NLR family member NLRC5 is a transcriptional regulator of MHC class I

Torsten B. Meissnera,b, Amy Lia, Amlan Biswasa,b, Kyoung-Hee Leea,b, Yuen-Joyce Liua, Erkan Bayira, Dimitrios Iliopoulosc, Peter J. van den Elsend,e, and Koichi S. Kobayashia,b,1

aDepartment of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02115; bDepartment of Pathology, Harvard Medical School, Boston, MA 02115; cDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; dDepartment of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands; and eDepartment of Pathology, VU University Medical Center, 1081 HV Amsterdam, The Netherlands

Communicated by Jack Strominger, Harvard University, Cambridge, MA, June 18, 2010 (received for review June 1, 2010) MHC class I plays a critical role in the immune defense against plexes (7, 8). These factors include the X-box binding and tumors by presenting antigens to CD8 T cells. An NLR , trimeric RFX protein complex (composed of RFX5, RFXAP, and class II transactivator (CIITA), is a key regulator of MHC class II RFXANK), the ×2-box binding cAMP response element binding expression that associates and cooperates with transcription factors protein/activating transcription factor (CREB/ATF), and the Y-box in the MHC class II promoter. Although CIITA also transactivates MHC binding nuclear factor-Y protein (composed of NF-YA, NF-YB, class I gene promoters, loss of CIITA in humans and mice results in the and NF-YC) (9). Together, they form a macromolecular nucleo- severe reduction of only MHC class II expression, suggesting that protein complex called the MHC enhanceosome (10). additional mechanisms regulate the expression of MHC class I. Here, Class II transactivator (CIITA), a member of the NLR or nu- we identify another member of the NLR protein family, NLRC5, as cleotide binding domain (NBD) leucine-rich repeat (LRR) family a transcriptional regulator of MHC class I genes. Similar to CIITA, of (11, 12), regulates the transcription of MHC class II by NLRC5 is an IFN-γ–inducible nuclear protein, and the expression of associating with the MHC enhanceosome (10, 13). The expression NLRC5 resulted in enhanced MHC class I expression in lymphoid as of CIITA is induced in B cells and dendritic cells as a function of well as epithelial cell lines. Using chromatin immunoprecipitation developmental stage and is inducible by IFN-γ in most cell types and reporter gene assays, we show that NLRC5 associates with and (14–16). There are 22 NLR proteins in humans, which share three activates the promoters of MHC class I genes. Furthermore, we show characteristic functional domains: an N-terminal protein–protein γ– that the IFN- induced up-regulation of MHC class I requires NLRC5, interaction domain such as a caspase recruitment domain (CARD) NLRC5 fi because knockdown of speci cally impaired the expression of or a , a centrally located NBD (or NACHT, NAIP, MHC class I. In addition to MHC class I genes, NLRC5 also induced the β CIITA, HET-E and TP1 domain), and C-terminal LRRs (11, 12). expression of 2-microglobulin, transporter associated with antigen Aside from CIITA, NLR proteins are localized in the cytoplasm processing, and large multifunctional protease, which are essential and contribute to the innate immune response by recognizing mi- for MHC class I antigen presentation. Our results suggest that NLRC5 crobial products and exogenous danger signals, leading to inflam- is a transcriptional regulator, orchestrating the concerted expression mation and/or cell death (11, 12). of critical components in the MHC class I pathway. Previous studies have shown that, although to a lesser extent, CIITA also has a role in the transactivation of MHC class I genes antigen presentation | class II transactivator | IFN-γ (6–9, 17). However, the expression of CIITA is generally restricted to and professional antigen-presenting cells and thus, HC class I and class II play essential roles in the activation of is unlikely to account for the ubiquitous expression of MHC class I Madaptive immune responses by presenting antigens to T lym- (6, 18). Furthermore, although mutations of the CIITA gene can phocytes. MHC class I molecules are composed of MHC-encoded cause bare syndrome (BLS), an immunodeficiency β β heavy chains and the invariant subunit 2-microglobulin ( 2M) (1). characterized by the lack of MHC class II expression, a subgroup of Humans have three classical MHC class Ia molecules (HLA-A, BLS patients that lack CIITA retains the expression of MHC class I HLA-B, and HLA-C), which are vital to the detection and elimina- but not MHC class II (19, 20). Similarly, in mice deficient for tion of viruses, cancerous cells, and transplanted cells. In addition, CIITA, both constitutive and IFN-γ–induced expression of MHC there are three nonclassical MHC class Ib molecules (HLA-E, HLA- class I molecules is intact (21–23). These findings indicate that, in F, and HLA-G), which have immune regulatory functions (2, 3). addition to CIITA, other molecules or mechanisms are involved in Antigen-derived peptides are presented by MHC class I–β2M the regulation of MHC class I expression. complexes at the cell surface to CD8 T cells carrying an antigen- Here, we show that another NLR protein, NLRC5 (NLR family, specific receptor. Peptides are mostly produced from the CARD domain containing 5/NOD27/CLR16.1), regulates the ex- degradation of cytoplasmic proteins by a specialized proteasome or pression of MHC class I. Similar to CIITA, NLRC5 is highly in- immunoproteasome, which is optimized to generate MHC class I ducible by IFN-γ and can translocate into the nucleus. We show peptides and contains several IFN-γ–inducible subunits, such as large that NLRC5 activates the promoters of MHC class I genes and multifunctional protease 2 (LMP2) and LMP7 (4). Peptide loading onto MHC class I is carried out by the peptide-loading complex (PLC), which includes the MHC class I heavy chain, β2M, tapasin, Author contributions: T.B.M., A.L., and K.S.K. designed research; T.B.M., A.L., A.B., K.-H.L., endoplasmic reticulum (ER)p57, calreticulin, and transporter asso- Y.-J.L., E.B., and D.I. performed research; P.J.v.d.E. contributed new reagents/analytic ciated with antigen processing 1 (TAP1)/TAP2, a transporter that tools; T.B.M., A.L., A.B., K.-H.L., Y.-J.L., E.B., D.I., and K.S.K. analyzed data; and T.B.M., A.L., and K.S.K. wrote the paper. translocates peptides from the cytoplasm into the ER (4, 5). fl Unlike MHC class II, which is found mainly in antigen-pre- The authors declare no con ict of interest. Data deposition: The data reported in this paper have been deposited in the Gene Ex- senting cells, MHC class Ia is ubiquitously expressed in almost all pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE22064). nucleated cells (1, 6). Both MHC class I and class II genes are highly 1 γ To whom correspondence should be addressed. E-mail: [email protected]. inducible by IFN- stimulation and share similar cis-regulatory edu. elements in their promoters, termed W/S, X1, X2, and Y-box This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. motifs, which also associate with similar transcription factor com- 1073/pnas.1008684107/-/DCSupplemental.

13794–13799 | PNAS | August 3, 2010 | vol. 107 | no. 31 www.pnas.org/cgi/doi/10.1073/pnas.1008684107 Downloaded by guest on October 1, 2021 induces the transcription of MHC class I as well as related genes obtained using the murine Nlrc5, which can also be trapped in the involved in MHC class I antigen presentation, suggesting that nucleus on LMB treatment (Fig. S2). NLRC5 is a critical regulator of MHC class I-mediated immu- Given the predicted size of the NLRC5 fusion protein (∼230 ne responses. kDa), passive diffusion through the nuclear pore is not possible. Active transport, however, requires the presence of an NLS that is Results recognized by nuclear import receptors (27). To identify the NLS NLRC5 Contains a Nuclear Localization Signal and Shuttles Between of NLRC5, we performed deletion mutant analysis. As depicted in the Cytosol and Nucleus. To study the function of NLRC5, we in- Fig. 1C, we expressed the deletion mutants of NLRC5 as GFP vestigated its cellular distribution using a GFP fusion protein. To fusion proteins. Although all fusion constructs containing an intact our surprise, NLRC5 was found not only in the cytosol but also in N-terminal CARD (WT, CARD, and ΔLRR) were found in the the nucleus (Fig. 1A Upper). We checked the stability of the fusion nucleus, deletion of the CARD (ΔCARD and LRR) resulted in protein by Western blot analysis, confirming that its nuclear lo- a strictly cytosolic localization (Fig. 1D). Similar to free GFP, the calization was not caused by a smaller, GFP-containing cleavage NACHT domain fusion protein was found in both the nucleus and product (Fig. S1A). It has been shown that CIITA, which also cytosol, presumably because of passive diffusion as a result of its displays a heterogeneous steady-state distribution, shuttles be- smaller size. These results suggested that an NLS may be located in tween the nucleus and cytosol as a result of nuclear localization the N-terminal CARD. Indeed, sequence analysis of NLRC5 signal (NLS)-mediated nuclear import and exportin-1 (CRM1)- revealed a putative bipartite NLS at the transition between the dependent nuclear export (24–26). Similar to CIITA, which is CARD and the NBD (Fig. 1E) (25, 26). As predicted, mutation of a closely related member of the NLR protein family (Fig. 1B), the NLS abolished nuclear localization under steady-state con- NLRC5 could be trapped in the nucleus on treatment with the ditions, and treatment of the cells with LMB failed to trap the NLS CRM1 inhibitor leptomycin B (LMB) (Fig. 1A Lower and Fig. mutants of NLRC5 in the nucleus (Fig. 1F). Taken together, our S1B). Quantification of the cellular distribution in a blind manner results show that, similar to the transcriptional coregulator CIITA, revealed that under steady-state conditions, NLRC5 localized NLRC5 shuttles between the cytosol and nucleus and thus, is likely exclusively in the cytosol in ∼15% of the cells. The majority of the to have a nuclear function. cells showed an intermediate distribution (80%), and about 5% of the cells displayed an exclusively nuclear localization (Fig. S1B). NLRC5 Transcriptionally Induces the Expression of MHC Class I and LMB treatment resulted in nuclear localization of NLRC5 in more Functionally Related Genes. The observation that NLRC5 is also fi than 75% of the cells. Of note, we observed that in cells highly found in the nucleus and shares signi cant sequence similarity to the expressing the protein, NLRC5 was predominantly localized to the transcriptional coregulator CIITA (Fig. 1 A and B)promptedusto cytosol, whereas NLRC5 was found more frequently in the nucleus perform a gene array to identify putative target genes of NLRC5. in cells with lower expression levels, indicating that the nuclear For this purpose, we generated Jurkat T cell lines that stably express either the wild-type or mutant forms of NLRC5 harboring muta- localization of NLRC5 is not a result of overexpression (Fig. 1A fi Upper). In addition to human NLRC5, similar results were tions in the NBD: Walker A (de cient in nucleotide binding), Walker B (deficient in nucleotide hydrolysis), and the combined Walker AB, carrying both mutations (Fig. S3A) (28). Gene-chip analysis using these mutant Jurkat T cells showed that a surprisingly limited number of genes were differentially regulated (Fig. S3). As predicted, clustering analysis grouped the active forms of NLRC5 (WT and Walker B) together, and they show a strikingly different pattern of compared with cells expressing either GFP alone or the catalytically inactive forms of NLRC5 (Walker A and Walker AB). Among the genes most up-regulated by the active forms of NLRC5 were the various members of the MHC class I (HLA-A, -B, -C,and-E) family as well as other genes involved in class I antigen presentation and processing, such as β2M, LMP2,and TAP1 (Fig. S3 B and C). Quantitative real-time PCR and Western blot analysis confirmed elevated levels of the corresponding tran- scripts and proteins, respectively, in cells expressing the WT and Walker B mutant NLRC5, but not GFP alone, or the inactive forms of NLRC5 (Walker A and Walker AB) (Fig. 2 A and B). Further- fl

more, ow-cytometry analysis using a pan HLA-A, -B, or -C anti- IMMUNOLOGY body and an antibody specific for HLA-E confirmed an increase in MHC class I surface expression in cells expressing NLRC5 WT or the Walker B mutant (Fig. 2CLeft). As previously shown, MHC class I and related genes are inducible by IFN-γ (Fig. 2C bottom γ Fig. 1. NLRC5 contains an N-terminal bipartite NLS and can translocate into row ) (5, 29). However, we did not observe elevated levels of IFN- the nucleus. (A, D, and F) HEK293T cells were transfected with expression expression in our gene-array analysis, and the expression level of plasmids coding for GFP or the indicated GFP fusion proteins; 48 h post- signal transducer and activator of transcription (STAT1), an IFN-γ– transfection, cells were treated with 10 nM leptomycin B (LMB) for 90 min or inducible gene, did not vary between the different cell lines (Fig. left untreated. Fixed cells were stained with Hoechst 33342 to indicate the 2A). These findings, along with the observation that overexpres- nuclei. (Scale bar: 10 μm.) (A) Cellular localization of NLRC5 and CIITA on sion of NLRC5 does not activate NF-κB-, activator protein 1 (AP-1), LMB treatment. (B) Phylogenetic tree of CARD-containing NLRs. (C) Sche- -sensitive response element (ISRE), or interferon regula- matic representation of the NLRC5 deletion mutant constructs used to map tory factor 3 (IRF3)-dependent reporter genes or the promoters the nuclear localization signal. The position of the NLS is indicated by an α β asterisk. (D) Cellular localization of NLRC5 deletion mutants on LMB treat- of IFN- and IFN- (Fig. S4), rule out the role of these other path- ment. (E) Sequence of the bipartite NLS found in the N terminus of NLRC5. ways in NLRC5-mediated MHC class I induction. Instead, NLRC5 Alanine substitution of the right or left arm of the NLS was used to construct might directly regulate the expression of MHC class I genes. the NLSI and NLSII import mutant-expression plasmids. (F) Cellular localiza- Because MHC class I is ubiquitously expressed in all nucleated tion of the NLSI and NLSII mutant forms of NLRC5 on LMB treatment. cells, we sought to determine whether the observed up-regulation

Meissner et al. PNAS | August 3, 2010 | vol. 107 | no. 31 | 13795 Downloaded by guest on October 1, 2021 Fig. 2. Induction of MHC class I and functionally related genes by NLRC5. (A) RNA isolated from Jurkat T cells stably expressing the indicated GFP fusion proteins was analyzed by quantitative real-time PCR for the expression of the indicated genes. GFP, empty vector; WT, wild-type NLRC5; A, Walker A mutant; B, Walker B mutant; AB, Walker AB mutant. (B) The same Jurkat T cell lines were examined for the expression of MHC class I heavy chain (HC), β2M, TAP1, and LMP2 by Western blot analysis. Actin levels are shown as a loading control. (C) Surface expression of MHC class I in Jurkat T cell lines expressing GFP (gray line) or the indicated GFP-NLRC5 fusion proteins (black line) was examined by flow cytometry using anti–pan-MHC class I (HLA-A, -B, and -C) and HLA-E antibodies. IFN-γ (100 U/mL) treatment was used as a positive control. Data obtained with an isotype control antibody are indicated by the shaded area. (D) HEK293T cells were tran- siently transfected with the expression plasmids for GFP fused to NLRC5, CIITA (black line), or GFP only (gray line). The expression of MHC class I (HLA-A, -B, and -C) or class II (HLA-DR) was analyzed by flow cytometry 48 h posttransfection. Data obtained with an isotype control antibody are indicated by the shaded area.

of MHC class I genes was limited to lymphoid cells or could be NLRC5 Binds to MHC Class I Promoters and Induces Their Expression. extended to other cell types. As shown in Fig. 2D, transient ex- To investigate whether NLRC5 directly acts on the promoters of pression of NLRC5 in an epithelial cell line (HEK293T cells) also the MHC class I genes, we performed luciferase-reporter gene increased MHC class I expression nearly 4-fold. In comparison, assays with the promoters of the corresponding genes. Transient expression of CIITA only moderately increased MHC class I ex- expression of NLRC5 in HEK293T cells is sufficient to induce pression but in agreement with previous reports (6, 9), strongly luciferase expression from the promoters of HLA-A,-B,-C,-F,-G, induced the expression of MHC class II. Expression of the Walker and β2M (Fig. 3A). Similar levels of induction on the same pro- A and B mutants in HEK293T cells (Fig. S5B) again showed that moters were observed when CIITA was overexpressed as has been nucleotide binding, but not nucleotide hydrolysis, was required for reported previously (8, 29–31). Only a minor induction was ob- the activity of NLRC5 and the induction of MHC class I. Impor- served on the promoter of TAP1, and NLRC5 failed to induce tantly, this transcriptional effect seems to be specific for the nu- luciferase expression on the TAP2 promoter and any of the MHC clear NLRs, because none of the cytosolic CARD-containing class II reporter constructs analyzed (HLA-DRA, -DQA,and NLRs tested (NOD1, NOD2, and NLRC3) increased the ex- -DPA). In contrast, CIITA transfection strongly activated the pression of MHC class I as shown by flow cytometry and Western promoters of MHC class II genes. Next, we examined if NLRC5 blot analysis (Fig. S5 A and B). In summary, these data indicate also physically associates with the MHC class I promoters using the that NLRC5 induces the expression of MHC class I and related stable Jurkat T cell lines described earlier in a ChIP assay. We genes involved in MHC class I antigen presentation and thus, can immunoprecipitated the corresponding wild-type and mutant substitute for IFN-γ stimulation of cells. NLRC5 proteins, and the associated DNA fragments were quan-

13796 | www.pnas.org/cgi/doi/10.1073/pnas.1008684107 Meissner et al. Downloaded by guest on October 1, 2021 NLRC5 and HLA-A expression were observed in Jurkat T cells (Fig. S7 A and B). Next, we analyzed the effect of NLRC5 depletion by RNA in- terference on the expression of MHC class I after IFN-γ stimula- tion. We had observed earlier that surface expression of MHC class I is readily induced on IFN-γ stimulation (Fig. S7C), and in agreement with our hypothesis, transfection of HeLa cells with two different NLRC5-specific siRNAs, but not a scrambled control siRNA, significantly reduced the IFN-γ–induced up-regulation of MHC class I (Fig. 4C Left and Fig. S8). In contrast, an unrelated but IFN-γ–inducible surface receptor, β1-integrin, was not affected by the depletion of NLRC5 (Fig. 4C Right). Similarly, the IFN-γ– induced expression of CIITA and HLA-DR was not affected by the depletion of NLRC5 (Fig. S8), strongly suggesting that NLRC5 is required for the efficient induction of MHC class I observed on IFN-γ stimulation. Discussion Since the complementation cloning of CIITA from MHC class II- deficient patients in 1993, CIITA has been often referred to as a master regulator of MHC class II expression, because CIITA is required for both constitutive and IFN-γ–inducible transcription Fig. 3. NLRC5 binds and transactivates MHC class I gene promoters. (A) of MHC class II genes (15, 20, 32). However, the contribution of NLRC5-mediated transactivation of MHC class I and functionally related CIITA to MHC class I expression is less clear. In this study, we genes. HEK293T cells were transiently transfected with either expression identify NLRC5 as a regulator of MHC class I genes in addition to vectors for GFP, GFP-NLRC5, or GFP-CIITA, along with luciferase reporter CIITA. NLRC5 and CIITA share important characteristics in constructs of the indicated gene promoters. Cell lysates were analyzed 48 h their structure and function. First, as related members of the NLR posttransfection by dual-luciferase assay. Data are a representative of three family (Fig. 1B), both have the same tripartite architecture, al- ± independent experiments performed in duplicates, and error bars represent though expression of the CARD-containing isoform of CIITA is SD. (B) Schematic representation of the W/SXY module found in the pro- moters of MHC class I and class II genes. The position of the primers used in the limited to dendritic cells (34). Interestingly, both proteins require ChIP assay is indicated with arrows (P1 and P2). (C) NLRC5 occupancy, in terms an active NBD for their function. It has been shown that the NTP of fold enrichment, at the HLA-A,-B,or-DRA promoters as determined by binding motif in CIITA is essential for transactivation of MHC ChIP. Jurkat T cells stably expressing the indicated GFP fusion proteins were class II genes (28, 35, 36). Similarly, the Walker A mutation, which analyzed by ChIP assay using an anti-GFP antibody for immunoprecipitation prevents NTP binding, but not the Walker B mutation, which and the indicated qPCR primers (B). GFP, empty vector; WT, wild-type NLRC5; abolishes NTP hydrolysis, resulted in a loss of NLRC5 function A, Walker A mutant; B, Walker B mutant; AB, Walker AB mutant. Error bars (Fig. 2). Second, both proteins can localize to the nucleus. CIITA indicate SEM (± SEM) from three independent experiments. carries three NLSs, including an N-terminal NLS, which is found at a similar position to that required for NLRC5 nuclear trans- tified by qPCR using gene-specific primers covering the immediate location (Fig. 1 E and F) (24–26). In addition, multiple nuclear upstream region of the HLA genes (Fig. 3B). As seen in Fig. 3C, export signals (NES) are predicted in the C-terminal LRRs of a 6- to 8-fold enrichment in promoter occupancy was observed for CIITA, and our deletion mutant analysis suggests that the NLRC5 WT and the Walker B mutant on the promoters of HLA-A C-terminal LRRs of NLRC5 are also involved in the regulation of and HLA-B compared with the cell line expressing GFP only. In nuclear export, although we have not further mapped the exact agreement with the data obtained from the gene-expression anal- position of the NES (26). Recently, it was shown that cytosolic yses, no promoter binding was detected for the inactive forms of NLRC5 negatively regulates the NF-κB and type I IFN signal- NLRC5 (Walker A and Walker AB) as well as on the promoter of ing pathway by direct binding to IκB kinase (IKK) and retinoic the MHC class II (HLA-DRA) and an unrelated gene (GAPDH). acid-inducible gene I (RIG-I) (37). Our findings do not rule out Furthermore, ChIP analysis in nonhematopoietic cells using tran- a function of NLRC5 in the cytoplasm but rather show its role in siently transfected HEK293T cells revealed that NLRC5 can as- the nucleus as a transcriptional regulator of MHC class I genes. sociate with HLA-A and -B promoters to a similar extent as CIITA Third, despite the lack of a DNA binding domain, both NLRC5

(Fig. S6), which has previously been reported to bind to MHC class and CIITA can associate with and transactivate MHC class I IMMUNOLOGY I promoters (10). Taken together, the luciferase assay and the ChIP promoters (Fig. 3 A and C and Fig. S6) (10, 17, 29). CIITA is experiment show that NLRC5 not only associates with the pro- known to associate with a set of transcription-factor complexes, or moters of the MHC class I genes with remarkable specificity but MHC enhanceosome, on the WXY motif of the MHC class I and also has the capacity to transactivate their expression. class II gene promoters. The results of our ChIP and reporter gene assays indicate that NLRC5 may use a similar platform to NLRC5 Is Rapidly Induced by IFN-γ and Is Required for IFN-γ–Induced activate MHC class I gene promoters. Finally, both NLRC5 and Expression of MHC Class I. It has been shown that rapid induction of CIITA are highly inducible on IFN-γ stimulation (Fig. 4A) (15, 32, CIITA mediates the up-regulation of MHC class II on IFN-γ 33), and binding sites for STAT1, which are activated on IFN-γ stimulation (15, 32). Because NLRC5 is also an IFN-γ–inducible stimulation, have been mapped in the promoters of both genes gene (33), we explored the possibility that NLRC5 may mediate (33, 38–40). This suggests that both proteins are involved in me- the IFN-γ–induced transcription of MHC class I genes. First, we diating IFN-γ–induced changes in gene expression. In particular, compared the expression kinetics of NLRC5 and a MHC class I CIITA and NLRC5 seem to orchestrate the concerted expression gene on IFN-γ treatment. HLA-A transcript levels reach a maxi- of sets of functionally related genes critical for antigen pre- mum only 12–24 h after IFN-γ stimulation in HeLa cells, but sentation. CIITA, in addition to the classical MHC class II genes, similar to the IFN-γ–response gene STAT1, NLRC5 is induced induces the invariant chain Ii and the nonclassical MHC class II early after IFN-γ treatment (Fig. 4A), which is also a character- genes HLA-DM and HLA-DO, which play accessory roles in MHC istic of CIITA induction by IFN-γ (15, 32). Similar kinetics of class II antigen presentation (16). We show here that NLRC5,

Meissner et al. PNAS | August 3, 2010 | vol. 107 | no. 31 | 13797 Downloaded by guest on October 1, 2021 Fig. 4. Knockdown of NLRC5 results in decreased up-regulation of MHC class I on IFN-γ treatment. (A) HeLa cells were stimulated with IFN-γ (100 U/mL) for the indicated time points, and the kinetics of NLRC5, HLA-A,andSTAT1 expression were analyzed by quantitative real-time PCR. (B and C) HeLa cells were transfected with NLRC5-specific or control siRNAs; 16 h posttransfection, cells were stimulated with IFN-γ for 24 h. (B) Knockdown efficiency of NLRC5 was determined by quantitative real-time PCR using gene-specific primers, and data were normalized to the expression of the GAPDH gene. Scr, control scrambled siRNA. Error bars represent the ± SD from one representative of three independent experiments performed in duplicates. *P < 0.05. (C) Surface expression of MHC class I and β1-integrin was analyzed by flow cytometry. (D) Model depicting the role of NLRC5 in the IFN-γ–induced up-regulation of MHC class I genes. Discussion has further details.

beyond the induction of MHC class I genes, up-regulates β2M, our findings show that NLRC5 is necessary and sufficient for the TAP1, and LMP2, which are essential for antigen presentation by induction of MHC class I expression. NLRC5 may, thus, act as the MHC class I pathway (Fig. 2A). a counterpart to CIITA in its function as an MHC class I trans- However, in spite of these similarities and overlapping func- activator (CITA). Future analyses of the in vivo function of NLRC5 tions, there are also noticeable differences between NLRC5 and are required to reveal if these two molecules play redundant or CIITA. A unique feature of NLRC5 is its striking specificity for more exclusive roles in MHC class I-dependent immune responses. the induction of genes involved in the MHC class I pathway as We, thus, propose the following model of NLRC5 function in opposed to CIITA, which can induce both MHC class I and class II the expression of MHC class I genes. On IFN-γ stimulation, ac- genes. In this study, we found that expression of NLRC5 in epi- tivated STAT1 acts on the promoters of NLRC5 and CIITA and thelial and lymphoid cells was sufficient to induce MHC class I but rapidly induces these genes (Fig. 4D). Subsequently, CIITA may not MHC class II genes, despite their similar promoter architec- activate the promoters of both MHC class I and class II genes by ture (Fig. 2 C and D). Furthermore, our findings also suggest that associating with the MHC enhanceosome, which includes the NLRC5 is exclusively associated with the promoters of MHC class RFX, CREB/ATF, and NF-Y protein complexes on the con- I (Fig. 3C) and NLRC5 transactivated promoters of MHC class I served WXY module in the MHC promoters (Fig. 4D). NLRC5 and related genes but not those of MHC class II genes (Fig. 3A). A may also associate with a similar enhanceosome on the MHC class possible explanation for this specificity could lie in the structural I promoter, consisting of the same or similar components as those described for the CIITA enhanceosome. However, unlike the differences between the two proteins. NLRC5, unlike CIITA, lacks fi N-terminal acidic and proline/serine/threonine-rich domains, CIITA enhanceosome, the NLRC5 enhanceosome is speci cto which are required for MHC class II promoter activation (41). promoters of MHC class I and related genes (Fig. 4D). In conclusion, we have found that an NLR protein, NLRC5, acts NLRC5 will, thus, require additional cofactors to interact with and as a transactivator for MHC class I and related genes. Because activate the enhanceosome found on the MHC class I promoters. MHC class I is crucial for the activation of CD8 T cells, NLRC5 Given its specificity for MHC class I induction, it is also possible may play an important role in the immune response against viral that NLRC5 plays a dominant role in the regulation of MHC class infection or cancers. Future studies are required to unveil the I gene expression. This view is supported by the results of our function of NLRC5 in vivo under these pathological conditions. knockdown analyses, which clearly show that the IFN-γ–induced up-regulation of CIITA cannot compensate for the reduction in Materials and Methods MHC class I expression observed on NLRC5 depletion (Fig. 4C and Cell Lines and Reagents. Human embryonic kidney 293T (HEK293T) cells (CRL- Fig. S8). Furthermore, no reduction in MHC class I expression has 11268; ATCC) and HeLa cells (CCL-2; ATCC) were cultured in DMEM supple- been observed in CIITA-deficient mice (21–23). Taken together, mented with 10% FBS and penicillin (100 U/mL)/streptomycin (100 μg/mL;

13798 | www.pnas.org/cgi/doi/10.1073/pnas.1008684107 Meissner et al. Downloaded by guest on October 1, 2021 Gibco). Jurkat T cells (TIB152; ATCC) were maintained in RPMI-1640 (Thermo Renilla luciferase vector (pRL-null; Promega) were included for normaliza- Scientific) supplemented with 10% FBS and penicillin/streptomycin. HEK293T tion of transfection efficiency. Cells were harvested 48 h posttransfection, was transiently transfected using FuGENE 6 Transfection Reagent (Roche) in and cell lysates were analyzed using the Dual-Luciferase Reporter Assay ’ serum-free media according to the manufacturer s protocol. Recombinant System (Promega) according to the manufacturer’s instructions. The reporter γ human IFN- is from BioLegend. LMB was obtained from LC Laboratories. gene constructs were previously described (31).

Quantitative Real-Time PCR Analysis. Quantitative real-time PCR analysis was ChIP Assay. Chromatin immunoprecipitation of NLRC5 was performed as performed as described and is detailed in SI Materials and Methods (42). previously described and is detailed in SI Materials and Methods (43).

Flow Cytometry. Antibodies against human HLA-A, -B, -C (W6/32), HLA-E Statistical Analysis. Data were subjected to Student t test for analysis of sta- (3D12), HLA-DR (L243; Biolegend), and β1-integrin (TS2/16; Martin Hemler, tistical significance, and a P value of <0.05 was considered to be significant. DFCI, Boston, MA) were used in this study. Cells were stained, washed, resus-

pended in PBS/1% FBS/0.05% NaN3, and analyzed by FACSCalibur (Becton Dickinson) followed by analysis using FlowJo software. ACKNOWLEDGMENTS. We thank Peter Cresswell (Yale University, New Haven, CT), Martin Hemler (DFCI, Boston, MA), Marja van Eggermond (Leiden Univer- sity, Leiden, The Netherlands) and Cheong-Hee Chang (University of Michigan, NLRC5 × 6 Knockdown of by RNA Interference. HeLa cells (0.5 10 /well) were Ann Arbor, MI) for providing reagents, Kai Wucherpfennig and Diane Mathis transfected with 20 nM siRNA using Hyperfect (Qiagen) according to the for critically reading the manuscript, Carl Novina, Shannon Turley, and Harvey ’ manufacturer s instructions. Cells were stimulated 16 h posttransfection with Cantor for helpful discussions, Bettina Franz, Etienne Gagnon, and Maja Janas γ 100 U/mL IFN- (BioLegend). After 24 h of stimulation, cells were harvested and for technical advice, and Norman Lautsch, Amelia Chen, and Andrea Dearth for fl analyzed by ow cytometry and quantitative real-time PCR. The control siRNA general assistance. This work was supported by grants from the National Insti- (scrambled) as well as siRNAs targeting NLRC5 were obtained from Ambion. tutes of Health and the Crohn’s and Colitis Foundation of America (K.S.K.). T.B.M. is a recipient of the European Molecular Biology Organization (EMBO) Luciferase Assay. HEK293T cells were split into 24 wells and cotransfected with Long-Term Fellowship, and K.S.K. is a recipient of the Investigator Award from 300 ng of GFP-, GFP-NLRC5–, or GFP-CIITA–expression plasmids and 100 ng of the Cancer Research Institute and the Claudia Adams Barr Award. The authors the indicated luciferase reporter constructs; 50 ng/well of promoterless have no conflicting financial interests.

1. Pamer E, Cresswell P (1998) Mechanisms of MHC class I—restricted antigen processing. 23. Williams GS, et al. (1998) Mice lacking the transcription factor CIITA—a second look. Annu Rev Immunol 16:323–358. Int Immunol 10:1957–1967. 2. Braud VM, Allan DS, McMichael AJ (1999) Functions of nonclassical MHC and non- 24. Cressman DE, Chin KC, Taxman DJ, Ting JP (1999) A defect in the nuclear translocation MHC-encoded class I molecules. Curr Opin Immunol 11:100–108. of CIITA causes a form of type II bare lymphocyte syndrome. Immunity 10:163–171. 3. Le Bouteiller P, Solier C (2001) Is antigen presentation the primary function of HLA-G? 25. Spilianakis C, Papamatheakis J, Kretsovali A (2000) Acetylation by PCAF enhances Microbes Infect 3:323–332. CIITA nuclear accumulation and transactivation of major histocompatibility complex 4. Shastri N, Cardinaud S, Schwab SR, Serwold T, Kunisawa J (2005) All the peptides that class II genes. Mol Cell Biol 20:8489–8498. fit: The beginning, the middle, and the end of the MHC class I antigen-processing 26. Cressman DE, O’Connor WJ, Greer SF, Zhu XS, Ting JP (2001) Mechanisms of nuclear pathway. Immunol Rev 207:31–41. import and export that control the subcellular localization of class II transactivator. 5. Peaper DR, Cresswell P (2008) Regulation of MHC class I assembly and peptide J Immunol 167:3626–3634. binding. Annu Rev Cell Dev Biol 24:343–368. 27. Lange A, et al. (2007) Classical nuclear localization signals: Definition, function, and 6. Reith W, Mach B (2001) The bare lymphocyte syndrome and the regulation of MHC interaction with importin alpha. J Biol Chem 282:5101–5105. expression. Annu Rev Immunol 19:331–373. 28. Hanson PI, Whiteheart SW (2005) AAA+ proteins: Have engine, will work. Nat Rev Mol 7. van den Elsen PJ, Gobin SJ, van Eggermond MC, Peijnenburg A (1998) Regulation of Cell Biol 6:519–529. MHC class I and II gene transcription: Differences and similarities. Immunogenetics 48: 29. Gobin SJ, Peijnenburg A, Keijsers V, van den Elsen PJ (1997) Site alpha is crucial for 208–221. two routes of IFN gamma-induced MHC class I transactivation: The ISRE-mediated 8. van den Elsen PJ, Peijnenburg A, van Eggermond MC, Gobin SJ (1998) Shared route and a novel pathway involving CIITA. Immunity 6:601–611. regulatory elements in the promoters of MHC class I and class II genes. Immunol 30. Girdlestone J (2000) Synergistic induction of HLA class I expression by RelA and CIITA. Today 19:308–312. Blood 95:3804–3808. 9. Boss JM, Jensen PE (2003) Transcriptional regulation of the MHC class II antigen 31. Gobin SJ, van Zutphen M, Westerheide SD, Boss JM, van den Elsen PJ (2001) The MHC- presentation pathway. Curr Opin Immunol 15:105–111. specific enhanceosome and its role in MHC class I and beta(2)-microglobulin gene 10. Masternak K, et al. (2000) CIITA is a transcriptional coactivator that is recruited to transactivation. J Immunol 167:5175–5184. MHC class II promoters by multiple synergistic interactions with an enhanceosome 32. Chang CH, Fontes JD, Peterlin M, Flavell RA (1994) Class II transactivator (CIITA) is complex. Genes Dev 14:1156–1166. sufficient for the inducible expression of major histocompatibility complex class II 11. Wilmanski JM, Petnicki-Ocwieja T, Kobayashi KS (2008) NLR proteins: Integral members genes. J Exp Med 180:1367–1374. of innate immunity and mediators of inflammatory diseases. J Leukoc Biol 83:13–30. 33. Kuenzel S, et al. (2010) The nucleotide-binding oligomerization domain-like receptor 12. Ting JP, Willingham SB, Bergstralh DT (2008) NLRs at the intersection of cell death and NLRC5 is involved in IFN-dependent antiviral immune responses. JImmunol184:1990–2000. immunity. Nat Rev Immunol 8:372–379. 34. Nickerson K, et al. (2001) Dendritic cell-specific MHC class II transactivator contains 13. Beresford GW, Boss JM (2001) CIITA coordinates multiple histone acetylation a caspase recruitment domain that confers potent transactivation activity. J Biol Chem modifications at the HLA-DRA promoter. Nat Immunol 2:652–657. 276:19089–19093. 14. DeSandro A, Nagarajan UM, Boss JM (1999) The bare lymphocyte syndrome: Molecular 35. Wright KL, et al. (1998) CIITA stimulation of transcription factor binding to major clues to the transcriptional regulation of major histocompatibility complex class II histocompatibility complex class II and associated promoters in vivo. Proc Natl Acad genes. Am J Hum Genet 65:279–286. Sci USA 95:6267–6272. IMMUNOLOGY 15. Steimle V, Siegrist CA, Mottet A, Lisowska-Grospierre B, Mach B (1994) Regulation of 36. Bewry NN, Bolick SC, Wright KL, Harton JA (2007) GTP-dependent recruitment of CIITA MHC class II expression by interferon-gamma mediated by the transactivator gene to the class II major histocompatibility complex promoter. JBiolChem282:26178–26184. CIITA. Science 265:106–109. 37. Cui J, et al. (2010) NLRC5 negatively regulates the NF-kappaB and type I interferon 16. LeibundGut-Landmann S, et al. (2004) Mini-review: Specificity and expression of signaling pathways. Cell 141:483–496. CIITA, the master regulator of MHC class II genes. Eur J Immunol 34:1513–1525. 38. Muhlethaler-Mottet A, Di Berardino W, Otten LA, Mach B (1998) Activation of the MHC 17. Martin BK, et al. (1997) Induction of MHC class I expression by the MHC class II class II transactivator CIITA by interferon-gamma requires cooperative interaction transactivator CIITA. Immunity 6:591–600. between Stat1 and USF-1. Immunity 8:157–166. 18. Ting JP, Trowsdale J (2002) Genetic control of MHC class II expression. Cell 109 (Suppl): 39. Piskurich JF, Wang Y, Linhoff MW, White LC, Ting JP (1998) Identification of distinct S21–S33. regions of 5′ flanking DNA that mediate constitutive, IFN-gamma, STAT1, and TGF- 19. Bénichou B, Strominger JL (1991) Class II-antigen-negative patient and mutant B-cell beta-regulated expression of the class II transactivator gene. J Immunol 160:233–240. lines represent at least three, and probably four, distinct genetic defects defined by 40. Piskurich JF, Linhoff MW, Wang Y, Ting JP (1999) Two distinct gamma interferon- complementation analysis. Proc Natl Acad Sci USA 88:4285–4288. inducible promoters of the major histocompatibility complex class II transactivator 20. Steimle V, Otten LA, Zufferey M, Mach B (1993) Complementation cloning of an MHC gene are differentially regulated by STAT1, interferon regulatory factor 1, and class II transactivator mutated in hereditary MHC class II deficiency (or bare transforming growth factor beta. Mol Cell Biol 19:431–440. lymphocyte syndrome). Cell 75:135–146. 41. Chin KC, Li GG, Ting JP (1997) Importance of acidic, proline/serine/threonine-rich, and 21. Chang CH, Guerder S, Hong SC, van Ewijk W, Flavell RA (1996) Mice lacking the MHC GTP-binding regions in the major histocompatibility complex class II transactivator: class II transactivator (CIITA) show tissue-specific impairment of MHC class II Generation of transdominant-negative mutants. Proc Natl Acad Sci USA 94:2501–2506. expression. Immunity 4:167–178. 42. Petnicki-Ocwieja T, et al. (2009) Nod2 is required for the regulation of commensal 22. Itoh-Lindstrom Y, et al. (1999) Reduced IL-4-, lipopolysaccharide-, and IFN-gamma- microbiota in the intestine. Proc Natl Acad Sci USA 106:15813–15818. induced MHC class II expression in mice lacking class II transactivator due to targeted 43. Iliopoulos D, Hirsch HA, Struhl K (2009) An epigenetic switch involving NF-kappaB, Lin28, deletion of the GTP-binding domain. J Immunol 163:2425–2431. Let-7 MicroRNA, and IL6 links inflammation to cell transformation. Cell 139:693–706.

Meissner et al. PNAS | August 3, 2010 | vol. 107 | no. 31 | 13799 Downloaded by guest on October 1, 2021