Novel Mutations Within the RFX-B Gene and Partial Rescue of MHC and Related Genes Through Exogenous Class II Transactivator in RFX-B-Deficient Cells This information is current as of October 1, 2021. Uma M. Nagarajan, Ad Peijnenburg, Sam J. P. Gobin, Jeremy M. Boss and Peter J. van den Elsen J Immunol 2000; 164:3666-3674; ; doi: 10.4049/jimmunol.164.7.3666 http://www.jimmunol.org/content/164/7/3666 Downloaded from References This article cites 58 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/164/7/3666.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 1, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Novel Mutations Within the RFX-B Gene and Partial Rescue of MHC and Related Genes Through Exogenous Class II Transactivator in RFX-B-Deficient Cells1 Uma M. Nagarajan,2* Ad Peijnenburg,2† Sam J. P. Gobin,† Jeremy M. Boss,3,4* and Peter J. van den Elsen3† MHC class II deficiency or bare lymphocyte syndrome is a severe combined immunodeficiency caused by defects in MHC-specific regulatory factors. Fibroblasts derived from two recently identified bare lymphocyte syndrome patients, EBA and FZA, were found to contain novel mutations in the RFX-B gene. RFX-B encodes a component of the RFX transcription factor that functions in the assembly of multiple transcription factors on MHC class II promoters. Unlike RFX5- and RFXAP-deficient cells, trans- fection of exogenous class II transactivator (CIITA) into these RFX-B-deficient fibroblasts resulted in the induction of HLA-DR ␤ Downloaded from and HLA-DP and, to a lesser extent, HLA-DQ. Similarly, CIITA-mediated induction of MHC class I, 2-microglobulin, and invariant chain genes was also found in these RFX-B-deficient fibroblasts. Expression of wild-type RFX-B completely reverted the noted deficiencies in these cells. Transfection of CIITA into Ramia cells, a B cell line that does not produce a stable RFX-B mRNA, resulted in induction of an MHC class II reporter, suggesting that CIITA overexpression may partially override the RFX-B defect. The Journal of Immunology, 2000, 164: 3666–3674. ajor histocompatibility complex class II genes encode indirectly to conserved regulatory elements in the proximal pro- http://www.jimmunol.org/ cell-surface glycoproteins that fulfill a crucial function moter of these genes (reviewed in Refs. 6–10). M in the immune response and in T cell development The conserved S, X (comprising X1 and X2), and Y box DNA (1, 2). The importance of these molecules is illustrated in patients sequence regulatory elements that have been identified in the prox- suffering from MHC class II deficiency or bare lymphocyte syn- imal promoter of MHC class II, class I, and their accessory genes drome (BLS),5 a severe combined immunodeficiency. This dis- are bound by several DNA-binding protein complexes (reviewed ease, which is characterized by an absence of MHC class II ex- in Refs. 3, 6, 7, and 11–14). These include RFX, X2BP (cAMP pression and frequently also by a reduced level of MHC class I response element binding protein; CREB), and NF-Y, which bind expression (3), results in the inability to present antigenic peptides cooperatively to the X1 box (8, 15), X2 box (8, 9, 16, 17), and Y by guest on October 1, 2021 to CD4ϩ T cells for initiation of the immune response. BLS pa- box (18, 19), respectively. Together these proteins are engaged in tients manifest severely impaired humoral and cellular immune the formation of a highly stable multiprotein DNA complex (20– responses, as well as impaired development of CD4ϩ T cells (4, 5) 24). Although individual binding of RFX or X2BP to some of the and therefore are extremely susceptible to many opportunistic in- isotypic MHC class II-conserved elements has been shown to be fections (5). BLS is inherited in an autosomal recessive fashion weak or absent (8, 22), the RFX/X2BP/NF-Y complex is formed and results from mutations in the genes encoding transacting reg- on X-Y DNA of all MHC class II isotypic promoters (22). In a ulatory proteins. These transcription factors are critical to the ex- similar fashion, multiprotein complex formation also occurs on ␤ ␤ pression of MHC class II genes and play an ancillary role in the X-Y DNA of the MHC class I and 2-microglobulin ( 2m) pro- transcriptional control of MHC class I genes by binding directly or moters.6 This complex formation allows the MHC class II isotypes to be regulated in a coordinate fashion. The lack of MHC class II expression and the reduction of MHC class I expression in BLS *Department of Microbiology and Immunology, Emory University School of patient-derived cell lines with defects in one of the RFX sub- Medicine, Atlanta, GA 30322; †Division of Molecular Biology, Department of Im- munohematology and Blood Bank, Leiden University Medical Center, Leiden, The units is attributed to the failure to assemble this complex in vivo Netherlands (25, 26).6 Received for publication October 5, 1999. Accepted for publication January 19, 2000. Formation of this multiprotein complex on X-Y DNA is not The costs of publication of this article were defrayed in part by the payment of page sufficient for activation of MHC class II genes. Activation requires charges. This article must therefore be hereby marked advertisement in accordance the activity of the MHC class II transactivator (CIITA) (27). with 18 U.S.C. Section 1734 solely to indicate this fact. CIITA is believed to act as a transcriptional coactivator through 1 This research was supported by grants from the National Institutes of Health to J.M.B. (GM47310 and AI34000) and The Netherlands Organization for Research to protein/protein interactions with the RFX complex and as such P.J.v.d.E. (Grant 901-09-243). S.J.P.G. is a Fellow of the Royal Netherlands Acad- connects the MHC-specific transcription complex with the basal emy for Arts and Sciences. transcription initiation apparatus (28–30). CIITA has strong trans- 2 U.M.N. and A.P. contributed equally to the content of this manuscript. activation properties that are mediated through its acidic NH2-ter- 3 J.M.B. ans P.vD.E. contributed equally to the supervision of this manuscript. minal domain (31, 32). Whereas CIITA is critical for expression of 4 Address correspondence and reprint requests to Dr. Jeremy M. Boss, Department of Microbiology and Immunology, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322. E-mail address: [email protected] 5 Abbreviations used in this paper: BLS, bare lymphocyte syndrome; CREB, cAMP 6 Gobin, S. J. P., M. Van Zutphen, S. D. Westerheide, J. M. Boss, and P. J. van den ␤ ␤ response element binding protein; 2m, 2-microglobulin; CIITA, class II transacti- Elsen. MHC gene transactivation through the SXY regulatory module is controlled by vator; Ii, invariant chain; CAT, chloramphenicol acetyltransferase. a specific enhanceosome. Submitted for publication. Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 3667 MHC class II genes, CIITA augments IFN-␥ induction of MHC GAPDH-specific RT-PCR and hybridization was performed as described ␤ previously (44). class I and 2m genes (8–10). Four distinct genetic complementation groups have been de- RNA and DNA hybridization analysis scribed in BLS (A–D) (33), and the genes affected in these comple- mentation groups have been identified. BLS patients of comple- For Figs. 1 and 2, total cellular RNA was prepared using RNAzolB (Cinna/ Biotecx Laboratories, Houston, TX) following the manufacturer’s instruc- mentation group A are defective in CIITA (27, 34). The genes tions. Twenty micrograms of total RNA was separated on an 1.2% agarose affected in groups B, C, and D all encode subunits of the hetero- gel containing 2.2 M formaldehyde, transferred to a Hybond N membrane trimeric phosphoprotein RFX (35). BLS groups B, C, and D are (Amersham), and hybridized using probes that were labeled with 32 defective in RFX-B (also referred to as RFXANK) (36, 37), RFX5 [ P]dCTP by random priming (DuPont-NEN, Brussels, Belgium). Trans- fer and hybridization were performed according to the instructions of the (38, 39), and RFXAP (40, 41), respectively. RFX-B is the smallest manufacturer of the membranes. The human cDNA probes for MHC class ␤ (33 kDa) of the three subunits that constitute the RFX complex I, MHC class II, invariant chain (Ii), 2m, and GAPDH were described (36, 37). Despite evidence demonstrating that RFX-B interacts before (43–46). For Fig. 7, RNA was extracted from cells using the with DNA (42), the protein has no known DNA binding domain RNeasy kit (Qiagen, Valencia, CA). Total RNA (10 ␮g) was used for (36, 37). RFX-B contains three ankyrin repeats that may be im- Northern blot analysis. Random primed RFX-B cDNA (Rediprime II; Amersham) spanning exons 3–10 was used as a probe, and hybridization portant in the formation of the RFX complex and the assembly of was conducted in Ultrahyb (Ambion, Austin, TX).