Silencing by DNA Methylation MHC Class II Genes Against Epigenetic

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

This information is current as Queralt Seguín-Estévez, Raffaele De Palma, Michal of September 24, 2021. Krawczyk, Elisa Leimgruber, Jean Villard, Capucine Picard, Augusto Tagliamacco, Gianfranco Abbate, Jack Gorski, Arcangelo Nocera and Walter Reith J Immunol 2009; 183:2545-2553; Prepublished online 20

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

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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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

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

Queralt Seguín-Este´vez,2* Raffaele De Palma,2† Michal Krawczyk,3* Elisa Leimgruber,* Jean Villard,‡ Capucine Picard,§ Augusto Tagliamacco,¶ Gianfranco Abbate,† Jack Gorski,ʈ 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) and the transcriptional coactivator CIITA. RFX coordinates the assembly of a multiprotein “enhanceosome” complex on MHCII promoters. This enhanceosome serves as a docking site for the binding of CIITA. Whereas the role of the enhanceosome in recruiting CIITA is well established, little is known about its CIITA-independent functions. A novel role of the enhanceosome was revealed by the analysis of HLA-DOA expression in human MHCII-negative B cell lines lacking RFX or CIITA. HLA-DOA was found to be reactivated by complementation of CIITA-deficient but not RFX-deficient B cells. Silencing of HLA-DOA was asso- Downloaded from ciated with DNA methylation at its promoter, and was relieved by the demethylating agent 5-azacytidine. Surprisingly, DNA methylation was also established at the HLA-DRA and HLA-DQB loci in RFX-deficient cells. This was a direct consequence of the absence of RFX, as it could be reversed by restoring RFX function. DNA methylation at the HLA-DOA, HLA-DRA, and HLA-DQB promoters was observed in RFX-deficient B cells and fibroblasts, but not in CIITA-deficient B cells and fibroblasts, or in wild-type fibroblasts, which lack CIITA expression. These results indicate that RFX and/or enhanceosome assembly plays a key CIITA- http://www.jimmunol.org/ independent role in protecting MHCII promoters against DNA methylation. This function is likely to be crucial for retaining MHCII genes in an open chromatin configuration permissive for activation in MHCII-negative cells, such as the precursors of APC and nonprofessional APC before induction with IFN-␥. The Journal of Immunology, 2009, 183: 2545–2553.

olecules of MHC class II (MHCII)5 play pivotal roles selection processes that shape the TCR repertoire of the CD4ϩ T in the adaptive immune system because they present cell population. In the periphery, MHCII-mediated Ag presenta- ϩ M peptides to the Ag receptor (TCR) of CD4 T cells. In tion to CD4ϩ T cells is critical for the maintenance of peripheral the thymus, the recognition of MHCII-peptide complexes by the self-tolerance and for the initiation, regulation, and development of

TCR of developing thymocytes guides the positive and negative Ag-specific immune responses directed against infectious agents by guest on September 24, 2021 and tumors. To ensure these diverse functions, MHCII genes have to be expressed in a complex and tightly regulated cell type-spe- *University of Geneva, Faculty of Medicine, Geneva, Switzerland; †Department of cific and inducible pattern. Their expression is largely restricted to Clinical and Experimental Medicine, Second University of Naples, Naples, Italy; ‡Immunology and Transplant Unit, Geneva University Hospital, Geneva, Switzer- specialized epithelial cells in the cortex (cTEC) and medulla land; §Study Center of Primary Immunodeficiencies, Necker Hospital, Assistance (mTEC) of the thymus and dedicated APC, including various den- Publique–Hoˆpitaux de Paris, Paris, France; ¶Transplant Immunology Unit, Depart- ʈ dritic cell (DC) subsets, cells of the monocyte-macrophage lin- ment of Transplantation, S. Martino Hospital, Genoa, Italy; and Blood Research Institute, Blood Center of Wisconsin, Milwaukee WI 53201 eage, and B cells (1–4). Other cell types, such as endothelial cells, Received for publication February 4, 2009. Accepted for publication June 9, 2009. epithelial cells, and fibroblasts, generally do not express MHCII The costs of publication of this article were defrayed in part by the payment of page genes unless they are stimulated with IFN-␥ (1–4). IFN-␥-induced charges. This article must therefore be hereby marked advertisement in accordance MHCII expression on such nonhematopoietic cells is thought to with 18 U.S.C. Section 1734 solely to indicate this fact. allow them to participate as nonprofessional APC during ongoing 1 Work in the laboratory of W. Reith was supported by the Swiss National Science Foundation, The Swiss Multiple Sclerosis Society, The Geneva Cancer League, immune responses. and the National Center of Competence in Research–Neural Plasticity and Repair Humans have three “classical” MHCII isotypes: HLA-DR, (NCCR-NEURO). Work in the laboratory of R. de Palma was supported by Grants HLA-DP, and HLA-DQ. Intracellular routing and peptide loading 2005064784_004 and 2007XKCCWF_004 from the Ministero dell’Istruzione dell’Universitaè della Ricerca. Work in the laboratory of A. Nocera was in part of these classical MHCII molecules require several accessory mol- supported by a scholarship to A. Tagliamacco provided by the S. Martino Hos- ecules, including the invariant (Ii) chain and two “nonclassical” pital, Genoa, Italy. MHCII molecules called HLA-DM and HLA-DO (5–7). The genes 2 Q.S.-E., R.P., A.N., and W.R. contributed equally to this study. encoding the Ii chain and the ␣- and ␤-chains of HLA-DR, HLA- 3 Current address: Regulatory Biology Laboratory, Salk Institute for Biological Stud- ies, 10010 North Torrey Pines Road, La Jolla, CA 92037. DQ, HLA-DP, and HLA-DM are expressed in a tightly coregu- ␣ ␤ 4 Address correspondence and reprint requests to Dr. Walter Reith, Department of Pa- lated manner (1–4). The genes encoding the - and -chains of thology and Immunology, University of Geneva, Faculty of Medicine, 1 rue Michel- HLA-DO are also coregulated with MHCII genes (8–10), but in a Servet, CH-1211, Geneva, Switzerland. E-mail address: [email protected] more cell type-restricted pattern. HLA-DO expression is largely 5 Abbreviations used in this paper: MHCII, MHC class II; BLS, bare lymphocyte limited to B cells, mTEC, and certain DC subsets (7, 11–14). syndrome; ChIP, chromatin immunoprecipitation; CREB, cAMP responsive element- binding protein; cTEC, cortical thymic epithelial cells; DC, dendritic cell; 5AC, The coexpression of MHCII and accessory genes is coordinated 5-azacytidine; mTEC, medullary thymic epithelial cell; NF-Y, nuclear factor Y; RFX, by a conserved enhancer situated upstream of the transcription regulatory factor X; TSA, trichostatin A; TSS, transcription start site; WT, wild type. start site (TSS) of each gene (1–4). This enhancer functions as a Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 composite regulatory module consisting of four subsequences www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900376 2546 RFX PROTECTS MHC CLASS II GENES AGAINST DNA METHYLATION Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021 FIGURE 1. The HLA-DOA and HLA-DOB genes are coregulated with classical MHCII genes by RFX and CIITA. A, Schematic representation of the molecular machinery that controls MHCII gene expression. The transcription factors RFX, CREB, and NF-Y bind cooperatively to form an enhanceosome complex bound to the conserved S-Y enhancers found upstream of the TSS of each MHCII gene. RFX consists of three subunits, RFX5, RFXANK, and RFXAP. 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-DOB mRNAs were compared by semiquantitative RT-PCR between RFX5-deficient SJO B cells and control Raji B cells, splenic mononuclear cells (SMC), or blood mononuclear cells (BMC). C, The expression levels of HLA-DOA, HLA-DOB, HLA-DRA, and HLA-DPB mRNAs were measured by real-time RT-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, and RFXAP-deficient 6.1.6 and DA cells. Results are expressed relative to the Raji or B-EBV cells, and show the mean Ϯ SD derived from three independent experiments. 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 (3Ј) and in exon 6 of HLA-DOB (E6) were used as negative controls. A preimmune serum was used as control Ab. PCR fragments were quantified relative to the indicated dilutions of input chromatin. E, Binding of RFX and CIITA to the HLA-DOA, HLA-DOB, and HLA-DRA promoters were analyzed by quantitative ChIP in Raji, RJ2.2.5 (RJ), and BLS1 cells. Results are representative of three experiments and are expressed relative to Raji. called the S, X, X2, and Y boxes (Fig. 1A). The molecular ma- ment of CIITA (4, 24, 25). The latter is a non-DNA-binding tran- chinery that regulates MHCII expression via the S-X-X2-Y (S-Y) scriptional coactivator that binds to the enhanceosome complex by enhancer has been exceptionally well defined thanks to elucidation means of multiple protein-protein interactions with RFX, CREB, of the genetic defects responsible for the bare lymphocyte syn- and NF-Y (Fig. 1A) (24, 26, 27). drome (BLS), a hereditary immunodeficiency disease resulting Whereas RFX, CREB, and NF-Y are expressed widely in most from mutations in genes encoding transcription factors that are cell types, CIITA is expressed in a cell type-specific and IFN-␥- essential for MHCII expression (1). Most BLS patients carry mu- inducible manner that governs the constitutive and inducible pat- tations in the genes coding for an X box-binding complex called tern of MHCII expression (1–4). Due to this key role as the master regulatory factor X (RFX) (1). RFX is composed of three subunits regulator of MHCII genes, most studies on the molecular mecha- called RFX5, RFXAP, and RFXANK (or RFX-B). BLS can be due nisms controlling MHCII expression have concentrated on the to mutations in each of these subunits (15–18). RFX binds coop- mode of action of CIITA. Mechanisms implicated in MHCII gene eratively with the X2 box-binding factor cAMP responsive ele- activation by CIITA include the recruitment of chromatin remod- ment-binding protein (CREB) (19) and the Y box-binding protein eling factors, histone modifying complexes, components of the nuclear factor Y (NF-Y) (20) to generate a higher order multipro- general transcription machinery, and transcription elongation fac- tein enhanceosome complex on the S-Y module (Fig. 1A) (21–24). tors (28–33). In these studies, the enhanceosome was generally This enhanceosome complex then serves as a platform for recruit- attributed a more passive role, serving primarily as a docking The Journal of Immunology 2547

surface for CIITA. However, there is growing evidence that the amplified from BLS3 genomic DNA, cloned, and sequenced. All clones enhanceosome also makes key CIITA-independent contributions containedaGtoAmutation at the first nucleotide of the splice donor site to MHCII gene activation, such as nucleosome eviction from the situated downstream of exon 18. The same mutation was identified previously in another BLS patient (BCH.) and has been shown to lead to skip- TSS (34–36). ping of exon 18 and to a complete loss of function of CIITA (43). A detailed analysis of defective MHCII expression in mutant cells lacking RFX or CIITA indicated that HLA-DOA expression could be reactivated by complementation of CIITA-deficient cells Results but not RFX-deficient cells. The irreversible nature of impaired Irreversible loss of HLA-DOA expression in RFX-deficient cells HLA-DOA expression in RFX-deficient cells pointed to an epige- There have been conflicting reports on the relative dependence of netic silencing mechanism. Analysis of this mechanism revealed HLA-DOA and HLA-DOB expression on RFX and CIITA (8, 9). that a key function of RFX and/or the enhanceosome complex is to We therefore quantified HLA-DOA and HLA-DOB mRNAs in var- protect HLA-DOA and other MHCII genes against the establish- ious RFX- and CIITA-deficient B cell lines by semiquantitative ment of DNA methylation. Protection against DNA methylation RT-PCR and quantitative real-time RT-PCR (Fig. 1, B and C). was abrogated in RFX-deficient B cells and fibroblasts but re- Compared with wild type (WT) control cells, HLA-DOA and HLA- mained effective in CIITA-deficient B cells and fibroblasts. These DOB mRNA levels were strongly reduced in CIITA-deficient results thus reveal a novel CIITA-independent function of RFX RJ2.2.5 cells, RFX5-deficient SJO cells, RFXANK-deficient BLS1 and the enhanceosome complex. cells, and RFXAP-deficient 6.1.6 and DA cells. This reduction was in most cases almost as strong as that observed for classical MHCII

Materials and Methods genes such as HLA-DRA, HLA-DRB, and HLA-DPB.Asob- Downloaded from Cells and culture served previously by others, the dependence of HLA-DOB expression on CIITA was less strong than its dependence on RFX (9). A The Raji, HHK, RJ2.2.5, BLS1, SJO, 6.1.6, DA, and ABI cell lines, as well as CIITA-complemented RJ2.2.5 cells, RFXANK-complemented BLS1 residual level of HLA-DOA expression was evident in RFXANK- cells, RFX5-complemented SJO cells, and RFXAP-complemented 6.1.6 deficient BLS1 cells, but not in the other RFX-deficient cells. Sim- and DA cells, have all been described (37–40). The BLS3 cell line was ilar variations between different BLS cells in the degree of residual established from a skin biopsy of an MHCII-deficient BLS patient. Cells expression of specific MHCII genes have been reported previously http://www.jimmunol.org/ were grown in RPMI 1640 plus GlutaMAX medium (Invitrogen) supple- mented with 10% (15% for BLS3) FCS and antibiotics. Complemented (1). The explanation for such cell type-specific differences in leaky SJO cells were cultured for 48 h in the presence of 150 nM trichostatin A MHCII expression remains unknown, but has in certain cases been (TSA), 1, 2 or 5 ␮M 5-azacytidine (5AC), or a combination of 150 nM attributed to partial loss-of-function mutations (44). TSA with 1, 2, or 5 ␮M 5AC. Control primary B cells consisted of human These expression studies confirmed that the HLA-DOA and PBMC or spleen lymphocytes prepared from a patient splenectomized for HLA-DOB genes are indeed regulated by both RFX and CIITA. To an abdominal trauma. All human cells were obtained with consent under accepted protocols. verify this further we performed semiquantitative and quantitative ChIP experiments with Abs directed against RFX and CIITA (Fig. RNA expression analysis 1, D and E). In WT B cells, both RFX and CIITA were indeed RNA extraction and cDNA synthesis were performed as described (41). found to bind specifically to the HLA-DOA and HLA-DOB pro- by guest on September 24, 2021 mRNAs were quantified by semiquantitative RT-PCR or real-time PCR as moters (Fig. 1D). As observed for the control HLA-DRA promoter described (41, 42). Primer sequences will be made available upon request. (34, 41, 45), binding of RFX to the HLA-DOA and HLA-DOB Results were normalized using 18S rRNA. promoters was abolished in RFXANK-deficient BLS1 cells but Chromatin immunoprecipitation (ChIP) was retained in CIITA-deficient RJ2.2.5 cells, whereas binding of CIITA was lost in both mutants (Fig. 1E). ChIP experiments were performed as described (34, 41) using Abs specific for CIITA, RFX, H3-Ac, H4-Ac, H3-K27Me3 (all from Upstate Biotechnology), To confirm further that expression of the HLA-DOA and HLA- and anti-histone H3 (Abcam). Results were quantified by real-time PCR using DOB genes is activated directly by RFX and CIITA, we comple- the iCycler iQ real-time PCR detection system (Bio-Rad) and a SYBR Green- mented the RFX-deficient and CIITA-deficient cell lines with ex- based kit for quantitative PCR (iQ Supermix; Bio-Rad). pression vectors encoding the corresponding WT proteins. The Bisulfite sequencing expression of HLA-DOA and HLA-DOB was restored as efficiently as that of the control HLA-DRA and HLA-DPB genes in RJ2.2.5 ␮ Two to 6 g of genomic DNA was cut with HindIII, purified by phenol- cells complemented with CIITA. The expression of HLA-DOB was 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 overnight also restored efficiently in BLS1 cells complemented with RFX- incubation at 50°C in the dark in 218 ␮l of 3.6 M bisulfite (Sigma-Aldrich), ANK, 6.1.6 cells complemented with RFXAP, DA cells comple- 0.5 mM hydroquinone. The modified DNA was purified using PCR puri- mented with RFXAP, and SJO cells complemented with RFX5 ␮ fication columns (Qiagen) and eluted in 50 l of elution buffer. A second (Fig. 2A and data not shown). Surprisingly, the expression of HLA- denaturation step was then performed as above. Samples were neutralized DOA was not restored by complementation of any of the RFX- with NH4OAc, precipitated, and washed with ethanol. Bisulfite-modified DNA was amplified by PCR using the Expand High Fidelity enzyme deficient mutants (Fig. 2A and data not shown). Thus, compared (Roche). Amplification cycles were adapted to the lengths of the PCR with HLA-DOB and all classical MHCII genes, HLA-DOA is products and the melting temperature of the primers. PCR products were unique in that its expression is lost irreversibly in RFX-deficient purified (High Pure PCR product purification; Roche) and sequenced di- cells. rectly, or cloned in the pGEM-T Easy Vector (Promega) for sequencing of individual clones. Conversion of nonmethylated cytosines to thymidines We next performed ChIP experiments to exclude the possibility was Ͼ95% in all experiments. that the inability to restore HLA-DOA expression might be due to inefficient complementation. Occupation by RFX and CIITA were Identification of the mutation in BLS3 restored as efficiently at the HLA-DOA promoter as at the HLA- Treatment of the BLS3 cells with IFN-␥ demonstrated that MHCII expres- DOB and HLA-DRA promoters in the complemented BLS1 cells sion was not induced. In contrast, complementation of BLS3 with a CIITA (Fig. 2B). At all three promoters, binding of RFX and CIITA were expression vector induced MHCII expression. These results suggested that the defect lies in the CIITA gene. CIITA cDNAs from BLS3 were amplified restored to WT levels in the complemented cells. The inability to by RT-PCR, cloned, and sequenced. All cDNAs contained an in-frame restore HLA-DOA expression is thus not due to inefficient comple- deletion of exon 18. Exon 18 and its flanking intronic sequences were mentation or reduced promoter occupation by RFX and CIITA. 2548 RFX PROTECTS MHC CLASS II GENES AGAINST DNA METHYLATION

FIGURE 2. HLA-DOA is irreversibly silenced in RFX-deﬁcient B cells. A, The expression levels of HLA- DOA, HLA-DOB, HLA-DRA, and HLA-DPB mRNAs were measured by real-time RT-PCR in WT B-EBV cells, BLS1 cells, BLS1 cells complemented with RFX- ANK (BLS1c), 6.1.6 cells, 6.1.6 cells complemented with RFXAP (6.1.6c), DA cells, DA cells complemented 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 or Raji cells and show the mean Ϯ SD derived from three independent experiments. B, Binding of RFX and CIITA to the HLA-DOA, HLA-DOB, and HLA-DRA promoters were analyzed by quantitative ChIP in Raji, BLS1, and BLS1 complemented with RFXANK (BLS1c). Results are representative of three experiments and are expressed relative to Raji. Downloaded from

Epigenetic silencing of HLA-DOA in RFX-deficient cells dramatic increase in the level of H3-K27Me3 at the HLA-DOA http://www.jimmunol.org/ The inability to restore HLA-DOA expression by complemen- promoter in RFX-deficient cells relative to WT and CIITA-de- tation of RFX-deficient cells suggested that the absence of RFX ficient cells (Fig. 3B). Collectively, these results are consistent might lead to irreversible silencing of the HLA-DOA locus. In with the establishment of a closed chromatin environment char- other systems, irreversible gene silencing can often be attrib- acterized by typical heterochromatin-associated features—in- uted to the establishment of a closed heterochromatin structure. cluding DNA methylation, histone deacetylation, and H3-K27 Heterochromatin is typically associated with methylation of methylation—at the HLA-DOA gene in RFX-deficient cells. DNA at CpG dinucleotides, histone deacetylation, and/or the introduction of specific histone methylation marks (46–48). To determine whether the first two features might be implicated in by guest on September 24, 2021 silencing of the HLA-DOA gene, we examined whether HLA- DOA expression could be restored in complemented BLS1 cells by treating them with the methylation inhibitor 5AC and/or the deacetylase inhibitor TSA. HLA-DOA mRNA levels attaining 25–30% of that observed in WT Raji B cells were obtained in complemented BLS1 cells treated with various combinations of the two inhibitors (Fig. 3A). A significant, albeit more modest, increase in HLA-DOA expression was also observed upon the addition of each inhibitor on its own. These results suggested that both DNA methylation and histone deacetylation are implicated in rendering the HLA-DOA gene refractory to reactivation in RFX-deficient cells. The fact that HLA-DOA expression was restored only incompletely by the two inhibitors suggests that additional epigenetic mechanisms, such as histone methylation, also contribute to HLA-DOA silencing. To examine further the involvement of histone modifications in HLA-DOA silencing, we performed ChIP experiments with Abs directed against acetylated histone H3 (H3-Ac), acetylated histone H4 (H4-Ac) and trimethylated lysine 27 of H3 (H3- K27Me3), a modification that is frequently associated with silenced genes (48). Compared with WT and CIITA-deficient cells, the H4-Ac and H3-Ac marks were markedly reduced at the HLA-DOA promoter in RFX-deficient cells (Fig. 3B). This FIGURE 3. The HLA-DOA gene is epigenetically silenced in RFX-de- is consistent with the finding that the deacetylase inhibitor TSA ficient B cells. A, The expression level of HLA-DOA mRNA was measured by real-time RT-PCR in control Raji cells and in complemented BLS1 cells could partially reactivate HLA-DOA expression (Fig. 3A). How- (BLS1c) treated for 48 h with the indicated concentrations of TSA and/or ever, histone acetylation could be restored partially by comple- 5AC. Results are representative of two experiments and are expressed rel- mentation of the RFX-deficient cells (data not shown), indicat- ative to Raji. B, The levels of H4-Ac, H3-Ac, and H3-K27Me3 were an- ing that mechanisms other than histone deacetylation are also alyzed by quantitative ChIP in Raji, RJ2.2.5 (RJ), SJO, and BLS1 cells. implicated in maintaining the HLA-DOA gene repressed. In Results were normalized to TBP and show the mean Ϯ SD derived from contrast to the reduction in histone acetylation, there was a three independent experiments. The Journal of Immunology 2549

FIGURE 4. DNA methylation is established at MHCII promoters in RFX-deficient B cells. A, PCR products corresponding to the HLA-DOA, HLA-DRA, and HLA-DQB promoters were amplified from bisulfite- treated DNA derived from BLS1, SJO, RJ2.2.5 (RJ), and Raji. The amplification products were sequenced directly. The sequence profiles focus on the regions con- Downloaded from taining 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 and HLA-DRA promoters were amplified from bisulfite-treated DNA derived from BLS1, SJO, and RJ2.2.5 (RJ). The amplification products were

cloned and 20 individual clones were sequenced for http://www.jimmunol.org/ each promoter and cell type. Each sequence is represented 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) are indicated. by guest on September 24, 2021

Increased DNA methylation at MHCII promoters in peared to be a general increase in the level of methylation at RFX-deficient B cells MHCII promoters in RFX-deficient cells. The aforementioned experiments with 5AC suggested that the To determine whether MHCII promoter methylation in RFX- HLA-DOA gene is maintained silent by DNA methylation in deficient cells is a direct consequence of the deficiency in RFX, we RFX-deficient cells. To document this directly we performed assessed whether this methylation could be reversed by comple- bisulfite sequencing experiments to measure the extent of CpG mentation. Methylation at the HLA-DRA and HLA-DQB promoters methylation at the HLA-DOA promoter in WT and mutant B was completely eliminated following complementation of BLS1 cells. The results were analyzed either by direct sequencing of cells with RFXANK (Fig. 5A). These results demonstrated that the the PCR products (Fig. 4A) or by cloning of the PCR fragments increase in methylation observed at MHCII promoters in BLS1 and sequencing of individual clones (Fig. 4B). Methylation of cells is a direct consequence of the loss of RFX rather than an specific CpG dinucleotides within the HLA-DOA promoter was unrelated or nonspecific characteristic of these cells. Interestingly, readily detectable in RFX-deficient BLS1 and SJO cells, but methylation at the HLA-DOA promoter was erased only partially absent in WT Raji cells and CIITA-deficient RJ2.2.5 cells. and remained significantly above that observed in RJ2.2.5 and Raji Methylation was most evident at a CpG dinucleotide situated cells (Fig. 5). Sequencing of individual clones indicated that the just downstream of the TSS. Surprisingly, a similar cell type- overall extent of conversion of methylated to nonmethylated CpG specific pattern of CpG methylation was also revealed at other dinucleotides in the HLA-DOA gene was only ϳ50%. The fact that MHCII genes; specific CpG dinucleotides were strongly meth- methylation at HLA-DOA was not removed completely in comple- ylated within the HLA-DRA and HLA-DQB promoters in BLS1 mented BLS1 cells is consistent with the finding that HLA-DOA and SJO cells, but not in Raji or RJ2.2.5 cells. There thus ap- expression is not restored in these cells (Fig. 2A). 2550 RFX PROTECTS MHC CLASS II GENES AGAINST DNA METHYLATION

FIGURE 5. DNA methylation is reverted completely at classical MHCII promoters but only partially at the HLA-DOA promoter by complementation of RFX-deficient B cells. A, PCR products corresponding to the HLA-DOA, HLA-DRA, and HLA-DQB promoters were amplified from bisulfite-treated DNA derived from BLS1 and BLS1 complemented with RFXANK (BLS1c). The amplification products were sequenced directly. The sequence profiles focus on the 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 complemented with RFXANK (BLS1c), and RJ2.2.5

(RJ). The ampliﬁcation products were cloned Downloaded from and 20 individual clones were sequenced for each cell type. Sequences are represented as in Fig. 4B. http://www.jimmunol.org/

Increased DNA methylation at MHCII promoters in has so far been attributed to only very few transcription factors. RFX-deficient fibroblasts CTCF has been reported to confer protection against de novo To extend our analysis to another cell type, we compared the meth- methylation of a specific domain in the imprinted H19 gene (49). ylation status of the HLA-DRA and HLA-DOA genes between WT, Binding of SP1 has been associated with protection against meth- RFX-deficient, and CIITA-deficient fibroblasts. As RFX-deficient fi- ylation at the human RIL and mouse Aprt genes (50, 51). Stat4 has broblasts we used a cell line established from a BLS patient (ABI.) been implicated in preventing methylation at the mouse Il18r1 carrying a mutation in RFXAP (38). As CIITA-deficient fibroblasts gene (52). we used a new cell line established from a BLS patient (BLS3) that Methylation of the CpG dinucleotide present in the S-Y en- by guest on September 24, 2021 we have recently shown to carry a severe loss-of-function mutation hancer of the HLA-DRA gene had previously been documented in in the CIITA gene (Fig. 6; see Materials and Methods). The mu- 6.1.6 and SJO cells (53). This earlier report had not examined the tation in BLS3 lies in the splice donor site situated downstream of methylation status of CpG dinucleotides situated elsewhere in the exon 18 of the CIITA gene (Fig. 6). Strong methylation was ob- HLA-DRA promoter, or in the promoters of other MHCII genes. served at specific CpG dinucleotides in the HLA-DRA and HLA- Our finding that DNA methylation is established in several inde- DOA genes in the RFX-deficient ABI fibroblasts (Fig. 7A). This pendent RFX-deficient cell lines at multiple positions in all MHCII methylation was not observed in WT fibroblasts or in the CIITA- loci examined indicates that methylation at MHCII genes is a gen- deficient BLS3 fibroblasts (Fig. 7). As in B cells, therefore, MHCII eral feature of RFX-deficient cells, and it rules out the possibility promoter methylation is observed in RFX-deficient fibroblasts but that it might simply represent and artifact associated with one par- not in WT- or CIITA-deficient fibroblasts. ticular MHCII gene or cell line. The regions subjected to methylation at MHCII genes are not Discussion restricted to their S-Y modules, but instead extend further up- Our results show that MHCII promoters become heavily methyl- stream and downstream, including positions situated down- ated at specific CpG dinucleotides in cells that lack an intact RFX stream of the TSS. The CpG dinucleotides that are methylated complex. This MHCII promoter methylation is observed in cells most strongly are not situated at identical positions in different carrying mutations in each of the three subunits of RFX, including MHCII genes. Furthermore, the precise methylation pattern at a RFX5-deficient SJO cells, RFXANK-deficient BLS1 cells, and given MHCII gene can vary between different RFX-deficient RFXAP-deficient ABI cells. It is furthermore a direct consequence cells. Taken together, these observations suggest that it may be of the absence of RFX because it can be completely reversed at the the global methylation status of an extended region, rather than HLA-DRA and HLA-DQB promoters by restoring RFX function in methylation at specific positions in defined regulatory elements, the RFX-deficient cells. The presence of a functional RFX com- that is responsible for transcriptional silencing of MHCII genes. plex thus protects MHCII promoters against the establishment, ac- RFX is a critical factor for the activation of MHCII genes be- cumulation, or maintenance of CpG methylation. This function is cause it nucleates assembly of the enhanceosome complex on the independent of CIITA, since MHCII promoter methylation is not S-Y enhancer by promoting reciprocal cooperative binding inter- observed in WT cells that do not express CIITA (fibroblasts) or in actions with CREB and NF-Y (1–4, 21–24). These cooperative mutant cells having a defective CIITA gene (RJ2.2.5 and BLS3). binding interactions are absolutely essential for promoting enhan- Protecting MHCII promoters against DNA methylation is thus a ceosome assembly. MHCII promoters consequently remain en- novel CIITA-independent function associated with the RFX com- tirely unoccupied, even by CREB and NF-Y, in cells that lack RFX plex. This mechanism is rather unusual, since providing protection (36, 54, 55). Protecting MHCII promoters against DNA methyl- against the establishment of DNA methylation is a property that ation could thus be either a direct function of RFX itself, or an The Journal of Immunology 2551 Downloaded from

FIGURE 7. DNA methylation is established at MHCII promoters in RFX-deficient but not CIITA-deficient fibroblasts. A, PCR products corresponding to the HLA-DRA and HLA-DOA promoters were amplified from bisulfite-treated DNA derived from RFXAP-deficient ABI fibroblasts http://www.jimmunol.org/ and control fibroblasts (WT). The amplification products were cloned and 16 individual clones were sequenced for each promoter and cell type. Se- FIGURE 6. Identification of a novel CIITA-deficient BLS fibroblast quences are represented as in Fig. 4B. B, PCR products corresponding to cell line. A, MHCII and MHCI expression were analyzed by flow cytom- the HLA-DRA and HLA-DOA promoters were amplified from bisulfite- ␥ etry on BLS3 cells, BLS3 cells induced with IFN- , and BLS3 cells trans- treated DNA derived from RFXAP-deficient ABI fibroblasts and CIITA- duced with a lentiviral CIITA expression vector. The percentages of deficient BLS3 fibroblasts. The amplification products were sequenced di- MHCII-positive cells are indicated. B, CIITA cDNAs were amplified from rectly. The sequence profiles focus on the regions containing the CpG BLS3 by RT-PCR, cloned, and sequenced. All clones contained an in- dinucleotides (*) indicated in the schematic maps shown below. frame deletion (boxed sequence) corresponding precisely to exon 18. The by guest on September 24, 2021 nucleotide and predicted amino acid sequences of the region affected by the deletion are shown for WT and BLS3 cells. The position of the deletion is shown relative to the map of the CIITA protein. Acidic (DE) and proline- erased by restoring a functional RFX complex by stable comple- serine-threonine-rich regions (PST), the nucleotide (GTP) binding and oli- mentation of the cells. A related mechanism could be that RFX gomerization domain (NOD), and the leucine rich repeat (LRR) domain of and/or the enhanceosome complex can inhibit the recruitment of CIITA are indicated. C, PCR amplification and sequencing of genomic methylated DNA-binding proteins, which are involved in inter- DNA from BLS3 revealed the presence ofaGtoAmutation at the first preting the information encoded by DNA methylation and recruit- nucleotide of the splice donor site following exon 18. This splice site mu- ing enzymes, including histone deacetylases and methylases, re- tation (*) results in the exclusion of exon 18 from the spliced transcript. sponsible for establishing and maintaining a silenced chromatin The splicing patterns for the WT (solid line) and mutant (dashed line) genes are shown. conformation (46–48, 58). Alternative mechanisms could be that binding of RFX and/or enhanceosome assembly inhibit the establishment of methylation by de novo DNA methylases or recruit indirect consequence of its key role in promoting enhanceosome mechanism leading to active demethylation (57). The former assembly by stabilizing binding of CREB and NF-Y. In the latter mechanism seems less likely because it would not explain how scenario, the protective effect could be conferred by binding of restoring RFX function by complementation of the mutant cells CREB, NF-Y, or even the entire enhanceosome complex. could erase methylation. However, recruiting active demethylating The mechanisms via which RFX and/or the enhanceosome com- mechanism would again be able to account for demethylation in plex confer protection against DNA methylation and silencing re- the complemented cells. main to be established. One possibility is that continuous binding Methylation at the HLA-DOA promoter in RFX-deficient cells is of RFX and/or enhanceosome assembly could somehow inhibit or associated with a marked reduction in the levels of histone acet- block access of the maintenance methylase DNMT1 (56) during ylation and a strong increase in the H3-K27Me3 mark. These DNA replication. The loss of RFX could thus lead to a progressive changes in histone modification are typical of repressive hetero- accumulation and maintenance during successive cell divisions of chromatin, suggesting that the HLA-DOA gene is epigenetically sporadically introduced cytosine methylation. The maintenance of silenced in the RFX-deficient cells. In agreement with this inter- randomly introduced methylations would be consistent with the pretation, methylation at the HLA-DOA promoter cannot be fully fact that the methylation pattern observed at a given MHCII gene reverted, and HLA-DOA expression cannot be restored to normal, varies between different RFX-deficient cells. An RFX-dependent when RFX function is reestablished by stable complementation of block in DNMT1-mediated transmission of the methylated state the RFX-deficient cells. The continued presence of a functional (passive demethylation) (57) would also explain the finding that RFX complex is thus required to avoid irreversible epigenetic si- the high level of methylation established at the HLA-DRA and lencing of the HLA-DOA gene. It has been well established in HLA-DQB promoters in RFX-deficient cells can be completely numerous systems that there is a tight relationship between DNA 2552 RFX PROTECTS MHC CLASS II GENES AGAINST DNA METHYLATION methylation and gene silencing (56, 58, 59). It is therefore highly cells with IFN-␥ activates CIITA expression, thereby inducing likely that the establishment of methylation also leads to silencing them to become MHCII positive and allowing them to be recruited of MHCII genes other than HLA-DOA, although in this case si- into immune responses as nonprofessional APC. It is tempting to lencing can be reverted when RFX function is restored by stable speculate that the mechanism we have uncovered is critical for complementation of the RFX-deficient cells. This is consistent avoiding epigenetic silencing of MHCII genes in potential non- with the finding that binding of RFX to the methylated X box of professional APC, such that these cells retain their MHCII genes in the HLA-DRA promoter can reactivate expression in reporter gene a configuration that is permissive for activation whenever this assays (53). might be required. The same mechanism may be operating to pro- It is currently not clear why HLA-DOA differs from other tect MHCII genes against the establishment of methylation and MHCII genes in that it is refractory to RFX-dependent reversion of silencing in the progenitors of TEC and in hematopoietic precursor DNA methylation and gene reactivation. One possibility is that cells (such as pre-B cells and monocytes), which lack MHCII ex- RFX binding and enhanceosome assembly are less stable or more pression but will give rise to mature cells (cTEC, mTEC, B cells, transient at the HLA-DOA promoter than at other MHCII genes. In macrophages, and DC) that are strictly dependent on MHCII ex- this respect, it has been observed that the levels of RFX and CIITA pression for their function. binding detected by ChIP are indeed very low at the HLA-DOA promoter (37, 55). A second explanation could reside in a specific Acknowledgments peculiarity of the HLA-DOA gene itself or of the chromatin domain We thank all members of the laboratory for valuable discussions, and Dio- in which it is situated. For instance, in contrast to other MHCII nisio Martín-Zanca and Laura Andre´s-Martín for help with the DNA meth- genes, HLA-DOA contains a dense CpG island within the body of ylation experiments. Downloaded from the gene. It may also be relevant that HLA-DO is the only MHCII isotype for which the ␣- and ␤-chain genes are separated in the Disclosures genome by a series of intervening genes, rather than being found The authors have no financial conflicts of interest. immediately adjacent to each other (60). The HLA-DOA locus could thus be situated in a distinct chromatin domain that is more References 1. Reith, W., and B. Mach. 2001. The bare lymphocyte syndrome and the regulation susceptible to epigenetic silencing than its partner HLA-DOB or http://www.jimmunol.org/ of MHC expression. Annu. Rev. Immunol. 19: 331–373. other MHCII genes. 2. Ting, J. P., and J. Trowsdale. 2002. Genetic control of MHC class II expression. Although the HLA-DO genes are regulated by RFX and CIITA, Cell 109(Suppl.): S21–S33. they are expressed in a more cell type-restricted pattern than other 3. Boss, J. M., and P. E. Jensen. 2003. Transcriptional regulation of the MHC class II antigen presentation pathway. Curr. Opin. 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