The MHC-Specific Enhanceosome and Its Role in MHC Class I and β2-Microglobulin Transactivation

This information is current as Sam J. P. Gobin, Marlijn van Zutphen, Sandy D. of October 1, 2021. Westerheide, Jeremy M. Boss and Peter J. van den Elsen J Immunol 2001; 167:5175-5184; ; doi: 10.4049/jimmunol.167.9.5175 http://www.jimmunol.org/content/167/9/5175 Downloaded from

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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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The MHC-Specific Enhanceosome and Its Role in MHC Class I ␤ 1 and 2-Microglobulin Gene Transactivation

Sam J. P. Gobin,2* Marlijn van Zutphen,* Sandy D. Westerheide,† Jeremy M. Boss,† and Peter J. van den Elsen* ␤ ␤ The regions of MHC class I and 2-microglobulin ( 2m) posses a regulatory module consisting of S, X, and Y boxes, which is shared by MHC class II and its accessory genes. In this study we show that, similar to MHC class II, the SXY ␤ module in MHC class I and 2m promoters is cooperatively bound by a multiprotein complex containing regulatory factor X, CREB/activating factor, and nuclear factor Y. Together with the coactivator class II transactivator this multiprotein complex drives transactivation of these genes. In contrast to MHC class II, the multiprotein complex has an additional function ␤ in the constitutive transactivation of MHC class I and 2m genes. The requirement for all transcription factors in the complex and correct spacing of the binding sites within the SXY regulatory module for complex formation and functioning of this multiprotein Downloaded from complex strongly suggests that this complex can be regarded as a bona fide enhanceosome. The general coactivators CREB binding , p300, general control nonderepressible-5, and p300/CREB binding protein-associated factor exert an ancillary function ␤ in MHC class I and 2m transactivation, but exclusively through the class II transactivator component of this enhanceosome. Thus, the SXY module is the basis for a specific enhanceosome important for the constitutive and inducible transactivation of ␤ MHC class I and 2m genes. The Journal of Immunology, 2001, 167: 5175Ð5184. http://www.jimmunol.org/ ight control of MHC class I and II expression is crucial exact role in transactivation is not fully understood. MHC class for an adequate immune response. The expression of II genes are fully dependent on the coactivator class II trans- T MHC class II and functionally related genes (invariant activator (CIITA) for their transactivation. This non-DNA bind- chain, HLA-DM) is regulated by a group of conserved regula- ing protein requires the multiprotein complex consisting of tory elements, referred to as the S, X (comprising the X1 and RFX, CREB/ATF, and NFY, to drive MHC class II expression. X2 sequences), and Y boxes (1, 2). Together these form a reg- The SXY regulatory module was initially considered to be spe- ulatory module, cooperatively bound by regulatory factor X cific for MHC class II and functionally related genes. The recent (RFX), X2 box-binding protein (X2BP), and nuclear factor Y discovery that the coactivator CIITA plays an important role in by guest on October 1, 2021 (NFY) to the X1, X2, and Y boxes, respectively. RFX is a MHC class I expression was the first line of evidence to suggest a trimer consisting of RFX5, RFX-associated protein (RFXAP), common pathway of transcriptional regulation of MHC class I and and RFXB/RFX containing three ankyrin repeats (RFXANK). II genes (3, 4). Additional research has indicated that this pathway X2BP is a complex of CREB/activating is not isolated and has revealed the existence of the SXY module 3 ␤ (ATF) factors and NFY is a trimer consisting of the NFYa, also in the proximal promoter regions of MHC class I and 2- ␤ NFYb, and NFYc subunits (1, 2). In vivo, the X1, X2, and Y microglobulin ( 2m) genes (5). Taking advantage of type III bare boxes, but not the S box, are occupied. Although the S box lymphocyte syndrome (BLS) patient cell lines, which are defective appears to be important for MHC class II transactivation, its in subunits of the RFX complex, it was demonstrated that the SXY ␤ module in MHC class I and 2m promoters mediates several trans- *Department of Immunohematology and Blood Transfusion, Leiden University Med- activation pathways in which the RFX complex is pivotal; RFX ical Center, Leiden, The Netherlands; and †Department of Microbiology and Immu- regulates the constitutive expression and is crucial for CIITA-in- nology, Emory University School of Medicine, Atlanta, GA 30322 ␤ duced transactivation of MHC class I and 2m genes (6). Received for publication November 1, 2000. Accepted for publication September There has been increasing evidence that the SXY module of 4, 2001. MHC class I interacts with the same as the SXY module The costs of publication of this article were defrayed in part by the payment of page of MHC class II. In MHC class I, the X1 box mediates transacti- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. vation by the RFX complex (6, 7), and is bound by the DNA- 1 This work was supported by The Netherlands Foundation for the Support of Mul- binding subunit RFX5 (6). The X2 box is bound by several mem- tiple Sclerosis Research (96-248 MS) and the Dr. Gisela Thier Foundation (to bers of the CREB/ATF family of transcription factors, including P.J.v.d.E.), and National Institutes of Health Grants AI34000 and GM47310 (to J.M.B.). S.J.P.G. is a fellow of the Royal Netherlands Academy of Arts and Sciences. CREB1, cAMP response element modulator 1, and ATF1 (3, 6), and the Y box is bound by an NFY-like complex (8). Despite the 2 Address correspondence and reprint requests to Dr. Sam J. P. Gobin, Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, functional importance in transactivation of the proteins acting Building 1, E3-Q, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. E-mail address: ␤ through the SXY module of MHC class I and 2m, the individual [email protected] binding of these proteins to their target boxes has been variable 3 ␤ ␤ Abbreviations used in this paper: ATF, activating transcription factor, 2m, 2- and sometimes difficult to determine. This could be explained by microglobulin; BLS, bare lymphocyte syndrome; CBP, CREB binding protein; CIITA, class II transactivator; ISRE, IFN-stimulated response element; PCAF, p300/ the requirement for cooperative binding, which could compensate CBP-associated factor; PKA, A; IVGF, in vivo genomic footprinting; for low affinity protein/DNA interaction to the individual boxes C, coding; NC, noncoding; RFX, regulatory factor X; X2BP, X2 box-binding protein; NFY, nuclear factor Y; RFXAP, RFX-associated protein; RFXANK, RFX containing due to locus-specific nucleotide variation, as is demonstrated for three ankyrin repeats. MHC class II genes (1, 2, 9, 10). Furthermore, the involvement of

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 5176 THE MHC-SPECIFIC ENHANCEOSOME AND MHC CLASS I TRANSACTIVATION

the coactivator CIITA lead to the hypothesis that all these proteins binding buffer (12 mM HEPES (pH 7.9), 12% v/v glycerol, 0.3 mM DTT, 1 form a multiprotein complex that functions as an enhanceosome mM EDTA, 0.24 mM MgCl2, 0.1% v/v Nonidet P-40), with 100 ng poly(dI- ␮ driving transactivation (6, 11). dC), 100 ng sonicated herring sperm ssDNA, 50 ng methylated pBR322, 5 g BSA, and 50,000 cpm 32P-radiolabeled probe for 30 min. Incubation of the In this study, we investigated the formation and composition of protein/DNA reaction was performed on ice to favor RFX binding or at room the multiprotein complex interacting with the SXY module in the temperature to favor CREB/ATF binding and multiprotein complex formation ␤ ␤ promoters of MHC class I and 2m and its role in gene activation. (9). The nucleotide sequence of the X (X1X2) and XY probes is: X- 2m, The requirement for all transcription factors in the complex and GCACTGCGTCGCTGGCTTGGAGACAGGTGACGGTCCCTGCGGGCC TTGTCCTG; X-B7, TGTCGGGTCCTTCTTCCAGGATACTCGTGACGC correct spacing of the binding sites within the regulatory module ␤ GTCCCCACTTCCCACTCCC; XY- 2m, GCACTGCGTCGCTGGCTTGG for complex formation and functioning of this multiprotein com- AGACAGGTGACGGTCCCTGCGGGCCTTGTCCTGATTGGCTGGGCA plex strongly suggests that this complex can be regarded as a bona CGCGTT; XY-B7, TGTCGGGTCCTTCTTCCAGGATACTCGTGACGCG fide enhanceosome. The presence of this regulatory module exclu- TCCCCACTTCCCACTCCCATTGGGTATTGGATATCT; XY-A2, TGTA sively in MHC genes and the participation of MHC-specific tran- GGGTCCTTCTTCCTGGATACTCACGACGCGGACCCAGTTCTCACTC CCATTGGGTGTCGGGTTTCC; and XY-DRA, TTGCAAGAACCCTTC scription factors in this multiprotein complex determine the spec- CCCTAGCAACAGATGCGTCATCTCAAAATATTTTTCTGATTGGCCAA ificity of this enhanceosome for this family of molecules critical AGAGTAATT. for Ag presentation. The HLA-DRA X1X2, X1 (X1-⌬X2), X2 (⌬X1-X2) and Y probes that were used as competitor have been described previously (9). The samples were run on a 5% nondenaturing polyacrylamide gel (69:1) in 1ϫ GTG Materials and Methods buffer (0.5 mM EDTA; 90 mM Tris; 28.5 mM taurine) for 2 h, at 200 V and Cell culture 4¡C. For DNA competition assays, protein extracts were incubated with competitor DNA for 30 min. before adding radiolabeled probe. For the The cell lines used in this study were the Burkitt’s lymphoma B cell line supershift assays, 1 ␮g of each Ab was added 30 min. after adding the Downloaded from Raji, the Raji derived CIITA-deficient cell line RJ225, the RFX5-deficient probe and incubated for an additional 30 min. The RFX (anti-RFX5) and EBV-transformed B cell line SJO, the CIITA-deficient fibroblast cell line CREB (anti-CREB1) specific antisera have been previously described (9). ATU (12), the RFXB/RFXANK-deficient fibroblast cell line EBA (7), the The antisera against NFYa (200-401-100) and NF1 (sc-870x) were from RFX5-deficient fibroblast cell line OSE (13), the RFXAP-deficient fibro- Rockland (Gilbertsville, PA) and Santa Cruz Biotechnology (Santa Cruz, blast cell line ABI (14), the teratocarcinoma cell line Tera-2, and the mon- CA), respectively. key fibroblast-like kidney cell line COS-1. These cell lines were grown in

IMDM (Life Technologies, Paisley, Scotland) supplemented with 10% Plasmids http://www.jimmunol.org/ (v/v) heat-inactivated FCS (Life Technologies), penicillin (100 IU/ml), and streptomycin (100 ␮g/ml). Freshly isolated T cells from ATU (comple- Luciferase reporter plasmids used were generated by cloning genomic pro- mentation group A; Ref. 12), EBA (group B; Ref. 7), OSE (group C; Ref. moter fragments into pGL3-Basic (Promega, Madison, WI). These constructs ␤ 13), and ABI (group D; Ref. 14) were cultured in the presence of PHA (10 contain, respectively, a 302-bp 2m PCR-generated promoter fragment ␮ ␤ g/ml) and IL-2 (10 IU/ml) in RPMI 1640 medium (Life Technologies) (pGL3- 2m), a 228-bp BglI-AhaII HLA-A2 promoter fragment (pGL3-HLA- supplemented with 10% human serum. Stable transfectant of the BLS- A), a 269-bp AspI-AhaII HLA-B7 promoter fragment (pGL3-HLA-B), a derived cell lines complemented with their missing factor were grown on 281-bp HLA-Cw3 PCR-generated promoter fragment (pGL3-HLA-C), medium with 40 mg/ml hygromycin (3, 6). a 261-bp HLA-E PCR-generated promoter fragment (pGL3-HLA-E), a 265-bp HLA-F PCR-generated promoter fragment (pGL3-HLA-F), FACS analysis a 217-bp HLA-G PCR-generated promoter fragment (pGL3-HLA-G), a 690-bp TAP1/LMP2 PCR-generated promoter fragment (pGL3-TAP1), by guest on October 1, 2021 T cells from BLS complementation groups A, B, C, and D, and T cells and a 594-bp TAP2 PCR-generated promoter fragment (pGL3-TAP2). from the corresponding parents were stained by indirect immunofluores- The SV40 promoter-driven plasmid pGL3-control (Promega) was used cence. MHC class I and MHC class II were detected with the mAbs W6/32 as control. and B8.11.2, respectively. Cells were also stained with anti-CD3 (BD Bio- ␤ The S, X1, X2, and Y box mutant promoter constructs of 2m, HLA-B7, sciences, Woerden, The Netherlands) as control. FITC-conjugated anti- ␤ and HLA-A2, and the spacing mutant promoter constructs of 2m and mouse IgG was used as second Ab. The acquisition was performed on a HLA-B7 were generated by overlap extension PCR (3). These mutant pro- FACScan (BD Biosciences, Mountain View, CA), using a CellQuest pro- moter constructs are identical to the wild-type constructs (pGL3-HLA-A, gram for analysis. ␤ pGL3-HLA-B, and pGL3- 2m) except for a 4- to 5-bp mutation in the core sequence of the individual boxes (6) or a 5- or 10-bp insertion (GATCG or In vivo genomic footprinting (IVGF) GATCGATCGA) between the S and X or X and Y boxes. All plasmids In vivo methylation, preparation of DNA, and ligation-mediated PCR for were verified by sequence analysis (T7-polymerase sequence kit; Amer- IVGF was performed as described by Mueller and Wold (15) with minor sham, Little Chalfont, Buckinghamshire, U.K.). ␤ The expression vector pREP4-CIITA is described previously (3). The modifications (16). The promoter regions of 2m and HLA-DRA were analyzed with the following sets of primers for the coding (C) strand (C1, PRc/RSV expression vectors containing CREB binding protein (CBP), ␤ p300, CREB1, and kCREB, a CREB variant that lacks the DNA-binding C2, and C3) and the noncoding (NC) strand (NC1, NC2, and NC3): 2m- ␤ domain, were a kind gift of Dr. R. H. Goodman. The expression vectors C1, GCGAGCACAGCTAAGGCCA; 2m-C2, GCGAGACATCTCGGC ␤ ␤ pECE/RSV-ATF1, pRcRSV-hGCN5, and pCX-p300/CBP-associated fac- CCGAAT; 2m-C3, TCTCGGCCCGAATGCTGTCAGC; 2m-NC1, ␤ tor (PCAF) were a kind gift of Dr. M. Green, Dr. S. Berger, and Dr. Y. CTAGAATGAGCGCCCGGTGT; 2m-NC2, CCGGAGGGCGCCGAT ␤ Nakatani, respectively. The PCAF insert was cloned into pRc/RSV for GTA; 2m-NC3, AGGGCGCCGATGTACAGACAGCAAACT; DRA- C1, CGCTCATCAGCACAGCTATGATG; DRA-C2, GCCATTTTCTT transfection experiments. The expression vector pCMV-S12E1A and CTTGGGCGCTCT; DRA-C3, TCTTCTTGGGCGCTCTTTTGGGAGT pCMV-S12E1A2-36 were a kind gift of Dr. T. Collins. The expression CA, DRA-NC1, TCCATTGATTCTATTCTCACTAATGTGCTTC; DRA- vectors pMT-protein kinase A (PKA) and pMT-PKAmut were a kind gift NC2, TCCCTGTCTAGAAGTCAGATTGGGGTTAAAG; and DRA-NC3, of Dr. S. McKnight. CAGATTGGGGTTAAAGAGTCTGTCCGTGATTTGA. The Renilla luciferase constructs pRL-SV40 (Promega) and pRL-actin were used as internal control for transfection efficiency. pRL-actin was Nuclear extracts generated by cloning a PCR-generated 1-kb human ␤-actin promoter frag- ment into pRL-null (Promega). Crude and purified nuclear extracts were prepared from the Burkitt’s lym- phoma B cell line Raji as described (9). Binding activity of the protein fractions was monitored by EMSA. The fractions containing RFX, CREB/ Transient transfection ATF or both were selected for study. NFY co-eluted with RFX from the Adherent cells were transfected by the calcium phosphate coprecipitation Hi-Trap Q column (Amersham Pharmacia Biotech, Piscataway, NJ). method as described previously (6). In each of four wells of a six-well 6 EMSA plate, 0.2 ϫ 10 cells were transfected with a DNA mix containing 1 ␮g firefly luciferase pGL3 reporter plasmid and 0.2 ␮g Renilla luciferase pRL- Protein binding reactions were performed essentially as described previously SV40 control plasmid (Tera-2), or with 0.5 ␮g of pRSV-LacZ (COS-1). (9). Purified nuclear protein extract (1.25 ␮g) was incubated in protein/DNA For cotransfection 0.5 ␮g of pREP4-CIITA, or 1 ␮g of Rc/RSV-CREB1, The Journal of Immunology 5177 pRc/RSV-kCREB, pRc/RSV-ATF1, pMT-PKA, pMT-PKAmut, pRc/ class II genes. Despite this general conservation, nucleotide vari- RSV-CBP, pRc/RSV-p300, pRc/RSV-GCN5, pRc/RSV-PCAF, pCMV- ation within the boxes of the different loci of MHC class I and class S12E1A, and pCMV-S12E1A2-36 was used. II genes are noted which could influence protein binding and trans- Nonadherent cells (Raji, RJ225) were transfected by electroporation (3), with 10 ␮g firefly luciferase pGL3 reporter plasmid and 1 ␮g Renilla activation by CIITA. ␤ luciferase pRL-actin control plasmid. The SV40 promoter-driven pGL3 In contrast to MHC class II promoters, MHC class I and 2m control plasmid (Promega) was used in the electroporation experiments as promoters contain additional regulatory elements, such as an IFN- reference for promoter activity in the different cell lines. stimulated response element (ISRE) and ␬B binding sites. These To measure promoter activity, cells were harvested 3 days after calcium phosphate transfection or 2 days after electroporation. Luciferase activity regulatory elements (positioned upstream of the SXY module) are was determined using the (dual)-luciferase reporter assay system (Pro- the mediators of alternative transactivation routes that provide for mega) and a luminometer (Tropix, Badford, MA). the constitutive and inducible MHC class I expression (17, 18). There are several lines of evidence in support for a physiolog- Results ically important role of CIITA and RFX in MHC class I expres- CIITA and RFX regulate MHC class I genes sion. This is evident in cell material from BLS patients, which The regulation of MHC class II gene transcription by CIITA has have a gene defect in either CIITA (complementation group A), been well studied. Only recently, it has been shown that MHC RFXB/RFXANK (group B), RFX5 (group C), or RFXAP (group ␤ class I and 2m genes are also regulated by CIITA (3, 4). These D). Activated T cells lacking either CIITA, RFXB/RFXANK, genes share a regulatory module in their promoter consisting of S, RFX5, or RFXAP displayed a significant reduction in the level of X (comprising X1 and X2 halves), and Y boxes, in which X1 is an MHC class I expression at the cell surface (Fig. 1A). Because

RFX binding site, X2 a CREB/ATF binding site and Y an NFY MHC class II promoters do not have any additional regulatory Downloaded from binding site. The SXY regulatory module shows considerable se- element, they have become fully dependent on the SXY module quence homology and conserved spacing between the S and X for their transactivation and, therefore, MHC class II is not ex- boxes and the X and Y boxes among the various MHC class I and pressed in these activated BLS-derived T cells (Fig. 1A). Further

FIGURE 1. CIITA and RFX are important http://www.jimmunol.org/ ␤ in the transactivation of MHC class I and 2m genes. A, FACS analysis showing a reduced MHC class I cell surface expression on PHA/ IL-2 stimulated T cells from BLS patients de- ficient in CIITA, RFXB/RFXANK, RFX5, or RFXAP (dotted line). Activated T cells from their respective parents served as controls (sol- id line). Note that the level of CD3 was unaf- fected in activated BLS-derived T cells. B, by guest on October 1, 2021 Transient transfection assay of MHC class I promoter-driven luciferase constructs (10 ␮g/ 106 cells) in Raji and CIITA-deficient RJ225 cells showing the reduced promoter activity of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, ␤ and 2m. TAP1 and TAP2 promoter activity was not compromised by the CIITA deficiency in RJ225. The percentage of promoter activity in RJ225 compared with Raji is indicated on the right. The SV40 promoter-driven lucif- erase vector pGL3-control (Control) served as positive control. Cotransfected pRL-actin was used as control for transfection efficiency. Nor- malized luciferase activity values are ex- pressed as mean Ϯ SD of n ϭ 4. C, Reduced MHC class I expression in fibroblast cell lines. FACS analysis showing reduced MHC class I cell surface expression on fibroblast cell lines from BLS patients deficient in CIITA, RFX5, or RFXAP (thin line, open profile) which was restored in complemented fibroblast cell lines (bold line, shaded). D, Transient cotransfection assay of MHC class I promoter-driven lucif- erase constructs (1 ␮g/well) with empty pREP4 vector or pREP4-CIITA (0.5 ␮g/well) in Tera-2 cells. HLA-A, HLA-B, HLA-C, ␤ HLA-E, HLA-F, and 2m are induced by CI- ITA, whereas HLA-G, TAP1, and TAP2 were unaffected by CIITA induction. Cotransfected pRL-SV40 plasmid was used as control for transfection efficiency. Normalized luciferase activity values are expressed as mean Ϯ SD of n ϭ 4. 5178 THE MHC-SPECIFIC ENHANCEOSOME AND MHC CLASS I TRANSACTIVATION

support for a physiological role of CIITA in MHC class I expres- sion comes from promoter activation studies in CIITA-deficient ␤ RJ225 cells. MHC class I and 2m promoter activity was signif- icantly reduced compared with CIITA expressing parental Raji cells, whereas the promoter activity of TAP1 and TAP2 was not compromised by the lack of CIITA (Fig. 1B). The residual MHC ␤ class I and 2m promoter activity is due to other regulatory ele- ments (such as ␬B and ISRE boxes) providing alternative trans- activation pathways. Next, taking advantage of BLS derived fibroblast cell lines, expression of exogenous CIITA in CIITA- deficient fibroblast cells resulted in an elevated MHC class I cell surface expression (Fig. 1C). Similarly, expression of exogenous RFX5 or RFXAP in RFX5- or RFXAP-deficient fibroblasts also resulted in an increase of MHC class I cell surface expression (Fig. 1C). Notably, complementation with the RFX subunits also lead to ␤ an enhanced endogenous MHC class I and 2m genes transcript levels (6). Together, these results show that CIITA and RFX have ␤ a physiological role in MHC class I and 2m transactivation and expression. In addition, we evaluated transactivation of the differ- Downloaded from ent MHC class I loci by CIITA, because the SXY regulatory mod- FIGURE 2. CIITA-induced transactivation is dependent on the X1, X2, ule is conserved in most MHC class I promoters (with the excep- and Y boxes and on proper spacing and alignment of the X and Y boxes. tion of the HLA-G promoter). As illustrated in Fig. 1D, CIITA was A, Transient cotransfection assay of wild-type and S, X1, X2, or Y box ␤ able to activate the promoters of the HLA-A, HLA-B, HLA-C, mutated 2m and HLA-B7 promoter-driven luciferase reporter constructs HLA-E, and HLA-F genes and not the promoters of HLA-G in (1 ␮g/well) with empty pREP4 vector or pREP4-CIITA (0.5 ␮g/well) in ␤ Tera-2 cells. The MHC class I L chain 2m was also induced by Tera-2 cells. Similar results were obtained with HLA-A2 promoter-driven CIITA, albeit to a lesser extent. The promoter activity of the MHC constructs. B, Transient cotransfection assay of wild-type and S5X, X5Y, http://www.jimmunol.org/ ␤ class I accessory genes TAP1 and TAP2 was unaffected by CIITA or X10Y spacing mutants of 2m and HLA-B7 promoter-driven luciferase ␮ (Fig. 1D; Refs. 3 and 4). Thus all MHC class I H chains (except reporter constructs (0.5 g/well) with empty pREP4 vector or pREP4- ␮ HLA-G) and the class I L chain ␤ m are transcriptionally con- CIITA (0.5 g/well) in Tera-2 cells. Normalized luciferase activity values 2 are expressed as mean Ϯ SD of n ϭ 4. trolled by CIITA. In contrast to MHC class II gene transactivation, little is known about the role of the regulatory elements of the SXY module and ␤ induced transactivation of the 2m and MHC class I promoters their interacting proteins in the CIITA route of transactivation for (Fig. 2B). However, insertion of a half-helical turn between the X ␤ 2m and MHC class I genes. Therefore, we embarked on the char- and Y box, abolished (␤ m) or reduced (MHC class I) CIITA- by guest on October 1, 2021 ␤ 2 acterization and functioning of the SXY module of 2m and MHC induced transactivation (Fig. 2B). Using promoter constructs in class I genes. which an additional 10 nucleotides introduced one helical turn be- tween the X and Y boxes, thereby changing spacing but conserving The X1, X2, and Y boxes and their stereospecific alignment are the box alignment, had little effect on MHC class I induction by important for CIITA-induced transactivation of ␤ m and MHC CIITA, whereas it still affected the CIITA-mediated induction of 2 ␤ class I genes 2m (Fig. 2B). These results indicate that the X-Y spacing and box alignment, but not the S-X spacing and alignment, is crucial for To investigate the importance of the S, X1, X2, and Y boxes in CIITA-induced transactivation of ␤ m and MHC class I genes. ␤ 2 CIITA-induced transactivation of 2m and MHC class I genes, we This further argues that the factors binding these boxes form a tested promoter activation in transient transfection assays using cooperatively functioning multiprotein complex to mediate CIITA promoter constructs with mutations in either the S, X1, X2, or Y transactivation. box. These reporter assays showed that mutation of the X1, X2, or Y box strongly reduced or even abolished CIITA-induced trans- ␤ ␤ Formation of a multiprotein complex on the SXY module of 2m activation of 2m and MHC class I, whereas mutation of the S box had relatively little effect on CIITA-induced transactivation (Fig. and MHC class I genes 2A). These results show that the X1, X2, and Y boxes are each Previously, we demonstrated the in vitro binding of CREB/ATF crucial in the CIITA-induced transactivation of MHC class I and proteins to a probe encompassing the X1X2 region of MHC class ␤ ␤ 2m, and that the S box is not critical for this route of transacti- I, but not to the X1X2 probe of 2m (3, 6, 19). Furthermore, the ␤ vation. This strongly indicates that the factors binding these boxes binding of RFX to the X1X2 probe of 2m and MHC class I was jointly provide a platform for CIITA transactivation. difficult to demonstrate in this experimental set-up, despite a clear ␤ The spacing between the boxes of the SXY regulatory module role for RFX in MHC class I and 2m transactivation (6). Even of MHC genes is highly conserved, in particular between the X and using purified nuclear extracts and experimental conditions favor- Y boxes (5, 6). To investigate the importance of the spacing be- ing either RFX or CREB/ATF binding (see experimental proce- tween the S and X and the X and Y boxes on CIITA-induced dures) it was difficult to establish a strong binding of RFX (and ␤ transactivation of 2m and MHC class I, we tested promoter con- CREB/ATF) to the X-probe encompassing the X1X2 region of ␤ structs in which an additional 5 or 10 nucleotides introduced a half both 2m and MHC class I (Fig. 3, A and B). This prompted us to or full helical turn between the boxes in transient transfection as- use probes including the Y box and experimental conditions that says. These reporter assays showed that insertion of a half helical were known to favor multiprotein complex formation on XY DNA turn between the S and X boxes, thereby changing the spacing and of HLA-DRA (9). Using purified nuclear extracts and XY probes ␤ the alignment of the boxes, had no significant effect on the CIITA- encompassing the X1, X2, and Y boxes of 2m and MHC class I, The Journal of Immunology 5179

FIGURE 3. Complex formation of RFX, CREB/ ␤ ATF, and NFY on the XY sequence of 2m and MHC class I. A, EMSA showing binding of RFX to ␤ the X probe (comprising the X1X2 region) of 2m and HLA-B7. RFX binding was competed with cold X1 and X2 box probes and was supershifted with the anti-RFX5 Ab. B, EMSA showing binding of CREB ␤ to the X probe of 2m and HLA-B7. CREB binding was competed with cold X1 and X2 box probes and Downloaded from supershifted with the anti-CREB1 Ab. C, EMSA showing complex formation on the XY probe of ␤ 2m and HLA-B7. Complex formation was com- peted with cold X1X2, X2, X1, and Y box sequences of HLA-DRA. The complex was shown to contain RFX, CREB/ATF, and NFY using specific Abs (anti- RFX5, anti-CREB1, and anti-NFYa). Supershifted http://www.jimmunol.org/ complexes are indicated by an arrow; and free probe by an asterisk. Similar results were obtained with X and XY probes of HLA-A2 and HLA-DRA. by guest on October 1, 2021

a slow migrating complex was formed (Fig. 3C), similar to that RFX determines in vivo occupancy of the SXY module in MHC ␤ observed with the XY probe of HLA-DRA (data not shown). The class I and 2m gene promoters specificity of the complex was determined by competition with To investigate in vivo promoter occupancy of this regulatory re- cold probes containing the different boxes of HLA-DRA. This ␤ gion, we have chosen the nonpolymorphic gene 2m to perform an higher order complex was not formed when competitor DNA en- IVGF assay. In the B cell line Raji, the X1, X2, and Y boxes of coding the X1X2, X2 (⌬X1-X2), X1 (X1-⌬X2), or Y box se- ␤ 2m were occupied (Fig. 4) similar to the occupancy pattern of the quences of HLA-DRA was added (Fig. 3C), illustrated by an in- HLA-DRA promoter (data not shown). The lack of in vivo protein crease of free DNA probe and little or no protein/DNA complexes. ␤ binding to the S box in the 2m promoter suggests that the S box This strongly suggests that the higher order complex was only is not an important protein-binding element. To investigate formed when all the protein components and X1, X2, and Y whether RFX is crucial for protein complex formation and occu- boxes were present and that the build up is similar to HLA- pancy of this region, we took advantage of the RFX5-deficient B DRA. Supershift analysis revealed the presence of RFX, CREB/ cell line SJO (BLS group C). In the absence of RFX5, no occu- ␤ ATF, and NFY in the higher order complex (Fig. 3C), demon- pancy of the X1X2 box region of 2m was observed (Fig. 4). ␤ ␤ strating that the 2m and MHC class I XY DNA/multiprotein Interestingly, the Y box of 2m remained still occupied, in contrast complex resembles that of MHC class II (HLA-DRA). Similar to the lack of Y box occupation on the DRA promoter in RFX- results were obtained when using the XY probe of HLA-A (data deficient BLS patients (data not shown; Ref. 20). In addition, not shown). Because in these binding studies multiprotein com- complementation of SJO with RFX5 re-established in vivo occu- plex formation was found even in the absence of the S box, it pancy of the X1X2 region similar to that of control B cells (Raji). can be deduced that the presence of the S box is not critical for Because the lack of one RFX subunit, in this case RFX5, influ- ␤ complex formation in vitro. ences promoter occupancy of the X1X2 region of 2m, this 5180 THE MHC-SPECIFIC ENHANCEOSOME AND MHC CLASS I TRANSACTIVATION Downloaded from

FIGURE 4. RFX5 determines the in vivo occupancy of the SXY mod- ␤ ule of the 2m promoter. A, IVGF showing the in vivo occupancy of the SXY region in the Burkitt’s lymphoma Raji but not in the RFX5-deficient http://www.jimmunol.org/ B cell line SJO. RFX5 transfection in SJO restores the occupation. Note that the S box was not occupied. The Y box was always occupied inde- pendent of the presence of RFX5. In vitro methylated DNA of Raji is used as reference. Arrows indicate position of occupancy, open arrows indicate hypermethylation. B, Nucleotide sequence of the proximal promoter region ␤ of 2m and position of occupied sites. Bullets indicate position of occu- pancy, open bullets indicate hypermethylation. by guest on October 1, 2021 strongly indicates the importance of RFX for recruitment of CREB/ATF and cooperative protein complex assembly on the SXY module.

Contribution of RFX and CREB/ATF to constitutive and CIITA- ␤ FIGURE 5. CIITA-induced transactivation is dependent on the pres- induced 2m and MHC class I transactivation ␤ ence of the RFX complex. Transient cotransfection assay of 2m and To determine the interdependency of the CIITA and the RFX tran- HLA-B7 promoter-driven luciferase reporter constructs (1 ␮g/well) with ␤ ␮ scription factors for 2m and MHC class I transactivation, we per- pREP4-RFXB, pREP4-RFX5, pREP4-RFXAP (1 g/well), and/or pREP4- formed transient transfection experiments in BLS-derived fibro- CIITA (0.5 ␮g/well) in BLS fibroblast cells of complementation group A, blast cell lines. CIITA was only able to induce ␤ m and MHC class B, C, and D. Normalized luciferase activity values are expressed as 2 Ϯ ϭ I promoter activity when BLS cell lines were complemented for mean SD of n 4. the missing RFX subunit (Fig. 5). In the RFXB-deficient cell line, the MHC class I promoter could be weakly induced by CIITA (Fig. note that both CREB1 and ATF1 also enhanced the basal level of ␤ 5). This demonstrates the requirement of each of the RFX subunits 2m and MHC class I transactivation (Fig. 6A). for the CIITA route of transactivation. It is of note that RFX also Additional experiments were performed with kCREB, a variant ␤ contributed to the basal level of 2m and MHC class I promoter of CREB that is mutated in its DNA binding domain. Interestingly, activity. The CIITA-independent activation by RFX is not ob- cotransfection with kCREB did not prevent but rather enhanced ␤ served for MHC class II promoters (data not shown; Ref. 6). Pre- the level of transactivation of 2m and MHC class I by CIITA, viously it has been shown that the X2 box binding protein complex whereas the constitutive level of transactivation was unaffected of MHC class I contains CREB/ATF factors such as CREB1, (Fig. 6B). This could indicate that, in the presence of redundant cAMP response element modulator 1, and ATF1 (6, 19). To test CIITA, kCREB can still be part of the multiprotein complex, whether CREB1 and ATF1 are important for the CIITA-induced which as a whole is still functional. Together, this argues for a role ␤ MHC class I and 2m gene transcription, transient cotransfection of RFX and CREB/ATF in both the constitutive and CIITA-in- ␤ experiments were performed. It was shown that expression of ex- duced transactivation of 2m and MHC class I genes. ogenous CREB1 enhanced the CIITA-induced transactivation of ␤ m and MHC class I (Fig. 6A). Similarly, ATF1 lead to an en- General coactivators enhance the CIITA-induced transactivation 2 ␤ ␤ of MHC class I and 2m hanced CIITA-induced transactivation of 2m and MHC class I (Fig. 6A). This demonstrates that CREB1 and ATF1 are functional Although it is evident that for transactivation of MHC class I and ␤ partners of the CIITA route of transactivation. It is of interest to 2m the formation of the multiprotein/DNA complex is essential The Journal of Immunology 5181 Downloaded from http://www.jimmunol.org/

FIGURE 7. General coactivators CBP, p300, GCN5, and PCAF en- ␤ FIGURE 6. CREB1 and ATF1 are partners in the CIITA-induced trans- hance the CIITA-induced transactivation of 2m and MHC class I. A, Tran- ␤ ␤ activation of 2m and MHC class I. A, Transient cotransfection assay of sient cotransfection assay of 2m and HLA-B7 promoter-driven luciferase ␤ ␮ ␮ 2m and HLA-B7 promoter-driven luciferase reporter constructs (1 g/ reporter constructs with pRc/RSV-CBP (1 g/well) and/or pREP4-CIITA well) with pRc/RSV-CREB1, pRc/RSV-ATF1 (1 ␮g/well), and/or pREP4- (0.25 ␮g/well) in Tera-2 cells. Similar results were obtained with HLA-A2 CIITA (CIITA; 0.5 ␮g/well) in Tera-2 cells. B, The non-DNA binding and HLA-DRA promoter-driven luciferase reporter constructs and in by guest on October 1, 2021 ␤ CREB mutant kCREB helps in the CIITA-induced transactivation of 2m COS-1 cells (data not shown). B, Transient cotransfection assay of ␤ and MHC class I. Transient cotransfection assay of 2m and HLA-B7 HLA-B7 promoter-driven luciferase reporter constructs with pRc/RSV- promoter-driven luciferase reporter constructs with pRc/RSV-CREB1 (1 p300, pRc/RSV-GCN5, and pRc/RSV-PCAF (1 ␮g/well) in combination ␮g/well) or pRc/RSV-kCREB (1 ␮g/well) in combination with pREP4- with pREP4-CIITA (0.25 ␮g/well) in Tera-2 cells. Normalized luciferase CIITA (0.5 ␮g/well) in Tera-2 cells. Normalized luciferase activity values activity values are expressed as mean Ϯ SD of n ϭ 4. are expressed as mean Ϯ SD of n ϭ 4.

␤ phorylation by PKA, CBP was not able to transactivate 2m and for CIITA mediated transactivation, none of the proteins in this MHC class I in the absence of CIITA (data not shown). This im- complex (i.e., RFX, CREB/ATF, NFY, and CIITA) are known to plied that CBP and other coactivators act exclusively through CI- posses histone acetyltransferase properties. To investigate whether ITA of the multiprotein complex. general coactivators are involved which could fulfil this function, ␤ we first tested for the role of CBP in 2m and MHC class I trans- Discussion activation. In transient cotransfection experiments, the general co- The existence of a common promoter module (the SXY module) in activator CBP was shown to further enhance the CIITA-induced MHC and functionally related genes has suggested a joint regula- ␤ transactivation of 2m and MHC class I (Fig. 7A). The specificity tion pathway of these genes (5). CIITA regulates all MHC class I ␤ of the enhanced transactivation by CBP was determined by co- and Ib loci (except HLA-G) and 2m in addition to all isotypes and transfection of the early adenovirus S12E1A protein and a func- accessory genes involved in MHC class II Ag presentation (Fig. 1). tionally nonactive S12E1A mutant. Cotransfection of S12E1A Therefore, the SXY module is the basis for the CIITA route of blocked the enhanced transactivation by CBP, whereas cotransfec- transactivation of this large family of closely related genes. tion of the S12E1A mutant had little or no effect (data not shown). The SXY regulatory module shows considerable sequence ho- Similarly, p300, GCN5, and PCAF further enhanced transactiva- mology and conserved spacing between the S and X boxes and the tion by CIITA (Fig. 7B). It is remarkable that CBP (and the other X and Y boxes among the MHC class I, class II and accessory ␤ coactivators) could not transactivate 2m and MHC class I in the genes. The conservation of the nucleotide sequence and spatial absence of CIITA (Fig. 7, A and B), because the X2 box-binding requirement of the X and Y boxes together with the mutual de- CREB/ATF complex is a potential target for CBP recruitment. pendency of the individual proteins for assembly and transactiva- ␤ To test whether the lack of CBP-induced transactivation was tion in 2m and class I genes argues that the multiprotein complex caused by a lack of phosphorylation of CREB, we performed ad- of RFX, CREB/ATF, NFY, and CIITA functions as an enhanceo- ditional cotransfection experiments with cAMP-dependent PKA some (11). It is shown that the X1, X2, and Y boxes of the SXY ␤ and its functionally inactive mutant, PKAmut. Even after phos- module in 2m and MHC class I genes are crucial for CIITA- 5182 THE MHC-SPECIFIC ENHANCEOSOME AND MHC CLASS I TRANSACTIVATION induced transactivation. In addition, a conserved spacing and The importance of CIITA in MHC expression is of a different alignment of the X and Y boxes is required for optimal CIITA- nature in MHC class I and class II expression. This is most evident induced transactivation. This is particularly clear in the transacti- in lymphoid cells, which constitutively express CIITA. In this type ␤ vation of the 2m promoter. Similar to MHC class II promoters, of cells, the absence of CIITA results in a reduced MHC class I cell XY DNA is cooperatively bound by a multiprotein complex, con- surface expression, whereas MHC class II is absent. The residual sisting of RFX, CREB/ATF, and NFY. IVGF studies revealed that MHC class I expression is due to the fact that MHC class I genes ␤ ␬ occupancy of the X1 and X2 boxes of the 2m promoter in RFX5- possess additional upstream regulatory elements ( B site, ISRE), deficient cells can be restored by functional complementation. This which provide for alternative transactivation pathways that can strongly suggest that the other RFX subunits and CREB/ATF are compensate for the lack of CIITA-induced expression. This is in recruited by RFX5 binding to the SXY module. contrast to MHC class II genes, which (with the exception of the Multiprotein complex formation on XY DNA can still be ac- invariant chain) only posses an SXY module and, therefore, fully complished despite an apparent weak binding of the individual are dependent on CIITA. As a result of these compensatory path- ␤ proteins to their respective binding sites, as is known from studies ways of 2m and MHC class I transactivation, the absence of on MHC class II promoters (9, 10). This is illustrated by cotrans- CIITA only results in a reduction of MHC class I cell surface fection of CIITA with kCREB, a CREB variant that lacks its DNA expression in activated T cells (Fig. 1A), whereas MHC class II binding domain, which still amplified, albeit less than wild-type expression is absent. In EBV-transformed B cells, the upstream ␤ CREB, the CIITA-induced transactivation of 2m and MHC class regulatory elements provide for a more full compensation of a I (see Fig. 6B). A possible explanation is that kCREB dimerizes CIITA-deficiency, due to the (EBV-related) high expression of with other CREB/ATF proteins and is as such incorporated into the NF-␬B and IFN-regulatory factors. Therefore, CIITA-deficient Downloaded from multiprotein complex. Because there is no absolute requirement EBV-transformed B cells, displayed in general only a lack of MHC for strong DNA binding affinity of the individual proteins to their class II expression, and no change in MHC class I expression at the respective binding sites, in particular to the X2 box, it appears to cell surface (data not shown), despite the fact that promoter activ- ␤ be the DNA binding affinity of the complex as a whole, which ity of 2m and MHC class I was reduced (Fig. 1B). In EBV- determines the ability of the multiprotein complex to transactivate transformed B cell lines, a similar compensation is observed of the (this study; Ref. 9). Therefore, it can be envisaged that there exists expression of the invariant chain. Because the invariant chain con- a certain tolerance for weak protein/DNA interactions in multipro- tains also additional regulatory elements in its promoter (29), they http://www.jimmunol.org/ tein complex functioning and CIITA-induced transactivation. This may provide for alternative transactivation pathways resulting re- would imply that proteins with minor mutations could still be func- duced promoter activity and in only a marginal reduction of in- tional within the multiprotein complex and that minor MHC class variant chain expression in CIITA-deficient (EBVϩ) B cells I locus-specific nucleotide variation in the regulatory elements of (S. J. P. Gobin, P. Biesta, and P. J. Van den Elsen, unpublished ␤ the SXY module is tolerated. observations; Ref. 20). The role of RFX in 2m and MHC class I The Y box is perhaps the most important “anchor” point and its transactivation is bipartite; RFX (as part of the multiprotein com- protein/DNA interactions are the most stringent. In line with the plex) is the mediator for transactivation by CIITA and it is a me-

importance of the Y box is the fact that of all boxes of the SXY diator for constitutive expression. The importance in the CIITA by guest on October 1, 2021 ␤ module, the Y box-region in MHC class I and 2m promoters route of transactivation is best illustrated in CIITA expressing T displays the highest nucleotide conservation (5) and appears to be cells that are deficient in the RFX subunits RFXB/RFXANK, constitutively occupied in vivo even in the absence of RFX5 (Fig. RFX5, or RFXAP. In these activated BLS-derived T cells the de- 4). Moreover, individuals with a mutation in the Y box have a ficiency in RFX, which abrogates the transactivation by CIITA, greatly reduced level of constitutive MHC class I expression (21, resulted in a reduced MHC class I cell surface expression congru- 22). Recent studies demonstrated that the NFY complex, which is ent with a lack of MHC class II expression (Fig. 1A). Furthermore, an integral part of the multiprotein complex, is able to interact with both RFX and CREB/ATF contribute also to the constitutive ex- ␤ and disrupt nucleosomes (23). It is proposed that the histone-like pression of 2m and MHC class I genes (Figs. 5 and 6). This structures in the NFY components are essential for this function. In second role for RFX is revealed in nonlymphoid cells, which only addition, DNA bending studies strongly suggest a major role for express CIITA upon induction by IFN-␥. BLS-derived fibroblasts NFY in promoter architecture (23). Furthermore, it has been deficient in one of the RFX subunits display a reduced MHC class shown that NFY increases the affinity for other transcription fac- I expression, which can be restored by complementation with the tors to their, sometimes poor, binding sites. As such, NFY could missing RFX subunit (6). This role in the constitutive expression fulfil an essential role in remodeling (23). Together, this of MHC class I is most probably related to the upstream cluster of ␤ argues for an essential role of NFY in multiprotein complex assembly regulatory elements in 2m and MHC class I genes, because nei- and functioning, rendering the Y box an important anchor point of the ther RFX nor CREB could induce MHC class I promoter con- XY module, as has been suggested for MHC class II (24, 25). structs lacking the upstream promoter elements (S. J. P. Gobin, M. No evidence was found for a direct role of the S box in the Van Zutphen, and P. J. Van den Elsen, unpublished observations). ␤ ␤ CIITA route of 2m and MHC class I transactivation, neither for Thus, CIITA provides for an ancillary route in 2m and MHC class multiprotein complex formation nor for promoter occupancy (Figs. I gene transactivation and requires the RFX-CREB/ATF-NFY 2Ð4). Therefore, it is possible that the S box does not act as a complex. Of this multiprotein complex, RFX and CREB have a genuine transcription factor-binding site, but rather plays a role in dual function; they have an ancillary function in the constitutive ␤ promoter architecture. The lack of occupancy of the S box in MHC 2m and MHC class I activation driven by the upstream regulatory class II would also suggest a minor role of the S box in the tran- elements, and provide, as part of the RFX-CREB/ATF-NFY com- scriptional control of MHC class II genes (this study; Refs. 16 and plex, the platform for CIITA transactivation. ␤ 20). However, other studies have shown that the S box could play For 2m and MHC class I, the multiprotein complex is crucial a role in the CIITA-mediated route of transactivation through the for CIITA-induced transactivation and is likely to have a similar SXY module (26Ð28), which may reflect a different role of the S build up as for MHC class II genes. Recent studies have revealed ␤ box in transactivation of 2m and MHC class I, vs MHC class II many aspect of the assembly of the RFX-CREB/ATF-NFY mul- genes. tiprotein complex on SXY DNA and interactions with CIITA (25, The Journal of Immunology 5183

26, 28, 30Ð32). In addition, CIITA can form a link to the basal 4. Martin, B. K., K. C. Chin, J. C. Olsen, C. A. Skinner, A. Dey, K. Ozato, and transcription initiation complex by interaction with its components J. Y. P. Ting. 1997. Induction of MHC class I expression by the MHC class II transactivator CIITA. Immunity 6:591. (29, 33Ð35). Together, this has lead to view this multiprotein com- 5. Van den Elsen, P. J., A. Peijnenburg, M. C. J. A. Van Eggermond, and plex as an enhanceosome (11, 32). S. J. P. Gobin. 1998. Shared regulatory elements in the promoters of MHC class I and class II genes. Immunol. Today 19:308. Histone acetylation is proposed to be an important mechanism 6. Gobin, S. J. P., A. Peijnenburg, M. Van Eggermond, M. Van Zutphen, to facilitate the recruitment of the basal transcription initiation R. Van den Berg, and P. J. Van den Elsen. 1998. The RFX complex is crucial for ␤ complex and binding of transcription factors to upstream regula- the constitutive and CIITA-mediated transactivation of MHC class I and 2- microglobulin genes. Immunity 9:531. tory elements; histone hyperacetylation is associated with tran- 7. Nagarajan, U. M., A. Peijnenburg, S. J. P. Gobin, J. M. Boss, and scriptional activity and hypoacetylation with transcriptionally si- P. J. Van den Elsen. 2000. Novel mutations within the RFX-B gene and partial lent chromatin (36). None of the proteins in the MHC-specific rescue of MHC and related genes through exogenous class II transactivator in RFX-B-deficient cells. J. Immunol. 164:3666. multiprotein complex (i.e., RFX, CREB/ATF, NFY, or CIITA) are 8. Schoneich, J., J. L. Lee, P. Mansky, M. Sheffery, and S. Y. Yang. 1997. The known to possess histone acetyltransferase properties. It can be pentanucleotide ATTGG, the “inverted CCAAT,” is an essential element for envisaged that the enhanceosome, to fulfil these functions, requires HLA class I gene transcription. J. Immunol. 158:4788. 9. Louis-Plence, P. L., C. S. Moreno, and J. M. Boss. 1997. Formation of a regu- histone acetyltransferases, such as the coactivators CBP, p300, latory factor X/X2 Box-binding protein nuclear factor-Y multiprotein complex on GCN5, and PCAF (36). Recently, functional interactions of CIITA the conserved regulatory regions on HLA class II genes. J. Immunol. 159:3899. 10. Fontes, J. D., N. Jabrane-Ferrat, and B. M. Peterlin. 1997. Assembly of functional with CBP, and of NFY with p300, GCN5, and PCAF have been regulatory complexes on MHC class II promoters in vivo. J. Mol. Biol. 270:336. demonstrated (23, 37, 38), which all have intrinsic histone acety- 11. Carey, M. 1998. The enhanceosome and transcriptional synergy. Cell 92:5. lation activity. 12. Peijnenburg, A., R. Van den Berg, M. J. C. A. Van Eggermond, O¬ . Sanal, J. M. J. J. Vossen, A.-M. Lennon, C. Alcaõ¬de-Loridan, and P. J. Van den Elsen. Here it is shown that CBP, p300, GCN5, and PCAF all enhance 2000. Defective MHC class II expression in an MHC class II deficiency patient Downloaded from ␤ the CIITA-induced transactivation of 2m and MHC class I. More- is caused by a novel deletion of a splice donor site in the MHC class II trans- activator gene. Immunogenetics 51:42. over, although CBP is a classic coactivator of CREB, in this ex- ¬ ␤ 13. Peijnenburg, A., M. C. J. A. Van Eggermond, R. Van den Berg, O. Sanal, perimental set-up CBP could not transactivate 2m and MHC class J. M. J. J. Vossen, and P. J. Van den Elsen. 1999. Molecular analysis of an MHC I genes in the absence of CIITA. Similarly, p300, GCN5 and class II deficiency patient reveals a novel mutation in the RFX5 gene. Immuno- PCAF exerted their ancillary function in ␤ m and MHC class I genetics 49:338. 2 14. Peijnenburg, A., S. J. P. Gobin, M. C. J. A. Van Eggermond, B. C. Godthelp, transactivation exclusively through CIITA. This shows that CIITA N. Graafeiland, and P. J. Van den Elsen. 1997. Introduction of exogenous class is the principal mediator for general coactivator activity. It is not II trans-activator in MHC class II-deficient ABI fibroblasts results in incomplete http://www.jimmunol.org/ ␤ rescue of MHC class II antigen expression. J. Immunol. 159:2720. clear why these coactivators could not transactivate 2m and MHC 15. Mueller, P. R., and B. Wold. 1989. In vivo footprinting of a muscle specific class I genes in the absence of CIITA. However, this could be enhancer by ligation mediated PCR. Science 246:780. explained by a recent report that the acetylation of CIITA by 16. Westerheide, S. D., P. Louis-Plence, D. Ping, X. F. He, and J. M. Boss. 1997. HLA-DMA and HLA-DMB functions through the conserved PCAF induces its nuclear accumulation and consecutive transac- SXY region. J. Immunol. 158:4812. tivation activity (39). This would imply that the enhanceosome is 17. Gobin, S. J. P., V. Keijsers, and P. J. Van den Elsen. 1998. The role of enhancer for its function dependent on CIITA acetylation by general coac- A in the locus-specific regulation of classical and non-classical HLA class I genes by NF-␬B. J. Immunol. 161:2276. tivators, but does not require the recruitment of the acetyltrans- 18. Gobin, S. J. P., M. Van Zutphen, A. M. Woltman, V. Keijsers, and P. J. Van den Elsen. 1999. Transactivation of classical and nonclassical HLA ferases to the protein/DNA complex. Still, it is possible that co- by guest on October 1, 2021 activators are recruited to the enhanceosome and form together class I genes through the IFN-stimulated response element. J. Immunol. 163: 1428. with CIITA a link with the basal transcription initiation complex. 19. Gobin, S. J. P., and P. J. Van den Elsen. 2001. Locus-specific regulation of In conclusion, in this study we show that the SXY module is the HLA-A and HLA-B expression is not determined by nucleotide variation in the X2 box promoter element. Blood 97:1518. basis for a cooperatively functioning multiprotein complex that 20. Kara, C. J., and L. H. Glimcher. 1993. Promoter accessibility within the envi- ␤ controls MHC class I and 2m, which is similar to that of MHC ronment of MHC is affected in class II-deficient combined immunodeficiency. class II and its accessory genes. Because of the spatial constraints EMBO J. 12:187. 21. Balas, A., F. Garcõ«a-Sa«nchez, F. Go«mez-Reino, and J. Vicario. 1994. HLA-A for the regulatory elements and the cooperative acting protein/ class I allele HLA-A2 expression defect associated with a mutation in its en- DNA it can be viewed as an MHC-specific enhanceosome (11). hancer B inverted CAT in two families. Hum. Immunol. 41:69. Although several components (CREB/ATF and NFY) of this en- 22. Lardy, N. M., N. Otting, A. R. Van der Horst, R. E. Bontrop, and L. P. De Waal. 1997. 5Ј regulatory nucleotide sequence of an HLA*0101 null allele. Immuno- hanceosome are implied in the transactivation of many other genes genetics 46:152. (23, 40), other components (RFX and CIITA) are, until now, only 23. Mantovani, R. 1999. The molecular biology of the CCAAT-binding factor NF-Y. known to be directly involved in transactivation of MHC and ac- Gene 239:15. 24. Linhoff, M. W., K. L. Wright, and J. P. Y. Ting. 1997. CCAAT-binding factor cessory genes. Thus, the specificity of this enhanceosome is de- NF-Y and RFX are required for in vivo assembly of a nucleoprotein complex that termined by the unique composition of the SXY module as well as spans 250 base pairs: the invariant chain as a model. Mol. Cell. Biol. 17:4589. 25. Zhu, X. S., M. W. Linhoff, G. Li, K. C. Chin, S. N. Maity, and J. P. Ting. 2000. the unique components and assembly of the multiprotein complex. Transcriptional scaffold: CIITA interacts with NF-Y, RFX, and CREB to cause stereospecific regulation of the class II major histocompatibility complex pro- moter. Mol. Cell. Biol. 20:6051. Acknowledgments 26. Nekrep, N., N. Jabrane-Ferrat, and B. M. Peterlin. 2000. Mutations in the bare We thank Dr. S. Berger, Dr. M. Green, Dr. T. Kouzarides, Dr. Y. Nakatani, lymphocyte syndrome define critical steps in the assembly of the regulatory factor Dr. S. McKnight, Dr. R. Goodman, and Dr. T. Collins for providing X complex. Mol. Cell. Biol. 20:4455. reagents. We also thank M. van Eggermond for excellent technical assis- 27. Brown, J. A., E. M. Rogers, and J. M. Boss. 1998. Mutational analysis of the MHC class II transactivator CIITA indicates a functional requirement for con- tance, and Dr. N. van der Stoep and Dr. J. P. Medema for critically reading served LCD motifs and for interactions with the conserved W-box promoter the manuscript. element. Nucleic Acids Res. 26:4128. 28. Westerheide, S. D., and J. M. Boss. 1999. Orientation and positional mapping of the subunits of the multicomponent transcription factors RFX and X2BP to the References major histocompatibility complex class II transcriptional enhancer. Nucleic Acids 1. Reith, W., and B. Mach. 2001. The bare lymphocyte syndrome and the regulation Res. 27:1635. of MHC expression. Annu. Rev. Immunol. 19:331. 29. Harton, J. A., and J. P.-Y. Ting. 2000. Class II transactivator: mastering the art 2. DeSandro, A., U. M. Nagarajan, and J. M. Boss. 1999. The bare lymphocyte of major histocompatibility complex expression. Mol. Cell. Biol. 20:6185. syndrome: molecular clues to the transcriptional regulation of major histocom- 30. DeSandro, A. M., U. M. Nagarajan, and J. M. Boss. 2000. Associations and patibility complex class II genes. Am. J. Hum. Genet. 65:279. interactions between bare lymphocyte syndrome factors. Mol. Cell. Biol. 20: 3. Gobin, S. J. P., A. Peijnenburg, V. Keijsers, and P. J. Van den Elsen. 1997. Site 6587. ␣ is crucial for two routes of IFN-␥-induced MHC class I transactivation: the 31. Villard, J., M. Peretti, K. Masternak, E. Barras, G. Caretti, R. Mantovani, and ISRE mediated route and a novel pathway involving CIITA. Immunity 6:601. W. Reith. 2000. A functionally essential domain of RFX5 mediates activation of 5184 THE MHC-SPECIFIC ENHANCEOSOME AND MHC CLASS I TRANSACTIVATION

major histocompatibility complex class II promoters by promoting cooperative 36. Sterner, D. E., and S. L. Berger. 2000. Acetylation of histones and transcription- binding between RFX and NF-Y. Mol. Cell. Biol. 20:3364. related factors. Microbiol. Mol. Biol. Rev. 64:435. 32. Masternak, K., A. Muhlethaler-Mottet, J. Villard, M. Zufferey, V. Steimle, and 37. Kretsovali, A., T. Agalioti, C. Spilianakis, E. Tzortzakaki, M. Merika, and W. Reith. 2000. CIITA is a transcriptional coactivator that is required to MHC J. Papamatheakis. 1998. Involvement of CREB binding protein in expression of class II promoters by multiple synergistic interactions with an enhanceosome major histocompatibility complex class II genes via interaction with the class II complex. Genes Dev. 14:1156. 33. Mahanta, S. K., T. Scholl, F. C. Yang, and J. L. Strominger. 1997. Transactiva- transactivator. Mol. Cell. Biol. 18:6777. tion by CIITA, the type II bare lymphocyte syndrome-associated factor, requires 38. Fontes, J. D., S. Kanazawa, D. Jean, and B. M. Peterlin. 1999. Interactions be- participation of multiple regions of the TATA box binding protein. Proc. Natl. tween the class II transactivator and CREB binding protein increase transcription Acad. Sci. USA 94:6324. of major histocompatibility complex class II genes. Mol. Cell. Biol. 19:941. 34. Scholl, T., S. K. Mahanta, and J. L. Strominger. 1997. Specific complex forma- 39. Spilianakis, C., J. Papamatheakis, and A. Kretsovali. 2000. Acetylation by PCAF tion between the type II bare lymphocyte syndrome-associated transactivators enhances CIITA nuclear accumulation and transactivation of major histocompat- CIITA and RFX5. Proc. Natl. Acad. Sci. USA 94:6330. 35. Fontes, J. D., B. Jiang, and B. M. Peterlin. 1997. The class II trans-activatorCI- ibility complex class II genes. Mol. Cell. Biol. 20:8489. ITA interacts with the TBP-associated factor TAFII33. Nucleic Acids Res. 25: 40. Montminy, M. 1997. Transcriptional regulation by cyclic AMP. Annu. Rev. Bio- 2522. chem. 66:807. Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021