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Silencing by DNA MethylationMHC Class II Genes against Epigenetic The Transcription Factor RFX Protects

Arcangelo Nocera and Walter ReithAugusto Tagliamacco, Gianfranco Abbate, Jack Gorski,Krawczyk, Elisa Leimgruber, Jean Villard, Capucine Picard, Queralt Seguín-Estévez, Raffaele De Palma, Michal

http://www.jimmunol.org/content/183/4/2545doi: 10.4049/jimmunol.0900376July 2009;

2009; 183:2545-2553; Prepublished online 20J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2009 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Transcription Factor RFX Protects MHC Class II Genesagainst Epigenetic Silencing by DNA Methylation1

Queralt Seguín-Estevez,2* Raffaele De Palma,2† Michal Krawczyk,3* Elisa Leimgruber,*Jean Villard,‡ Capucine Picard,§ Augusto Tagliamacco,¶ Gianfranco Abbate,† Jack Gorski,�

Arcangelo Nocera,2¶ and Walter Reith2,4*

Classical and nonclassical MHC class II (MHCII) genes are coregulated by the transcription factor RFX (regulatory factor X) andthe transcriptional coactivator CIITA. RFX coordinates the assembly of a multiprotein “enhanceosome” complex on MHCIIpromoters. This enhanceosome serves as a docking site for the binding of CIITA. Whereas the role of the enhanceosome inrecruiting CIITA is well established, little is known about its CIITA-independent functions. A novel role of the enhanceosome wasrevealed by the analysis of HLA-DOA expression in human MHCII-negative B cell lines lacking RFX or CIITA. HLA-DOA wasfound to be reactivated by complementation of CIITA-deficient but not RFX-deficient B cells. Silencing of HLA-DOA was asso-ciated with DNA methylation at its promoter, and was relieved by the demethylating agent 5-azacytidine. Surprisingly, DNAmethylation was also established at the HLA-DRA and HLA-DQB loci in RFX-deficient cells. This was a direct consequence of theabsence of RFX, as it could be reversed by restoring RFX function. DNA methylation at the HLA-DOA, HLA-DRA, and HLA-DQBpromoters was observed in RFX-deficient B cells and fibroblasts, but not in CIITA-deficient B cells and fibroblasts, or in wild-typefibroblasts, which lack CIITA expression. These results indicate that RFX and/or enhanceosome assembly plays a key CIITA-independent role in protecting MHCII promoters against DNA methylation. This function is likely to be crucial for retainingMHCII genes in an open chromatin configuration permissive for activation in MHCII-negative cells, such as the precursors ofAPC and nonprofessional APC before induction with IFN-�. The Journal of Immunology, 2009, 183: 2545–2553.

M olecules of MHC class II (MHCII)5 play pivotal rolesin the adaptive immune system because they presentpeptides to the Ag receptor (TCR) of CD4� T cells. In

the thymus, the recognition of MHCII-peptide complexes by theTCR of developing thymocytes guides the positive and negative

selection processes that shape the TCR repertoire of the CD4� Tcell population. In the periphery, MHCII-mediated Ag presenta-tion to CD4� T cells is critical for the maintenance of peripheralself-tolerance and for the initiation, regulation, and development ofAg-specific immune responses directed against infectious agentsand tumors. To ensure these diverse functions, MHCII genes haveto be expressed in a complex and tightly regulated cell type-spe-cific and inducible pattern. Their expression is largely restricted tospecialized epithelial cells in the cortex (cTEC) and medulla(mTEC) of the thymus and dedicated APC, including various den-dritic cell (DC) subsets, cells of the monocyte-macrophage lin-eage, and B cells (1–4). Other cell types, such as endothelial cells,epithelial cells, and fibroblasts, generally do not express MHCIIgenes unless they are stimulated with IFN-� (1–4). IFN-�-inducedMHCII expression on such nonhematopoietic cells is thought toallow them to participate as nonprofessional APC during ongoingimmune responses.

Humans have three “classical” MHCII isotypes: HLA-DR,HLA-DP, and HLA-DQ. Intracellular routing and peptide loadingof these classical MHCII molecules require several accessory mol-ecules, including the invariant (Ii) chain and two “nonclassical”MHCII molecules called HLA-DM and HLA-DO (5–7). The genesencoding the Ii chain and the �- and �-chains of HLA-DR, HLA-DQ, HLA-DP, and HLA-DM are expressed in a tightly coregu-lated manner (1–4). The genes encoding the �- and �-chains ofHLA-DO are also coregulated with MHCII genes (8–10), but in amore cell type-restricted pattern. HLA-DO expression is largelylimited to B cells, mTEC, and certain DC subsets (7, 11–14).

The coexpression of MHCII and accessory genes is coordinatedby a conserved enhancer situated upstream of the transcriptionstart site (TSS) of each gene (1–4). This enhancer functions as acomposite regulatory module consisting of four subsequences

*University of Geneva, Faculty of Medicine, Geneva, Switzerland; †Department ofClinical and Experimental Medicine, Second University of Naples, Naples, Italy;‡Immunology and Transplant Unit, Geneva University Hospital, Geneva, Switzer-land; §Study Center of Primary Immunodeficiencies, Necker Hospital, AssistancePublique–Hopitaux de Paris, Paris, France; ¶Transplant Immunology Unit, Depart-ment of Transplantation, S. Martino Hospital, Genoa, Italy; and �Blood Research Institute,Blood Center of Wisconsin, Milwaukee WI 53201

Received for publication February 4, 2009. Accepted for publication June 9, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 Work in the laboratory of W. Reith was supported by the Swiss National ScienceFoundation, The Swiss Multiple Sclerosis Society, The Geneva Cancer League,and the National Center of Competence in Research–Neural Plasticity and Repair(NCCR-NEURO). Work in the laboratory of R. de Palma was supported by Grants2005064784_004 and 2007XKCCWF_004 from the Ministero dell’Istruzionedell’Universita e della Ricerca. Work in the laboratory of A. Nocera was in partsupported by a scholarship to A. Tagliamacco provided by the S. Martino Hos-pital, Genoa, Italy.2 Q.S.-E., R.P., A.N., and W.R. contributed equally to this study.3 Current address: Regulatory Biology Laboratory, Salk Institute for Biological Stud-ies, 10010 North Torrey Pines Road, La Jolla, CA 92037.4 Address correspondence and reprint requests to Dr. Walter Reith, Department of Pa-thology and Immunology, University of Geneva, Faculty of Medicine, 1 rue Michel-Servet, CH-1211, Geneva, Switzerland. E-mail address: Walter.Reith@unige.ch5 Abbreviations used in this paper: MHCII, MHC class II; BLS, bare lymphocytesyndrome; ChIP, chromatin immunoprecipitation; CREB, cAMP responsive element-binding protein; cTEC, cortical thymic epithelial cells; DC, dendritic cell; 5AC,5-azacytidine; mTEC, medullary thymic epithelial cell; NF-Y, nuclear factor Y; RFX,regulatory factor X; TSA, trichostatin A; TSS, transcription start site; WT, wild type.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

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called the S, X, X2, and Y boxes (Fig. 1A). The molecular ma-chinery that regulates MHCII expression via the S-X-X2-Y (S-Y)enhancer has been exceptionally well defined thanks to elucidationof the genetic defects responsible for the bare lymphocyte syn-drome (BLS), a hereditary immunodeficiency disease resultingfrom mutations in genes encoding transcription factors that areessential for MHCII expression (1). Most BLS patients carry mu-tations in the genes coding for an X box-binding complex calledregulatory factor X (RFX) (1). RFX is composed of three subunitscalled RFX5, RFXAP, and RFXANK (or RFX-B). BLS can be dueto mutations in each of these subunits (15–18). RFX binds coop-eratively with the X2 box-binding factor cAMP responsive ele-ment-binding protein (CREB) (19) and the Y box-binding proteinnuclear factor Y (NF-Y) (20) to generate a higher order multipro-tein enhanceosome complex on the S-Y module (Fig. 1A) (21–24).This enhanceosome complex then serves as a platform for recruit-

ment of CIITA (4, 24, 25). The latter is a non-DNA-binding tran-scriptional coactivator that binds to the enhanceosome complex bymeans of multiple protein-protein interactions with RFX, CREB,and NF-Y (Fig. 1A) (24, 26, 27).

Whereas RFX, CREB, and NF-Y are expressed widely in mostcell types, CIITA is expressed in a cell type-specific and IFN-�-inducible manner that governs the constitutive and inducible pat-tern of MHCII expression (1–4). Due to this key role as the masterregulator of MHCII genes, most studies on the molecular mecha-nisms controlling MHCII expression have concentrated on themode of action of CIITA. Mechanisms implicated in MHCII geneactivation by CIITA include the recruitment of chromatin remod-eling factors, histone modifying complexes, components of thegeneral transcription machinery, and transcription elongation fac-tors (28–33). In these studies, the enhanceosome was generallyattributed a more passive role, serving primarily as a docking

FIGURE 1. The HLA-DOA and HLA-DOB genes are coregulated with classical MHCII genes by RFX and CIITA. A, Schematic representation of themolecular machinery that controls MHCII gene expression. The transcription factors RFX, CREB, and NF-Y bind cooperatively to form an enhanceosomecomplex bound to the conserved S-Y enhancers found upstream of the TSS of each MHCII gene. RFX consists of three subunits, RFX5, RFXANK, andRFXAP. The enhanceosome complex constitutes a platform to which CIITA is recruited by protein-protein interactions. Activation of transcription (arrow)is strictly dependent on both enhanceosome assembly and CIITA recruitment. B, The expression levels of actin, HLA-DRB, HLA-DOA, and HLA-DOBmRNAs were compared by semiquantitative RT-PCR between RFX5-deficient SJO B cells and control Raji B cells, splenic mononuclear cells (SMC), orblood mononuclear cells (BMC). C, The expression levels of HLA-DOA, HLA-DOB, HLA-DRA, and HLA-DPB mRNAs were measured by real-timeRT-PCR in WT Raji cells, CIITA-deficient RJ2.2.5 cells (RJ), the WT B cell line HHK, RFXANK-deficient BLS1 cells, RFX5-deficient SJO cells, andRFXAP-deficient 6.1.6 and DA cells. Results are expressed relative to the Raji or B-EBV cells, and show the mean � SD derived from three independentexperiments. D, Binding of RFX and CIITA to the HLA-DOA and HLA-DOB promoter regions (P) were analyzed in Raji cells by semiquantitative ChIP.Regions situated at the end of HLA-DOA (3�) and in exon 6 of HLA-DOB (E6) were used as negative controls. A preimmune serum was used as controlAb. PCR fragments were quantified relative to the indicated dilutions of input chromatin. E, Binding of RFX and CIITA to the HLA-DOA, HLA-DOB, andHLA-DRA promoters were analyzed by quantitative ChIP in Raji, RJ2.2.5 (RJ), and BLS1 cells. Results are representative of three experiments and areexpressed relative to Raji.

2546 RFX PROTECTS MHC CLASS II GENES AGAINST DNA METHYLATION

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surface for CIITA. However, there is growing evidence that theenhanceosome also makes key CIITA-independent contributionsto MHCII gene activation, such as nucleosome eviction from theTSS (34–36).

A detailed analysis of defective MHCII expression in mutantcells lacking RFX or CIITA indicated that HLA-DOA expressioncould be reactivated by complementation of CIITA-deficient cellsbut not RFX-deficient cells. The irreversible nature of impairedHLA-DOA expression in RFX-deficient cells pointed to an epige-netic silencing mechanism. Analysis of this mechanism revealedthat a key function of RFX and/or the enhanceosome complex is toprotect HLA-DOA and other MHCII genes against the establish-ment of DNA methylation. Protection against DNA methylationwas abrogated in RFX-deficient B cells and fibroblasts but re-mained effective in CIITA-deficient B cells and fibroblasts. Theseresults thus reveal a novel CIITA-independent function of RFXand the enhanceosome complex.

Materials and MethodsCells and culture

The Raji, HHK, RJ2.2.5, BLS1, SJO, 6.1.6, DA, and ABI cell lines, as wellas CIITA-complemented RJ2.2.5 cells, RFXANK-complemented BLS1cells, RFX5-complemented SJO cells, and RFXAP-complemented 6.1.6and DA cells, have all been described (37–40). The BLS3 cell line wasestablished from a skin biopsy of an MHCII-deficient BLS patient. Cellswere grown in RPMI 1640 plus GlutaMAX medium (Invitrogen) supple-mented with 10% (15% for BLS3) FCS and antibiotics. ComplementedSJO cells were cultured for 48 h in the presence of 150 nM trichostatin A(TSA), 1, 2 or 5 �M 5-azacytidine (5AC), or a combination of 150 nMTSA with 1, 2, or 5 �M 5AC. Control primary B cells consisted of humanPBMC or spleen lymphocytes prepared from a patient splenectomized foran abdominal trauma. All human cells were obtained with consent underaccepted protocols.

RNA expression analysis

RNA extraction and cDNA synthesis were performed as described (41).mRNAs were quantified by semiquantitative RT-PCR or real-time PCR asdescribed (41, 42). Primer sequences will be made available upon request.Results were normalized using 18S rRNA.

Chromatin immunoprecipitation (ChIP)

ChIP experiments were performed as described (34, 41) using Abs specific forCIITA, RFX, H3-Ac, H4-Ac, H3-K27Me3 (all from Upstate Biotechnology),and anti-histone H3 (Abcam). Results were quantified by real-time PCR usingthe iCycler iQ real-time PCR detection system (Bio-Rad) and a SYBR Green-based kit for quantitative PCR (iQ Supermix; Bio-Rad).

Bisulfite sequencing

Two to 6 �g of genomic DNA was cut with HindIII, purified by phenol-chloroform extraction, and denatured by adding NaOH to 0.3 M and in-cubating at 37°C for 20 min. DNA was then modified by an overnightincubation at 50°C in the dark in 218 �l of 3.6 M bisulfite (Sigma-Aldrich),0.5 mM hydroquinone. The modified DNA was purified using PCR puri-fication columns (Qiagen) and eluted in 50 �l of elution buffer. A seconddenaturation step was then performed as above. Samples were neutralizedwith NH4OAc, precipitated, and washed with ethanol. Bisulfite-modifiedDNA was amplified by PCR using the Expand High Fidelity enzyme(Roche). Amplification cycles were adapted to the lengths of the PCRproducts and the melting temperature of the primers. PCR products werepurified (High Pure PCR product purification; Roche) and sequenced di-rectly, or cloned in the pGEM-T Easy Vector (Promega) for sequencing ofindividual clones. Conversion of nonmethylated cytosines to thymidineswas �95% in all experiments.

Identification of the mutation in BLS3

Treatment of the BLS3 cells with IFN-� demonstrated that MHCII expres-sion was not induced. In contrast, complementation of BLS3 with a CIITAexpression vector induced MHCII expression. These results suggested thatthe defect lies in the CIITA gene. CIITA cDNAs from BLS3 were amplifiedby RT-PCR, cloned, and sequenced. All cDNAs contained an in-framedeletion of exon 18. Exon 18 and its flanking intronic sequences were

amplified from BLS3 genomic DNA, cloned, and sequenced. All clonescontained a G to A mutation at the first nucleotide of the splice donor sitesituated downstream of exon 18. The same mutation was identified previ-ously in another BLS patient (BCH.) and has been shown to lead to skip-ping of exon 18 and to a complete loss of function of CIITA (43).

ResultsIrreversible loss of HLA-DOA expression in RFX-deficient cells

There have been conflicting reports on the relative dependence ofHLA-DOA and HLA-DOB expression on RFX and CIITA (8, 9).We therefore quantified HLA-DOA and HLA-DOB mRNAs in var-ious RFX- and CIITA-deficient B cell lines by semiquantitativeRT-PCR and quantitative real-time RT-PCR (Fig. 1, B and C).Compared with wild type (WT) control cells, HLA-DOA and HLA-DOB mRNA levels were strongly reduced in CIITA-deficientRJ2.2.5 cells, RFX5-deficient SJO cells, RFXANK-deficient BLS1cells, and RFXAP-deficient 6.1.6 and DA cells. This reduction wasin most cases almost as strong as that observed for classical MHCIIgenes such as HLA-DRA, HLA-DRB, and HLA-DPB. As ob-served previously by others, the dependence of HLA-DOB expres-sion on CIITA was less strong than its dependence on RFX (9). Aresidual level of HLA-DOA expression was evident in RFXANK-deficient BLS1 cells, but not in the other RFX-deficient cells. Sim-ilar variations between different BLS cells in the degree of residualexpression of specific MHCII genes have been reported previously(1). The explanation for such cell type-specific differences in leakyMHCII expression remains unknown, but has in certain cases beenattributed to partial loss-of-function mutations (44).

These expression studies confirmed that the HLA-DOA andHLA-DOB genes are indeed regulated by both RFX and CIITA. Toverify this further we performed semiquantitative and quantitativeChIP experiments with Abs directed against RFX and CIITA (Fig.1, D and E). In WT B cells, both RFX and CIITA were indeedfound to bind specifically to the HLA-DOA and HLA-DOB pro-moters (Fig. 1D). As observed for the control HLA-DRA promoter(34, 41, 45), binding of RFX to the HLA-DOA and HLA-DOBpromoters was abolished in RFXANK-deficient BLS1 cells butwas retained in CIITA-deficient RJ2.2.5 cells, whereas binding ofCIITA was lost in both mutants (Fig. 1E).

To confirm further that expression of the HLA-DOA and HLA-DOB genes is activated directly by RFX and CIITA, we comple-mented the RFX-deficient and CIITA-deficient cell lines with ex-pression vectors encoding the corresponding WT proteins. Theexpression of HLA-DOA and HLA-DOB was restored as efficientlyas that of the control HLA-DRA and HLA-DPB genes in RJ2.2.5cells complemented with CIITA. The expression of HLA-DOB wasalso restored efficiently in BLS1 cells complemented with RFX-ANK, 6.1.6 cells complemented with RFXAP, DA cells comple-mented with RFXAP, and SJO cells complemented with RFX5(Fig. 2A and data not shown). Surprisingly, the expression of HLA-DOA was not restored by complementation of any of the RFX-deficient mutants (Fig. 2A and data not shown). Thus, comparedwith HLA-DOB and all classical MHCII genes, HLA-DOA isunique in that its expression is lost irreversibly in RFX-deficientcells.

We next performed ChIP experiments to exclude the possibilitythat the inability to restore HLA-DOA expression might be due toinefficient complementation. Occupation by RFX and CIITA wererestored as efficiently at the HLA-DOA promoter as at the HLA-DOB and HLA-DRA promoters in the complemented BLS1 cells(Fig. 2B). At all three promoters, binding of RFX and CIITA wererestored to WT levels in the complemented cells. The inability torestore HLA-DOA expression is thus not due to inefficient comple-mentation or reduced promoter occupation by RFX and CIITA.

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Epigenetic silencing of HLA-DOA in RFX-deficient cells

The inability to restore HLA-DOA expression by complemen-tation of RFX-deficient cells suggested that the absence of RFXmight lead to irreversible silencing of the HLA-DOA locus. Inother systems, irreversible gene silencing can often be attrib-uted to the establishment of a closed heterochromatin structure.Heterochromatin is typically associated with methylation ofDNA at CpG dinucleotides, histone deacetylation, and/or theintroduction of specific histone methylation marks (46 – 48). Todetermine whether the first two features might be implicated insilencing of the HLA-DOA gene, we examined whether HLA-DOA expression could be restored in complemented BLS1 cellsby treating them with the methylation inhibitor 5AC and/or thedeacetylase inhibitor TSA. HLA-DOA mRNA levels attaining25–30% of that observed in WT Raji B cells were obtained incomplemented BLS1 cells treated with various combinations ofthe two inhibitors (Fig. 3A). A significant, albeit more modest,increase in HLA-DOA expression was also observed upon theaddition of each inhibitor on its own. These results suggestedthat both DNA methylation and histone deacetylation are im-plicated in rendering the HLA-DOA gene refractory to reacti-vation in RFX-deficient cells. The fact that HLA-DOA expres-sion was restored only incompletely by the two inhibitorssuggests that additional epigenetic mechanisms, such as histonemethylation, also contribute to HLA-DOA silencing.

To examine further the involvement of histone modificationsin HLA-DOA silencing, we performed ChIP experiments withAbs directed against acetylated histone H3 (H3-Ac), acetylatedhistone H4 (H4-Ac) and trimethylated lysine 27 of H3 (H3-K27Me3), a modification that is frequently associated with si-lenced genes (48). Compared with WT and CIITA-deficientcells, the H4-Ac and H3-Ac marks were markedly reduced atthe HLA-DOA promoter in RFX-deficient cells (Fig. 3B). Thisis consistent with the finding that the deacetylase inhibitor TSAcould partially reactivate HLA-DOA expression (Fig. 3A). How-ever, histone acetylation could be restored partially by comple-mentation of the RFX-deficient cells (data not shown), indicat-ing that mechanisms other than histone deacetylation are alsoimplicated in maintaining the HLA-DOA gene repressed. Incontrast to the reduction in histone acetylation, there was a

dramatic increase in the level of H3-K27Me3 at the HLA-DOApromoter in RFX-deficient cells relative to WT and CIITA-de-ficient cells (Fig. 3B). Collectively, these results are consistentwith the establishment of a closed chromatin environment char-acterized by typical heterochromatin-associated features—in-cluding DNA methylation, histone deacetylation, and H3-K27methylation—at the HLA-DOA gene in RFX-deficient cells.

FIGURE 2. HLA-DOA is irreversibly silenced inRFX-deficient B cells. A, The expression levels of HLA-DOA, HLA-DOB, HLA-DRA, and HLA-DPB mRNAswere measured by real-time RT-PCR in WT B-EBVcells, BLS1 cells, BLS1 cells complemented with RFX-ANK (BLS1c), 6.1.6 cells, 6.1.6 cells complementedwith RFXAP (6.1.6c), DA cells, DA cells comple-mented with RFXAP (DAc), Raji cells, RJ2.2.5 cells(RJ), and RJ2.2.5 cells complemented with CIITA(RJc). Results are expressed relative to the WT HHK orRaji cells and show the mean � SD derived from threeindependent experiments. B, Binding of RFX andCIITA to the HLA-DOA, HLA-DOB, and HLA-DRApromoters were analyzed by quantitative ChIP in Raji,BLS1, and BLS1 complemented with RFXANK(BLS1c). Results are representative of three experi-ments and are expressed relative to Raji.

FIGURE 3. The HLA-DOA gene is epigenetically silenced in RFX-de-ficient B cells. A, The expression level of HLA-DOA mRNA was measuredby real-time RT-PCR in control Raji cells and in complemented BLS1 cells(BLS1c) treated for 48 h with the indicated concentrations of TSA and/or5AC. Results are representative of two experiments and are expressed rel-ative to Raji. B, The levels of H4-Ac, H3-Ac, and H3-K27Me3 were an-alyzed by quantitative ChIP in Raji, RJ2.2.5 (RJ), SJO, and BLS1 cells.Results were normalized to TBP and show the mean � SD derived fromthree independent experiments.

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Increased DNA methylation at MHCII promoters inRFX-deficient B cells

The aforementioned experiments with 5AC suggested that theHLA-DOA gene is maintained silent by DNA methylation inRFX-deficient cells. To document this directly we performedbisulfite sequencing experiments to measure the extent of CpGmethylation at the HLA-DOA promoter in WT and mutant Bcells. The results were analyzed either by direct sequencing ofthe PCR products (Fig. 4A) or by cloning of the PCR fragmentsand sequencing of individual clones (Fig. 4B). Methylation ofspecific CpG dinucleotides within the HLA-DOA promoter wasreadily detectable in RFX-deficient BLS1 and SJO cells, butabsent in WT Raji cells and CIITA-deficient RJ2.2.5 cells.Methylation was most evident at a CpG dinucleotide situatedjust downstream of the TSS. Surprisingly, a similar cell type-specific pattern of CpG methylation was also revealed at otherMHCII genes; specific CpG dinucleotides were strongly meth-ylated within the HLA-DRA and HLA-DQB promoters in BLS1and SJO cells, but not in Raji or RJ2.2.5 cells. There thus ap-

peared to be a general increase in the level of methylation atMHCII promoters in RFX-deficient cells.

To determine whether MHCII promoter methylation in RFX-deficient cells is a direct consequence of the deficiency in RFX, weassessed whether this methylation could be reversed by comple-mentation. Methylation at the HLA-DRA and HLA-DQB promoterswas completely eliminated following complementation of BLS1cells with RFXANK (Fig. 5A). These results demonstrated that theincrease in methylation observed at MHCII promoters in BLS1cells is a direct consequence of the loss of RFX rather than anunrelated or nonspecific characteristic of these cells. Interestingly,methylation at the HLA-DOA promoter was erased only partiallyand remained significantly above that observed in RJ2.2.5 and Rajicells (Fig. 5). Sequencing of individual clones indicated that theoverall extent of conversion of methylated to nonmethylated CpGdinucleotides in the HLA-DOA gene was only �50%. The fact thatmethylation at HLA-DOA was not removed completely in comple-mented BLS1 cells is consistent with the finding that HLA-DOAexpression is not restored in these cells (Fig. 2A).

FIGURE 4. DNA methylation is established atMHCII promoters in RFX-deficient B cells. A, PCR prod-ucts corresponding to the HLA-DOA, HLA-DRA, andHLA-DQB promoters were amplified from bisulfite-treated DNA derived from BLS1, SJO, RJ2.2.5 (RJ),and Raji. The amplification products were sequenceddirectly. The sequence profiles focus on the regions con-taining the CpG dinucleotides (*) indicated in the sche-matic maps shown above. Two different regions areshown for HLA-DQB. B, PCR products correspondingto the HLA-DOA and HLA-DRA promoters were ampli-fied from bisulfite-treated DNA derived from BLS1,SJO, and RJ2.2.5 (RJ). The amplification products werecloned and 20 individual clones were sequenced foreach promoter and cell type. Each sequence is repre-sented as a linear map showing the positions of unmeth-ylated (E) and methylated (F) CpG dinucleotides. Nu-cleotide coordinates of the CpG dinucleotides (bottom)and positions of the S-Y enhancer and TSS (top) areindicated.

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Increased DNA methylation at MHCII promoters inRFX-deficient fibroblasts

To extend our analysis to another cell type, we compared the meth-ylation status of the HLA-DRA and HLA-DOA genes between WT,RFX-deficient, and CIITA-deficient fibroblasts. As RFX-deficient fi-broblasts we used a cell line established from a BLS patient (ABI.)carrying a mutation in RFXAP (38). As CIITA-deficient fibroblastswe used a new cell line established from a BLS patient (BLS3) thatwe have recently shown to carry a severe loss-of-function mutationin the CIITA gene (Fig. 6; see Materials and Methods). The mu-tation in BLS3 lies in the splice donor site situated downstream ofexon 18 of the CIITA gene (Fig. 6). Strong methylation was ob-served at specific CpG dinucleotides in the HLA-DRA and HLA-DOA genes in the RFX-deficient ABI fibroblasts (Fig. 7A). Thismethylation was not observed in WT fibroblasts or in the CIITA-deficient BLS3 fibroblasts (Fig. 7). As in B cells, therefore, MHCIIpromoter methylation is observed in RFX-deficient fibroblasts butnot in WT- or CIITA-deficient fibroblasts.

DiscussionOur results show that MHCII promoters become heavily methyl-ated at specific CpG dinucleotides in cells that lack an intact RFXcomplex. This MHCII promoter methylation is observed in cellscarrying mutations in each of the three subunits of RFX, includingRFX5-deficient SJO cells, RFXANK-deficient BLS1 cells, andRFXAP-deficient ABI cells. It is furthermore a direct consequenceof the absence of RFX because it can be completely reversed at theHLA-DRA and HLA-DQB promoters by restoring RFX function inthe RFX-deficient cells. The presence of a functional RFX com-plex thus protects MHCII promoters against the establishment, ac-cumulation, or maintenance of CpG methylation. This function isindependent of CIITA, since MHCII promoter methylation is notobserved in WT cells that do not express CIITA (fibroblasts) or inmutant cells having a defective CIITA gene (RJ2.2.5 and BLS3).Protecting MHCII promoters against DNA methylation is thus anovel CIITA-independent function associated with the RFX com-plex. This mechanism is rather unusual, since providing protectionagainst the establishment of DNA methylation is a property that

has so far been attributed to only very few transcription factors.CTCF has been reported to confer protection against de novomethylation of a specific domain in the imprinted H19 gene (49).Binding of SP1 has been associated with protection against meth-ylation at the human RIL and mouse Aprt genes (50, 51). Stat4 hasbeen implicated in preventing methylation at the mouse Il18r1gene (52).

Methylation of the CpG dinucleotide present in the S-Y en-hancer of the HLA-DRA gene had previously been documented in6.1.6 and SJO cells (53). This earlier report had not examined themethylation status of CpG dinucleotides situated elsewhere in theHLA-DRA promoter, or in the promoters of other MHCII genes.Our finding that DNA methylation is established in several inde-pendent RFX-deficient cell lines at multiple positions in all MHCIIloci examined indicates that methylation at MHCII genes is a gen-eral feature of RFX-deficient cells, and it rules out the possibilitythat it might simply represent and artifact associated with one par-ticular MHCII gene or cell line.

The regions subjected to methylation at MHCII genes are notrestricted to their S-Y modules, but instead extend further up-stream and downstream, including positions situated down-stream of the TSS. The CpG dinucleotides that are methylatedmost strongly are not situated at identical positions in differentMHCII genes. Furthermore, the precise methylation pattern at agiven MHCII gene can vary between different RFX-deficientcells. Taken together, these observations suggest that it may bethe global methylation status of an extended region, rather thanmethylation at specific positions in defined regulatory elements,that is responsible for transcriptional silencing of MHCII genes.

RFX is a critical factor for the activation of MHCII genes be-cause it nucleates assembly of the enhanceosome complex on theS-Y enhancer by promoting reciprocal cooperative binding inter-actions with CREB and NF-Y (1–4, 21–24). These cooperativebinding interactions are absolutely essential for promoting enhan-ceosome assembly. MHCII promoters consequently remain en-tirely unoccupied, even by CREB and NF-Y, in cells that lack RFX(36, 54, 55). Protecting MHCII promoters against DNA methyl-ation could thus be either a direct function of RFX itself, or an

FIGURE 5. DNA methylation is revertedcompletely at classical MHCII promoters butonly partially at the HLA-DOA promoter bycomplementation of RFX-deficient B cells. A,PCR products corresponding to the HLA-DOA,HLA-DRA, and HLA-DQB promoters were am-plified from bisulfite-treated DNA derived fromBLS1 and BLS1 complemented with RFXANK(BLS1c). The amplification products were se-quenced directly. The sequence profiles focus onthe regions containing the CpG dinucleotides (*)indicated in the schematic maps shown above.Two different regions are shown for HLA-DQB.B, PCR products corresponding to the HLA-DOA promoter were amplified from bisulfite-treated DNA derived from BLS1, BLS1 comple-mented with RFXANK (BLS1c), and RJ2.2.5(RJ). The amplification products were clonedand 20 individual clones were sequenced foreach cell type. Sequences are represented as inFig. 4B.

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indirect consequence of its key role in promoting enhanceosomeassembly by stabilizing binding of CREB and NF-Y. In the latterscenario, the protective effect could be conferred by binding ofCREB, NF-Y, or even the entire enhanceosome complex.

The mechanisms via which RFX and/or the enhanceosome com-plex confer protection against DNA methylation and silencing re-main to be established. One possibility is that continuous bindingof RFX and/or enhanceosome assembly could somehow inhibit orblock access of the maintenance methylase DNMT1 (56) duringDNA replication. The loss of RFX could thus lead to a progressiveaccumulation and maintenance during successive cell divisions ofsporadically introduced cytosine methylation. The maintenance ofrandomly introduced methylations would be consistent with thefact that the methylation pattern observed at a given MHCII genevaries between different RFX-deficient cells. An RFX-dependentblock in DNMT1-mediated transmission of the methylated state(passive demethylation) (57) would also explain the finding thatthe high level of methylation established at the HLA-DRA andHLA-DQB promoters in RFX-deficient cells can be completely

erased by restoring a functional RFX complex by stable comple-mentation of the cells. A related mechanism could be that RFXand/or the enhanceosome complex can inhibit the recruitment ofmethylated DNA-binding proteins, which are involved in inter-preting the information encoded by DNA methylation and recruit-ing enzymes, including histone deacetylases and methylases, re-sponsible for establishing and maintaining a silenced chromatinconformation (46–48, 58). Alternative mechanisms could be thatbinding of RFX and/or enhanceosome assembly inhibit the estab-lishment of methylation by de novo DNA methylases or recruitmechanism leading to active demethylation (57). The formermechanism seems less likely because it would not explain howrestoring RFX function by complementation of the mutant cellscould erase methylation. However, recruiting active demethylatingmechanism would again be able to account for demethylation inthe complemented cells.

Methylation at the HLA-DOA promoter in RFX-deficient cells isassociated with a marked reduction in the levels of histone acet-ylation and a strong increase in the H3-K27Me3 mark. Thesechanges in histone modification are typical of repressive hetero-chromatin, suggesting that the HLA-DOA gene is epigeneticallysilenced in the RFX-deficient cells. In agreement with this inter-pretation, methylation at the HLA-DOA promoter cannot be fullyreverted, and HLA-DOA expression cannot be restored to normal,when RFX function is reestablished by stable complementation ofthe RFX-deficient cells. The continued presence of a functionalRFX complex is thus required to avoid irreversible epigenetic si-lencing of the HLA-DOA gene. It has been well established innumerous systems that there is a tight relationship between DNA

FIGURE 6. Identification of a novel CIITA-deficient BLS fibroblastcell line. A, MHCII and MHCI expression were analyzed by flow cytom-etry on BLS3 cells, BLS3 cells induced with IFN-�, and BLS3 cells trans-duced with a lentiviral CIITA expression vector. The percentages ofMHCII-positive cells are indicated. B, CIITA cDNAs were amplified fromBLS3 by RT-PCR, cloned, and sequenced. All clones contained an in-frame deletion (boxed sequence) corresponding precisely to exon 18. Thenucleotide and predicted amino acid sequences of the region affected by thedeletion are shown for WT and BLS3 cells. The position of the deletion isshown relative to the map of the CIITA protein. Acidic (DE) and proline-serine-threonine-rich regions (PST), the nucleotide (GTP) binding and oli-gomerization domain (NOD), and the leucine rich repeat (LRR) domain ofCIITA are indicated. C, PCR amplification and sequencing of genomicDNA from BLS3 revealed the presence of a G to A mutation at the firstnucleotide of the splice donor site following exon 18. This splice site mu-tation (*) results in the exclusion of exon 18 from the spliced transcript.The splicing patterns for the WT (solid line) and mutant (dashed line)genes are shown.

FIGURE 7. DNA methylation is established at MHCII promoters inRFX-deficient but not CIITA-deficient fibroblasts. A, PCR products cor-responding to the HLA-DRA and HLA-DOA promoters were amplifiedfrom bisulfite-treated DNA derived from RFXAP-deficient ABI fibroblastsand control fibroblasts (WT). The amplification products were cloned and16 individual clones were sequenced for each promoter and cell type. Se-quences are represented as in Fig. 4B. B, PCR products corresponding tothe HLA-DRA and HLA-DOA promoters were amplified from bisulfite-treated DNA derived from RFXAP-deficient ABI fibroblasts and CIITA-deficient BLS3 fibroblasts. The amplification products were sequenced di-rectly. The sequence profiles focus on the regions containing the CpGdinucleotides (*) indicated in the schematic maps shown below.

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methylation and gene silencing (56, 58, 59). It is therefore highlylikely that the establishment of methylation also leads to silencingof MHCII genes other than HLA-DOA, although in this case si-lencing can be reverted when RFX function is restored by stablecomplementation of the RFX-deficient cells. This is consistentwith the finding that binding of RFX to the methylated X box ofthe HLA-DRA promoter can reactivate expression in reporter geneassays (53).

It is currently not clear why HLA-DOA differs from otherMHCII genes in that it is refractory to RFX-dependent reversion ofDNA methylation and gene reactivation. One possibility is thatRFX binding and enhanceosome assembly are less stable or moretransient at the HLA-DOA promoter than at other MHCII genes. Inthis respect, it has been observed that the levels of RFX and CIITAbinding detected by ChIP are indeed very low at the HLA-DOApromoter (37, 55). A second explanation could reside in a specificpeculiarity of the HLA-DOA gene itself or of the chromatin domainin which it is situated. For instance, in contrast to other MHCIIgenes, HLA-DOA contains a dense CpG island within the body ofthe gene. It may also be relevant that HLA-DO is the only MHCIIisotype for which the �- and �-chain genes are separated in thegenome by a series of intervening genes, rather than being foundimmediately adjacent to each other (60). The HLA-DOA locuscould thus be situated in a distinct chromatin domain that is moresusceptible to epigenetic silencing than its partner HLA-DOB orother MHCII genes.

Although the HLA-DO genes are regulated by RFX and CIITA,they are expressed in a more cell type-restricted pattern than otherMHCII genes. HLA-DO expression is largely limited to B cells,mTEC, and certain DC subsets (7, 11–14). This differential regu-lation is likely to be critical because HLA-DO modulates the func-tion of HLA-DM and thus influences the population of peptidesthat are presented (7, 61). The mouse equivalent of HLA-DO (H-2O) has been implicated in controlling the balance between thepresentation of exogenous and endogenous peptides (7, 61). Thus,H-2O is expressed preferentially in mature APC, where it has beenreported to attenuate the presentation of endogenous self-peptidesin favor of exogenous Ags (61). H-2O-deficient mice are also im-paired in their ability to generate Ab responses, but with age theytend to develop autoimmunity associated with high titers of anti-nuclear Abs, including anti-double-stranded DNA Abs (61). Thesefindings emphasize that correctly regulated HLA-DO expression islikely to be critical for the immune system. It is tempting to spec-ulate that the increased susceptibility of HLA-DO to methylation-mediated silencing in the absence of RFX might play a biologi-cally important role in regulating the pattern of HLA-DOexpression. HLA-DO expression might, for instance, be preferen-tially switched off in cell types that exhibit low levels of RFX, orone of the other enhanceosome components, but retained in celltypes where expression of these factors is high, such as in B cells,mTEC, and DC.

We have identified and characterized a novel CIITA-deficientfibroblast cell line (BLS3). To our knowledge, BLS3 is the firstfibroblast cell line carrying a well-characterized loss-of-functionmutation in the CIITA gene. Until now, the lack of CIITA-deficientcells of nonhematopoietic origin has hampered studies on the roleof CIITA in mediating IFN-�-induced gene expression. BLS3should therefore represent a valuable new tool for analyzingCIITA-mediated functions in IFN-�-induced cells.

CIITA is generally not expressed in most cell types of nonhe-matopoietic origin, and MHCII genes consequently remain silentin these cells (4). MHCII promoters are nevertheless occupied, atleast partially, by RFX and the enhanceosome complex in suchMHCII-negative cells (36, 42). Stimulation of MHCII-negative

cells with IFN-� activates CIITA expression, thereby inducingthem to become MHCII positive and allowing them to be recruitedinto immune responses as nonprofessional APC. It is tempting tospeculate that the mechanism we have uncovered is critical foravoiding epigenetic silencing of MHCII genes in potential non-professional APC, such that these cells retain their MHCII genes ina configuration that is permissive for activation whenever thismight be required. The same mechanism may be operating to pro-tect MHCII genes against the establishment of methylation andsilencing in the progenitors of TEC and in hematopoietic precursorcells (such as pre-B cells and monocytes), which lack MHCII ex-pression but will give rise to mature cells (cTEC, mTEC, B cells,macrophages, and DC) that are strictly dependent on MHCII ex-pression for their function.

AcknowledgmentsWe thank all members of the laboratory for valuable discussions, and Dio-nisio Martín-Zanca and Laura Andres-Martín for help with the DNA meth-ylation experiments.

DisclosuresThe authors have no financial conflicts of interest.

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