the NF-Y band, fected the migration of the NF-Y band. In addition, Abs to NF- by guest on September 28, 2021 and Abs to NF-␬Bp50 had no effect (data not shown). ␬Bp50 had no affect (data not shown). To directly examine the expression of the RFX5 protein in G1B cells, we utilized the new Ab generated against the carboxyl-ter- minal RFX5 peptide (see Fig. 2, left, and Materials and Methods). Nuclear extracts from the G1B cell line do not contain an RFX This Ab was used for Western analysis of whole-cell extracts pre- DNA-binding activity pared from 2fTGH, G1B, and SJO cells. The Western blot shown We initially observed that extracts from G1B cells do not support in Fig. 3 indicates that the anti-RFX5 Ab detects a 75-kDa band in X box binding as demonstrated by the lack of formation of protein- both the mutant G1B and its wt parental line, 2fTGH. Furthermore, DNA complexes by gel-shift analysis (4). In this study, we com- G1B cells contain similar amounts of RFX5-specific protein as pared the X box binding activity of four cell lines defective in found in 2fTGH (compare lanes 4–6 with 1–3). This result sug- IFN-␥ induction of MHC class II genes, including the G1B, G2A, gests that there is no gross alteration or loss of RFX5 protein in the G3A, and G4A lines (1), as well as for other class II-deficient cell G1B mutant. In contrast, the 75-kDa protein in SJO detected by the lines (i.e., SJO and RJ2.2.5) (see Fig. 1B). To specifically visualize RFX5 Abs was greatly diminished (see Fig. 3, lanes 7–9), as ex- the RFX complex, a series of competitors including S/W, Y, X2, pected on the basis of earlier work (25). Although we routinely see and methylated DNA were used in this gel-shift experiment to a doublet at 75-kDa for 2fTGH and G1B cells and a faint band for eliminate the binding of proteins to the S/W, Y/CCAAT, and X2 SJO cells, the specificity of the second band is unknown. boxes and the binding of RFX1, respectively. Based on compari- sons to the band migration patterns in Fig. 1A, an RFX specific RFX5 from G1B contains two separate, single-base pair protein-DNA complex is missing in the G1B extract, while it is mutations present in the other IFN-␥ mutant cell lines, G2A, G3A, and G4A RFX5 cDNA clones from G1B and wt cells were prepared to in- (Fig. 1B, compare lane 2 with lanes 3, 4 and 5). The absence of vestigate molecular aberrations in the gene derived from G1B RFX binding activity in the G1B mutant is consistent with previ- cells. RFX5-specific primers for PCR amplification of reverse-tran- ous reports that the HLA-DRA promoter is bare in this line as scribed RNA were designed according to the published RFX5 se- shown by in vivo footprinting analyses (4, 44). Likewise, extracts quence (25). To eliminate PCR artifacts, several precautions were prepared from SJO cells that lack functional RFX protein do not taken. First, at least two different RT-PCR preparations were made bind to DNA containing the X1 box, whereas the binding in other using a mixture of Taq and Pwo DNA polymerases for higher B cells (i.e., Namalwa and Raji) is normal (Fig. 1B, compare lane fidelity. Subsequently, multiple clones were studied. As predicted 7 with lanes 6 and 8). On the other hand, RJ2.2.5 cells that lack a by the initial report characterizing RFX5 (25), RT-PCR amplifi- functional CIITA protein, and are thereby deficient in class II ex- cation gave rise to DNA fragments that were ϳ1900 bp in length pression, exhibit normal RFX binding activity (Fig. 1B, lane 9). for both wt and G1B. Finally, the entire length of multiple clones 6626 RFX5 MUTATION IN MHC CLASS II-DEFICIENT G1B CELLS

FIGURE 5. G1B cells are complemented by transfection with wt RFX5. G1B cells were stably transfected with plasmids encoding wt RFX5 (upper right) or vector alone (pREP8) (lower right). The G1B-transfected lines were treated either with IFN-␥ (500 U/ml) for 40 h (solid) or with un- supplemented medium (open). The transfected cell lines were stained with anti-HLA-DR Abs and analyzed by flow cytometry. FACScan analyses of

control class II-induced 2fTGH cells (upper left) and nontransfected G1B Downloaded from cells (lower left) are shown also.

activation and in promoting class II expression on the surfaces of cells. First, expression vectors encoding wt or G1B-RFX5 clones

were transiently transfected along with the class II DR promoter- http://www.jimmunol.org/ CAT reporter construct, pDRCAT300, in SJO cells, which is de- ficient of RFX5 protein (see Fig. 3). In the absence of expressed RFX5, the HLA-DR promoter is not active in SJO cells (25, 35). However, DR promoter induction is restored (nearly 4-fold over FIGURE 4. G1B RFX5 is defective in the production of HLA-DR. A, pcDNA3 vector alone) when wt RFX5 is introduced into SJO cells Diagram of mutations in G1B RFX5. The 358 and 1387 mutation sites are indicated on the linear representation of G1B RFX5 cDNA. These alter- (Fig. 4B). In contrast, G1B-RFX5 expression clones do not recon- ations in G1B RFX5 resulted in a leucine (L) to histidine (H) replacement stitute promoter activity when transfected into SJO cells (Fig. 4B), just upstream of the DBD (i.e., at amino acid 66) and a single-amino acid indicating noncomplementarity of RFX mutations in SJO and G1B change of a proline (P) to an arginine (R) at the border of the proline-rich cells. by guest on September 28, 2021 region (Pro; i.e., amino acid 409), respectively. B, G1B RFX5 does not Second, class II molecules do not appear on the surface of SJO transactivate the HLA-DRA promoter. wt or mutant G1B RFX5 expression cells that stably harbor G1B-RFX5 plasmid. In contrast, SJO cells plasmids were cotransfected with the pDRCAT300 reporter in SJO cells. stably transfected with wt RFX5 show a positive shift in HLA-DR CAT activity was measured in lysates prepared 48 h after transfection. The surface expression as determined by FACScan analyses (see Fig. average with SEM of results from five independent experiments is shown 4C). Taken together, these results confirm that the G1B-RFX5 that here. C, G1B RFX5 does not support the production of HLA-DR at the cell we isolated is unable to activate class II promoter function. surface. SJO cells were stably transfected with plasmids encoding vector alone (pREP4 or pcDNA3; open scans), wt RFX5 (upper right), or G1B Complementation of G1B with cloned RFX5 restores class II RFX5 (lower right). The transfected cell lines were stained with anti- surface expression HLA-DR Abs and analyzed by flow cytometry. FACScan analyses of con- trol class II-expressing Raji cells (upper left) and nontransfected SJO cells To underscore that the defect in G1B cells resides in RFX5 alone, (lower left) are shown also. we stably transfected G1B cells with the cloned wt RFX5 (or vec- tor alone as a negative control). Class II expression was induced in these stable lines by treating with IFN-␥, and then surface expres- derived from different RT-PCR preparations for both wt and G1B- sion was measured by flow cytometry. The results presented in RFX5 cDNAs was sequenced. Fig. 5 show a positive shift for G1B cells carrying wt RFX5, in- Comparison of mutant to wt RFX5 sequences by dideoxynucle- dicating that the lack of surface class II molecules in G1B cells is otide sequence analyses revealed the presence of single-base pair remedied by complementation with wt RFX5 (Fig. 5, compare up- point mutations in G1B. Unexpectedly, we discovered that RFX5 per right and left). Conversely, untransfected G1B or cells trans- from G1B is altered from wt at two positions, specifically at nu- fected with empty vector do not show a shift in class II expression cleotides 358 (T to A) and 1387 (C to G) (see Fig. 4A). This is true with IFN-␥ induction (Fig. 5, lower left and right, respectively). for multiple clones synthesized from different RT-PCR prepara- tions. Both of these transition mutations lead to the replacement of A single-base pair mutation accounts for the defective RFX5 nonpolar amino acids with basic positively charged amino acids: phenotype leucine to histidine (Leu66His) and proline to arginine (Pro409 To define the effect of each individual point mutation found in Arg) (Fig. 4A). This mutant RFX5 is hereafter referred to as G1B-RFX5, additional clones carrying either one or the other sin- G1B-RFX5. gle-base pair mutation were generated by exchanging restriction fragments between the wt and G1B mutant clones. The promoter- The mutant RFX5 from G1B lacks transactivating function activating function of each of these mutants was tested in the SJO To understand the consequences of these mutations, we examined transfection assay described above. Our results indicate that the the function of RFX5 isolated from G1B cells in promoter trans- Leu66His clone, having a mutation just upstream of the DBD The Journal of Immunology 6627

FIGURE 6. The Leu66His mutation is responsible for the defect in FIGURE 7. Alterations of the leucine-rich region in RFX5 abrogate Downloaded from HLA-DRA promoter activation in G1B cells. A, Diagram of nucleotide RFX transactivation of the HLA-DR promoter. A, Diagram of additional mutations in RFX5 isolated from G1B cells. Constructs containing either RFX5 mutations. Leucines at amino acid residues 62, 64, 66, and 68 were the Leu66His or Pro409Arg mutation are shown. The solid box denotes the individually altered to histidine, and cysteines 107, 126, 127, and 160 were DBD, and the shaded box represents the Pro of RFX5. B, SJO cells were each changed to serine residues, as indicated here. B, Transactivation func- electroporated with each mutant RFX5 construct along with pDRCAT300 tion of RFX is reduced with mutation of the leucine-rich region, but not by reporter plasmid DNA. The cells were harvested 48 h later, and CAT en- altering RFX5-unique cysteines in the DBD. wt or mutant RFX5 expres- zyme activity was assessed. The results shown here are the average and sion plasmids were cotransfected with the pDRCAT300 reporter in SJO http://www.jimmunol.org/ SEM of at least four transfected samples. Data for single-mutant constructs cells. CAT activity was measured in lysates prepared 48 h after transfec- Leu66His and Pro409Arg are shown in hatched and shaded boxes, tion. The average results and SEM from triplicate experiments are shown 62 64 66 respectively. here. Data for individual leucine mutants (Leu His, Leu His, Leu His, and Leu68His) are shown in the hatched boxes, and results for the cysteine mutants (Cys107Ser, Cys126, 127Ser, and Cys160Ser) are shown in the shaded boxes. homologous region, is unable to support the induction of the HLA-DR promoter (Fig. 6B). On the other hand, the Pro409Arg mutation present in the proline-rich region of RFX5 appears to have little to no adverse effect on activating function, as the pro- revealed that alteration of any of the leucine residues eliminated by guest on September 28, 2021 moter induction for this construct was approximately equivalent to transactivation, indicating that each leucine in this region is nec- that seen with wt RFX5 (Fig. 6B). essary for RFX function. Interestingly, this leucine-rich motif is only found in the RFX5 protein but not in the other RFX family A leucine-rich region in RFX5 is essential for transactivation members. Discovering that the nonfunctioning Leu66His mutation in G1B cells was positioned in an unusual sequence of RFX5 (62-LY- The unique cysteine residues in the DBD of RFX5 are LYLQL-68) rich in leucine residues (see Table I), we wondered dispensable for transactivation whether the leucines in this region were significant for the proper Finally, upon examination of the DBD sequence of RFX5 (30), we regulatory function of RFX. To test this possibility, each of four noted the presence of four cysteine residues that are uniquely leucines was mutagenized to histidines (similar to the original G1B found in RFX5 but not in the other RFX family members. This mutation). Then, SJO cells were transfected with the pDRCAT300 parallels the unique presence of the leucine-rich sequence in reporter plus expression constructs carrying each of these muta- RFX5. We questioned whether these unique cysteine residues were tions. Subsequent assays measuring CAT activity shown in Fig. 7B required either for supporting the intramolecular structure of RFX5

Table I. The leucine-rich sequence upstream of the DBD is unique to RFX5

Gene Species Amino Acid Sequence Upstream of the DBD Coding Regiona

RFX5 Human ...KLYLYLQLPSGPTTGDKSSEPSTLSNEEYMYAYRWIR... RFX1 Human . . .SGSGAGTYVIQGGYMLGSASQSYSHTTRASPATVQWLL... Mouse . . .SGSGAGTYVIQGGYMLGNASQSYSHTTRASPATVQWLL... RFX2 Human . . .SPIVSSAGAYLIHGGMDSTRHSLAHTARSSPAT lemaienlqksegitshksgllnsh LQWLL. . . Mouse . . .TPIVSGAGAYLIHGGMDGTRHSLAHTARSSPATLQWLL... RFX3 Human . . .QLISSSGGTYLIGNSMENSGHSVIHTTRASPAT iemaietlqksdglsthrssllnshLQWLL... Mouse . . .IGNSMENSGHSVTHTTRASPAT iemaietlqksdglsthrssllnshLQWLL... RFX4 Human . . .LRKCYEVGMMKGDEEKENNRASKPHSTPATLQWLE...

a Underlined bold lettering represents the RFX5 unique leucine-rich segment. b Italic lettering denotes the start of the DBD. c Lowercase lettering indicates the presence of an alternatively spliced segment. 6628 RFX5 MUTATION IN MHC CLASS II-DEFICIENT G1B CELLS by disulfide bonds or for regulation of RFX5 activity by cellular sheet. This potential diminishes when the histidine-altered se- redox cycles (45). To determine the significance of these amino quences are analyzed by these programs, suggesting that a change acids for the activity of RFX5, we altered each of the cysteines to in protein conformation occurs. serine residues by site-directed mutagenesis and then introduced There is a well-established precedence for leucine motifs play- the expression constructs along with the pDRCAT300 reporter into ing critical roles in regulatory proteins. Foremost, the leucine zip- SJO cells. HLA-DR promoter activation as assessed by CAT assays per motif present in transactivators such as , , and using extracts of transfected cells was not altered from wt levels C/EBP is required to direct protein dimerization through hydro- upon loss of any of the cysteine residues (see Fig. 7B). This indi- phobic interactions between opposing helical faces (51, 52). A cates that the cysteines, unlike the leucine residues, are dispensable growing family of proteins that participate in protein-protein in- for RFX5 activity in promoter transactivation. teractions, mediate hormone signals, regulate gene expression, and support processes requiring cell-to-cell adhesion contain leucine- rich repeats (LRR) (53). The LRR from these proteins share com- Discussion mon hydrophobic and hydrophilic features with each repeat, gen- The use of genetic mutants has been invaluable in discovering erally consisting of the following basic sequence: XLa/bXLa/bLS/ components of molecular pathways. One of the most successful TN/QXN/QalS/TXG/PG/PG/PXXalXXLX, where a/b is acidic/ examples is the elucidation of intracellular mediators and signal basic, al is aliphatic, and X is any amino acid. These proteins tend transducers, which contribute to an IFN response. For example, the to form ␤ sheets and amphipathic structures. A few notable ex- tyk kinase, which is associated with the for type I IFN, amples include CIITA with three LRR repeats (53); adenyl cyclase was first discovered with the use of a gene complementation ap- that interacts with Ras to transmit hormone signals (53); leucine- Downloaded from proach made possible by the availability of mutant cell lines de- rich proteoglycans that regulate the assembly of matrix proteins fective in their responses to type I IFN (46). Similarly, a number that affect cellular growth, repair, and differentiation (54); and the of mutant cell lines defective in either type I or II IFN signaling GPIb-V-IX protein, in which a point mutation that changes leucine have been instrumental in confirming the functional roles of Janus (at 129) to proline in a LRR reduces binding of von Willebrand kinases and STAT molecules in the IFN pathway (47, 48). factor and results in a bleeding disorder (55). A recent report char-

Along the same vein, four mutant cell lines, G1 to G4, were acterizing the structure and function of the novel transcriptional http://www.jimmunol.org/ genetically chosen on the basis of their selective loss of IFN-␥- coactivator p/CIP (56) describes a novel leucine motif. Through its induced MHC class II expression while retaining expression of interactions with CBP, the p/CIP transactivator mediates hormone- other IFN-␥-induced genes (1). Analyses of these and other mutant receptor signals to activate gene expression. This protein contains lines clearly indicate that the IFN-␥ induction of MHC class II six leucine-charged domains with a consensus sequence of genes requires additional molecules defined by the G1 to G4 mu- LX1X2LL, where X2 is Q or Y in four of six repeats, in addition tant cell lines which are not required for the IFN-␥ induction of to three other large protein-interacting domains. Finally, Rowland other genes such as IRF-1, TAP-1, and LMP-7 (1). Previously, we and Segall (57) recently described yet another unique leucine-rich performed the initial characterization of the G3A cell line, which sequence found in TFIIIA, specifically 343-LEKRLESGENGLN appears to lack the proper induction of the CIITA transcript (4). LLL-358. They showed that changing two or four of the leucines by guest on September 28, 2021 That report showed that CIITA is necessary for the IFN-␥ induc- to alanine in almost any combination resulted in a defective protein tion of MHC class II promoters as well as the Ii promoter. that was no longer able to transactivate a 5S RNA template, nor In this report, we correlate the loss of IFN-␥ induction of MHC was it able to support cell viability (57). In light of these examples, class II genes with the identification of a molecular defect in an the presence of the LYLYLQL leucine-rich motif in RFX5, which essential regulator, namely RFX5. Here, we present evidence that interacts with other proteins to regulate the constitutive and IFN- a nonconserved amino acid change in the RFX5 chain in the G1B ␥-inducible MHC class II gene expression (18, 32, 58, 59), is mutant cell line is sufficient to abolish the binding capacity of RFX significant. as well as the transactivating function of this essential regulator. Although the precise mechanism by which a leucine-to-histidine The mutated amino acid is located in a region that has not been change at residues 62, 64, 66, or 68 affects DNA binding remains previously recognized as important for the function of RFX5 and to be elucidated, there are several possibilities. First, mutations in may delineate a new leucine-rich functional domain. We have fur- the 62-LYLYLQL-68 region may affect the three-dimensional ther confirmed this finding by showing that new RFX5 leucine structure of either RFX5 or, specifically, the DBD defined by se- mutants created in vitro are incapable of transactivating a class II quence homology, thereby interfering with the binding of RFX to promoter, suggesting the identification of residues essential for the X1 element. As a result, the DBD might not be accessible or RFX activity. able to bind to the DNA. Second, it is possible that this region is The RFX5 protein from G1B contains two mutant residues; itself a DBD, required for optimal binding of RFX to its cognate however, only one of the two appears to contribute to the mutant class II promoter sequences. Finally, we favor the hypothesis that phenotype. The critical leucine-to-histidine mutation in G1B-RFX5 any change in this region may affect the local and/or overall struc- resides in amino acid residue 66, which is located upstream of the ture of RFX5, therefore preventing the proper assembly of the region of RFX5 defined by as the region re- subunits of RFX and/or the interactions with other proteins. To test sponsible for DNA binding activity (i.e., amino acids 93–167) (30, this idea, immunoprecipitation experiments were conducted to in- 31). An analysis of the region surrounding residue 66 shows that vestigate the proteins [RFX5 subunits, 36 kDa (26) or 41 kDa (15), it shares no homology with any of the previously defined RFX or other transactivating factors, i.e., CIITA, X2BP, and NF-Y] that family members (see Table I). Interestingly, this residue lies within are associated with wt and mutant RFX5 proteins in vivo (data not a short fragment that is rich in leucines (62-LYLYLQL-68). Cu- shown). Unfortunately, in our initial experiments analyzing wt and riously, we discovered that mutation of Leu62, Leu64,orLeu68 to G1B RFX5, we did not see additional proteins associated convinc- histidine also abrogates the transactivation potential of RFX5. ingly with the immunoprecipitated RFX5 (W. J. Brickey and Based on multiple algorithms for predicting protein secondary J. P.-Y. Ting, unpublished observation, and U. Nagarajan and J. M. structure, such as nnPredict and Secondary Structure Prediction Boss, unpublished observation). We interpret these negative re- (49, 50), the RFX5 leucine-rich region will very likely form a ␤ sults as due either to the unstable, low abundant, or transient nature The Journal of Immunology 6629 of protein-protein interactions or to a diminished affinity or spec- G3A IFN ␥ mutants reveals that defects in CIITA or RFX result in defective class ificity of the RFX5 Abs for native RFX5 protein, because the Abs II MHC and Ii gene induction. Immunity 1:687. 5. Chang, C. H., and R. A. Flavell. 1995. Class II transactivator regulates the ex- were generated against a peptide synthesized in vitro. It is notable pression of multiple genes involved in antigen presentation. J. Exp. Med. 181: that the analyses of protein-protein interactions with RFX5 up to 765. this point have been primarily performed with in vitro generated 6. Glimcher, L. H., and C. J. Kara. 1992. Sequences and factors: a guide to MHC class-II transcription. Annu. Rev. Immunol. 10:13. recombinant proteins (59) or highly enriched protein fractions (60). 7. Abdulkadir, S. A., and S. J. Ono. 1995. How are class II MHC genes turned on Understanding the critical role of RFX in regulating promoter and off? FASEB J. 9:1429. accessibility will be directly related to defining its interaction with 8. Reith, W., V. Steimle, and B. Mach. 1995. Molecular defects in the bare lym- phocyte syndrome and regulation of MHC class II genes. Immunol. Today 16: other regulatory factors, as well as its DNA binding activity. There 539. is strong evidence showing that the MHC class II promoters re- 9. Mach, B., V. Steimle, E. Martinez-Soria, and W. Reith. 1996. Regulation of quire and share a conserved, highly organized stereochemical ar- MHC class II genes: lessons from a disease. Annu. Rev. Immunol. 14:301. 10. Boss, J. M. 1997. Regulation of transcription of MHC class II genes. Curr. Opin. rangement of regulatory elements (12, 61) and multiple interacting Immunol. 9:107. proteins that bind to those elements (10, 62). Previous studies have 11. Brown, A. M., C. L. Barr, and J. P. Ting. 1991. Sequences homologous to class shown that RFX or an X1 box binding protein can interact with the II MHC W, X, and Y elements mediate constitutive and IFN-␥-induced expres- sion of human class II-associated invariant chain gene. J. Immunol. 146:3183. NF-Y transcription factor (18, 38), as well as the X2BP (58, 63, 12. Vilen, B. J., J. F. Penta, and J. P.-Y. Ting. 1992. Structural constraints within a 64). This was revealed using assays that measure direct biochem- trimeric transcriptional regulatory region: constitutive and interferon-␥ inducible ical interaction or the association-dissociation rates of protein- expression of the HLA-DRA gene. J. Biol. Chem. 267:23728. 13. Ting, J. P.-Y., K. L. Wright, K.-C. Chin, W. J. Brickey, and G. Li. 1997. The DNA interactions. In addition, an in vivo footprinting approach of DMB promoter: delineation, in vivo footprint, trans-activation, and trans-dom- transfected promoter constructs bearing mutations in the X1, X2, inant suppression. J. Immunol. 159:5457. Downloaded from or Y elements used in our laboratory showed interdependency of 14. Reith, W., E. Barras, S. Satola, M. Kobr, D. Reinhart, C. H. Sanchez, and B. Mach. 1989. Cloning of the major histocompatibility complex class II pro- protein-DNA interactions in intact cells (18, 19). Also, recent data moter binding protein affected in a hereditary defect in class II gene regulation. using a yeast two-hybrid system implicate an interaction of RFX Proc. Natl. Acad. Sci. USA 86:4200. with CIITA (59). 15. Moreno, C. S., E. M. Rogers, J. A. Brown, and J. M. Boss. 1997. Regulatory factor X, a bare lymphocyte syndrome transcription factor, is a multimeric phos- In contrast to the critical role of the leucine-rich region, altering phoprotein complex. J. Immunol. 158:5841. any of the RFX5-unique cysteines in the DBD has no adverse 16. Zeleznik-Le, N., J. C. Azizkhan, and J. P.-Y. Ting. 1991. Affinity-purified http://www.jimmunol.org/ effects on class II promoter transactivation in our system. Our re- CCAAT-box-binding protein (YEBP) functionally regulates expression of a hu- man class II major histocompatibility complex gene and the herpes simplex virus sults indicate that the possibility that RFX might be regulated by thymidine kinase gene. Proc. Natl. Acad. Sci. USA 88:1873. cellular reduction/oxidation systems at the cysteines in the DBD is 17. Hasegawa, S. L., and J. M. Boss. 1991. Two B cell factors bind the HLA-DRA diminished. Also, the Pro409Arg mutation found in G1B-RFX5 at X box region and recognize different subsets of HLA class II promoters. Nucleic Acids Res. 19:6269. the border of the proline-rich region in RFX5 does not diminish 18. Wright, K. L., B. J. Vilen, Y. Itoh-Lindstrom, T. L. Moore, G. Li, M. Criscitiello, transactivation function. These results indicate that these individ- P. Cogswell, J. B. Clarke, and J. P.-Y. Ting. 1994. CCAAT box binding protein ual amino acids do not play specific roles and/or do not alter pro- NF-Y facilitates in vivo recruitment of upstream DNA binding transcription fac- tors. EMBO J. 13:4042. tein structure or conformation to affect interactions of RFX with 19. Linhoff, M. W., K. L. Wright, and J. P.-Y. Ting. 1997. CCAAT-binding factor DNA or with other proteins. NF-Y and RFX are required for in vivo assembly of a nucleoprotein complex that by guest on September 28, 2021 In summary, this report identifies the molecular defect in the spans 250 base pairs: the invariant chain promoter as a model. Mol. Cell. Biol. ␥ 17:4589. G1B cell line that is selectively defective in the IFN- induction of 20. Calman, A. F., and B. M. Peterlin. 1987. Mutant human B cell lines deficient in MHC class II genes and their associated genes. Even though two class II major histocompatibility complex transcription. J. Immunol. 139:2489. different mutations in the G1B-RFX5 gene were found, only one of 21. Benichou, B., and J. L. Strominger. 1991. Class II-antigen-negative patient and mutant B-cell lines represent at least three, and probably four, distinct genetic the two contributes to the mutant phenotype. This single-residue defects defined by complementation analysis. Proc. Natl. Acad. Sci. USA 88: mutant (Leu66His) resulted in a drastic change in phenotype and 4285. the total loss of MHC class II expression, primarily through the 22. Loh, J. E., C. H. Chang, W. L. Fodor, and R. A. Flavell. 1992. Dissection of the interferon ␥-MHC class II signal transduction pathway reveals that type I and absence of promoter transactivation function. Consistent with this, type II interferon systems share common signalling component(s). EMBO J. 11: we found that mutants with neighboring leucines altered to histi- 1351. dine lacked transactivation function as well. This characterization 23. Seidl, C., C. Saraiya, Z. Osterweil, Y. P. Fu, and J. S. Lee. 1992. Genetic com- plexity of regulatory mutants defective for HLA class II gene expression. J. Im- should pave the way for further examination of this and other munol. 148:1576. regulators of MHC class II gene induction. 24. Steimle, V., L. A. Otten, M. Zufferey, and B. Mach. 1993. 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