PRDM1/BLIMP-1 Modulates IFN-γ -Dependent Control of the MHC Class I Antigen-Processing and Peptide-Loading Pathway This information is current as of September 26, 2021. Gina M. Doody, Sophie Stephenson, Charles McManamy and Reuben M. Tooze J Immunol 2007; 179:7614-7623; ; doi: 10.4049/jimmunol.179.11.7614 http://www.jimmunol.org/content/179/11/7614 Downloaded from

References This article cites 67 articles, 21 of which you can access for free at:

http://www.jimmunol.org/content/179/11/7614.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

PRDM1/BLIMP-1 Modulates IFN-␥-Dependent Control of the MHC Class I Antigen-Processing and Peptide-Loading Pathway1

Gina M. Doody,* Sophie Stephenson,* Charles McManamy,† and Reuben M. Tooze2*

A diverse spectrum of unique peptide-MHC class I complexes guides CD8 T cell responses toward viral or stress-induced Ags. Multiple components are required to process Ag and facilitate peptide loading in the endoplasmic reticulum. IFN-␥, a potent proinflammatory cytokine, markedly up-regulates transcription of involved in MHC class I assembly. Physiological mech- anisms which counteract this response are poorly defined. We demonstrate that promoters of functionally linked genes on this pathway contain conserved regulatory elements that allow antagonistic regulation by IFN-␥ and the B lymphocyte-induced maturation -1 (also known as PR domain-containing 1, with ZNF domain (PRDM1)). Repression of ERAP1, TAPASIN, MECL1, and LMP7 by PRDM1 results in failure to up-regulate surface MHC class I in response to IFN-␥ in Downloaded from human cell lines. Using the sea urchin ortholog, we demonstrate that the capacity of PRDM1 to repress the IFN response of such promoters is evolutionarily ancient and that dependence on the precise IFN regulatory factor element sequence is highly conserved. This indicates that the functional interaction between PRDM1 and IFN-regulated pathways antedates the evolution of the adaptive immune system and the MHC, and identifies a unique role for PRDM1 as a key regulator of Ag presentation by MHC class I. The Journal of Immunology, 2007, 179: 7614–7623. http://www.jimmunol.org/

he MHC class I system is first evident early in the evo- (MECL1), to generate the immunoproteasome (3). It acts to facil- lution of jawed vertebrates at the inception of the “adap- itate the transport of such peptides into the endoplasmic reticulum T tive” or “anticipatory” type immune response (1). The (ER), where the MHC class I peptide complex is assembled, by MHC class I Ag-processing and -presentation pathways co-opted inducing the expression of the peptide transporters TAP1 and and adapted existing cellular machinery to allow the sampling of TAP2 (4, 5). It enhances the expression of the TAP-associated during normal and altered cellular conditions and subse- chaperone protein TAPASIN which facilitates peptide loading into quent presentation to CD8-restricted T cells (2). The immune sys- the MHC class I-binding groove (6, 7) and ER amino peptidase 1

tem has evolved the ability to change the pattern of peptide pre- (ERAP1) (8, 9), which is responsible for trimming peptides to fit by guest on September 26, 2021 sentation in response to inflammatory cues by adapting the cellular the groove, a function vital to the generation of the mature peptide machinery engaged in the generation of the peptide repertoire. For repertoire (10). example, in the context of viral infection or inflammation, IFN-␥ The cumulative effects of IFN-␥ therefore provide the cell with modulates not only the transcription of the MHC class I genes, but both qualitatively different peptides and a quantitative increase in also multiple components of the Ag-processing and -loading path- surface MHC expression with which to elicit T cell activation. ways. IFN-␥ enhances the generation of peptides of an appropriate Changes in the peptide repertoire can lead to inappropriate de- length for presentation by inducing the expression of alternate pro- structive immune responses and contribute to the initiation of au- 3 teasome components, large multifunctional peptidase 2 (LMP2) , toimmune disease (11). The response to IFN-␥ provides a para- LMP7, and multicatalytic endopeptidase complex subunit-1 digm for the transcriptional control of MHC class I-dependent Ag presentation (3). Although the mechanisms for activation have *Section of Experimental Haematology, Leeds Institute of Molecular Medicine, and been extensively studied, the endogenous factors mediating oppos- †Academic Unit of Oncology and Haematology, Haematological Malignancy Diag- ing or repressive effects on IFN-␥-induced transcription to main- nostic Service, University of Leeds, Leeds, United Kingdom tain normal levels of MHC class I expression and unaltered peptide Received for publication February 16, 2007. Accepted for publication September 11, 2007. repertoires are ill-defined. Nevertheless, the tight control inherent in other aspects of the immune response suggests that these are The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance likely to exist. with 18 U.S.C. Section 1734 solely to indicate this fact. B lymphocyte-induced maturation protein-1 (BLIMP-1), 1 This work was supported by a Medical Research Council Clinician Scientist Fel- which is also known as PR domain-containing 1, with ZNF lowship (to R.M.T). domain (PRDM1), is an evolutionarily conserved transcrip- 2 Address correspondence and reprint requests to Dr. Reuben M. Tooze, Section of tional repressor of the Kru¨ppel family of zinc finger proteins Experimental Haematology, Leeds Institute of Molecular Medicine, Wellcome Trust Brenner Building, St. James’s University Hospital, Beckett Street, Leeds LS9 7TF, (12–15), which acts both through direct competition for pro- U.K. E-mail address: [email protected] moter occupancy, and by recruiting epigenetic modifiers (16– 3 Abbreviations used in this paper: LMP, large multifunctional peptidase; MECL-1, 20). It is best known as a regulator of terminal B cell differen- multicatalytic endopeptidase complex subunit-1; ER, endoplasmic reticulum; ERAP1, ER amino peptidase-1; BLIMP-1, B lymphocyte-induced maturation protein-1; tiation (21, 22), but additional roles for PRDM1 have also been PRDM1, PR domain-containing 1, with ZNF domain; IRF, IFN regulatory factor; identified in the control of T cell (23, 24), macrophage (25), and IRF-E, IRF element; ChIP, chromatin immunoprecipitation; EGFP, enhanced GFP; sebaceous gland differentiation (26). The original identification siRNA, short interfering RNA. of PRDM1 as a postinduction repressor of the IFN␤ promoter Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 during cellular viral infection suggested that PRDM1 is a key www.jimmunol.org The Journal of Immunology 7615 regulator of cellular responses to IFNs (12). The optimum plasmid preparations. The data are from a representative experiment dis- PRDM1 DNA-binding sequence overlaps with the IFN regula- played as fold increase in light units relative to unstimulated cells cotrans- tory factor-element (IRF-E) (27, 28), and we recently showed fected with the empty vector. that PRDM1 has the ability to regulate an IFN-␥-responsive Antibodies promoter by competing with IRFs for occupancy of an IRF-E Rabbit antisera to PRDM1 have been described previously (19, 32). (19). Given these characteristics, PRDM1 is a candidate to act Rabbit polyclonal Abs to IRF-1 and IRF-2 were obtained from Santa as a transcriptional repressor controlling MHC class I-depen- Cruz Biotechnology. Nonimmune rabbit IgG was obtained from Upstate dent Ag-presentation pathways. Biotechnology. Abs used for flow cytometry were mouse IgG2a con- Only a subset of IRF-E sequences, among all IFN-␥-responsive jugated to PE (BD Biosciences) and monoclonal anti-human HLA-ABC regulatory elements, are potential targets for PRDM1-depen- conjugated to PE (clone W6/32; DakoCytomation). Mouse mAb to TA- PASIN, clone 16, was obtained from BD Biosciences, goat polyclonal dent repression (19, 28). In this study, we demonstrate that the Ab to human keratin (GTX28572) was obtained from GeneTex, and promoters of multiple linked genes on the MHC class I Ag-pro- mouse mAb to ␤-actin, clone AC15, was obtained from Sigma-Aldrich. cessing and -loading pathway contain conserved IRF-E sequences Secondary Abs for immunofluorescence were Alexa Fluor 488 donkey which allow competitive binding by PRDM1 and IRFs. PRDM1 anti-mouse, Alexa Fluor 594 donkey anti-rabbit, and biotinylated don- key anti-goat coupled with AMCA-Avidin (Vector Laboratories) for represses the transcription of these genes and thus negatively con- blue fluorescence. trols MHC class I expression in response to IFN-␥. This reveals a unique function for PRDM1 as a repressor of MHC class I-depen- EMSA dent Ag presentation. Nuclear extracts were prepared as previously described (33) from COS

cells transfected with expression vectors for PRDM1, IRF-1, or IRF-2. Downloaded from Materials and Methods For EMSA, the double-stranded probes used contained the following sequences: ERAP F (5Ј-AGGACCGAAAGTGAAAGTGGAGCCCG Cell lines, expression vectors, and transfections GGGA-3Ј), ERAP R (5Ј-TCCCCGGGCTCCACTTTCACTTTCGGTC Ј Ј U266, H929, and HeLa cell lines were maintained in RPMI 1640 contain- CTG-3 ); TAPASIN F (5 -TTTGGAGGAAAGTGAAAGTGAAAGG Ј Ј ing 10% heat-inactivated FCS and COS cells were grown in DMEM con- AGGAAG-3 ), TAPASIN R (5 -CTTCCTCCTTTCACTTTCACTTT Ј Ј taining 10% FCS. HeLa or COS cells were seeded to reach 60% confluency CCTCCAAA-3 ); MECL1 F (5 -GAAGGGTAAAGGCGAAAGCGA AAGCAGGAAG-3Ј), MECL1 R (5Ј-CTTCCTGCTTTCGCTTTCGC on the day of transfection. Derivatives of bicistronic expression vector http://www.jimmunol.org/ Ј Ј pIRES2-EGFP (BD Clontech) encoding full-length (FL) PRDM1, the CTTTACCCTTC-3 ); LMP7 F (5 -CGGAGGAGGAAGTGAAAGCGA Ј Ј 528–789 C-terminal amino acids (⌬527) of PRDM1, IRF-1 and IRF-2, AAGCCACAGA-3 ), LMP7 R (5 -TCTGTGGCTTTCGCTTTCACT Ј have been described previously (19). Transfection of HeLa and COS cells TCCTCCTCCG-3 ). The underlined bases represent sites in which position 5 and 11 of the was performed with GeneJuice (Novagen) reagent according to the man- 32 ufacturer’s instructions. For RNA-interference experiments, U266 cells IRF-E were altered as described in Fig. 3. DNA probes, [ P]-labeled with were transfected with 400 pM short interfering RNA (siRNA) (200 pM of T4 polynucleotide kinase, were incubated with nuclear extract in the pres- each PRDM1 oligo or 400 pM control oligo) by electroporation using a ence of poly(dI:dC) (Amersham) for 30 min at room temperature. Super- Bio-Rad Gene Pulser with settings of 250 V and 960 ␮F. Twenty-four shift was performed by the addition of antisera to the extract before mixing hours posttransfection, cells were processed for protein and mRNA eval- with radioactive probe and competition assays included the addition of uation. The oligos used contained the following sequences: PRDM1 siRNA unlabeled probe to the reaction mixture. Ј Ј by guest on September 26, 2021 1: sense sequence, 5 -GGAAAGGACCUCUACCGUU-3 ; PRDM1 RT-PCR siRNA 2: sense sequence, 5Ј-GAUCUGACCCGAAUCAAUG-3Ј; control siRNA: sense sequence, 5Ј-CUACCUCUAGAACGGACGU-3Ј. RNA was isolated by the TRIzol method (Invitrogen Life Technologies) and subjected to DNase I treatment (DNAFree; Ambion). cDNA was gen- Luciferase vectors and assays erated using random hexamers and Superscript II reverse transcriptase (In- vitrogen Life Technologies). Conventional PCR was performed using Am- The following primers were used to amplify promoter sequences from pliTaq Gold (Applied Biosystems). Quantitative real-time PCR was human genomic DNA: ERAP1 forward (F) (5Ј-TATAAGATCTGGATC performed using SYBR Green MasterMix (Applied Biosystems) on an ABI CGCGTTCAGAAAGG-3Ј), ERAP1 reverse (R) (5Ј-AATTAAGCTTCTC Prism 7500 system (PerkinElmer) and evaluated with 7500 System Soft- ACCCTTGCGCCG-3Ј); TAPASIN F (5Ј-TCTAAAGCTTAAGGATG ware. Specificity of PCR was monitored with melting curve analysis, and CGCTCTTTATTTC-3Ј), TAPASIN R (5Ј-AAAACCATGGCGCTGCGA amplification of a single product of the expected size was verified on aga- CCTC-3Ј); MECL1 F (5Ј-TATAAGATCTCCTGAACAAGTCCAGAA- rose gels in initial experiments. Relative expression was normalized to 3Ј), MECL1 R (5Ј-AATTAAGCTTAGGCAGAGGGGATTAGG-3Ј); GAPDH and ACTIN. For quantitative RT-PCR, the following primers were LMP7 F (5Ј-TATAGGATCCGTACCTCTTACTGTAACC-3Ј), LMP7 R used: GAPDH F (5Ј-AACAGCGACACCCACTCCTC-3Ј), GAPDH R (5Ј- (5Ј-AATTAAGCTTATGACCGCCCAGCACCCA-3Ј). CATACCAGGAAATGAGCTTGACAA-3Ј); ACTIN F (5Ј-CATCGAG The amplicons were then cloned into the vector pXPG (29) generating CACGGCATCGTCA-3Ј), ACTIN R (5Ј-AGCACAGCCTGGATAGC promoter constructs spanning Ϫ248 to ϩ61 for ERAP1, Ϫ377 to ϩ174 for AAC-3Ј); ERAP1 F (5Ј-TCACCAGCAAATCCGACATG-3Ј), ERAP1 R TAPASIN, Ϫ203 to ϩ93 for MECL1, and Ϫ239 to ϩ45 for LMP7, rel- (5Ј-CCCACATTAAATTTGATCCATTCC-3Ј); TAPASIN F (5Ј-CAAG ative to the transcriptional start sites of reference sequences indicated in GATTCAAAGAAGAAAGCAGAGT-3Ј), TAPASIN R (5Ј-GGAGAGAG Table I. The TAPASIN promoter plasmid was cut with SmaI and religated, ATTGGAGGGATTAGG-3Ј); MECL1 F (5Ј-TGTGGACGCATGTGTG to contain the minimal IFN-responsive elements (30, 31). Mutations were ATCA-3Ј), MECL1 R (5Ј-GGTTCCAGGCACAAAGTGGTA-3Ј); LMP7 F introduced into the IRF-E site using the Gene-tailor site-directed mutagen- (5Ј-GGAGTGATTGCAGCAGTGGAT-3Ј), LMP7 R (5Ј-TGCCAAGCA esis kit (Invitrogen Life Technologies) using the following primers: GGTAAGGGTTAA-3Ј). ERAP1 mutant F (5Ј-CTGCCGCTCCCCGGGCTCCAGTTTCAGTTTC The primers used to PCR HLA-A, B, C have been published previously GGTCCTG-3Ј), ERAP1 mutant R (5Ј-TGGAGCCCGGGGAGCGG (34). The ACTIN primers were F (5Ј-AGAAAATCTGGCACCACACC-3Ј) CAGGCTGGCGCTG-3Ј); TAPASIN mutant F (5Ј-ATGCCGCCCTTTG andR(5Ј-CTCCTTAATGTCACGCACGA-3Ј). GAGGAAACTGAAACTGAAAGGAGG-3Ј), TAPASIN mutant R (5Ј- TTTCCTCCAAAGGGCGGCATGAGGGGCGGT-3Ј); MECL1 mutant F Chromatin immunoprecipitation (ChIP) (5Ј-GCCTTTGAGGAAGGGTAAAGCCGAAACCGAAAGCAGG-3Ј), MECL1 mutant R (5Ј-CTTTACCCTTCCTCAAAGGCCCGTGGCGGT- Chromatin prepared from myeloma or transfected HeLa cells was pre- 3Ј); LMP7 mutant F (5Ј-CCTTCGATCTGTGGCTTTCGGTTTCAGTT cleared with BSA saturated protein A-Sepharose followed by precipitation CCTCCTCC-3Ј), LMP7 mutant R (5Ј-CGAAAGCCACAGATCGAAGGG with 2 ␮g of Ab to PRDM1, IRF-1, IRF-2, or control rabbit Ab and protein GAGGGAACA-3Ј). A-Sepharose. DNA was eluted and cross-links reversed by overnight in- All wild-type and mutant constructs were sequence verified. For lucif- cubation at 65°C. Input DNA was prepared from an equal volume of chro- erase assays, three replicate transfections were performed for each condi- matin following RNase treatment and resuspended in the same final vol- tion. Experiments were done using the Promega luciferase assay system ume as the ChIP samples. A standard curve of input DNA was generated and analyzed on a Berthold Lumat LB Luminometer. Each experiment was from each chromatin sample. Target sequences were analyzed by real-time performed in duplicate with similar results and confirmed with separate PCR as described above. PCRs were monitored by melting curve analysis 7616 PRDM1/BLIMP-1 CONTROLS CLASS I Ag-PROCESSING PATHWAY

Table I. Overlapping PRDM1 and IRF-E consensus sequencesa

PRDM1/BLIMP-1 Consensus A/Ca A G T/C G A A A G T/C G/T

IRF-E G/C A A A G/C T/C G A A A G/C T/C

Gene Species Ref. Sequence Position

ERAP1 (ARTS1) Human NM_0016442.2 ϩ7toϪ13 C G A A A G T GAAA G T G GAGCCC Mouse C G A A A G T GAAA G T G AAGGTG Rat C G A A A G T GAAA G T G AAGCTG Rabbit C G A A A G T GAAA G T G CGGGCC

TAPASIN (TAPBP) Human NM_003190.3 ϩ114 to ϩ133 G G A A A G T GAAA G T G AAAGGA Mouse G G A A A A T GAAA G T G AAAGGG Rat G G A A A A T GAAA G T G AAAGGA Dog G G A A A G C GAAA G T G AAGGGG

MECL1 (PSMB10) Human NM_002801.2 Ϫ28 to Ϫ9TAAA G G C GAAA G C G AAAGCA Mouse T G G G G G T GAAA G C AAAGTA Rat T A A A G G T GAAA G C G AAAGTA Dog T A A A G G T GAAA G C G AAACCA

LMP7 (PSMB8) Human NM_148919.3 ϩ5toϪ15 A G G A A G T GAAA G C G AAAGCC Mouse A G G A A G T GAAA G C G AAAGCC Downloaded from Rat A G G A A G C GAAA G C G AAAGCC Dog A G G A A G T GAAA G C G AAAGCC

PA28␣ (PSME1) Human NM_006263.2 ϩ47 to ϩ28 G C G A A G G GAAA G C G AAAGCG Mouse GGGAAGGGA AA G C G AAAGCA Rat GGGAAGGG AAA G C G AAAGCA Dog G G G A A G A G A AA G C G AAAGCG http://www.jimmunol.org/ PA28␤ (PSME2) Human NM_002818.2 ϩ2toϩ21 G G G G A G T GAAA G C G AAAGCC Mouse G G A G A G T GAAA G C G AAAGC T Rat G G A G A G T GAAA G C G AAAGC T Dog G T G G A G T GAAA G C G AAAGGC

TAP2 Human NM_000544.3 Ϫ34 to Ϫ15 G G G A A G C GAAA G C G AAAGCT Mouse GTG A A G T GAAA G C G AAAGCC Dog GGG A A G C GAAA G T G AAAGC T Opossum CGG A A G C GAAA G T G AAAGGG

TAP1 Human NM_000593.5 ϩ157 to ϩ139G G A A A G C GAAA T C G AAAGCG

Mouse GGAAGAA GAAA C C G AAAGCC by guest on September 26, 2021 Rat G G A A G A A GAAA C C G AAAGCA Dog G G A A A G G GAAA C C G AAAGCG

HLA-A Human GAGAAAA GAAA C T G CGGAG T HLA-B Human AAGAAGT GAAA C T C AGGGGG HLA-C Human GAGAAGT GAAA C T C AGGGGG B2M Human GAAAACT GAAA A C G GGAAAG

aPRDM1 and IRF-E consensus sequence alignment: position shown relative to transcriptional start site of indicated reference sequence; letters shown in bold identify PRDM1 consensus; boxed sequences identify IRF-E consensus.

and representative products were verified by sequencing. The amount of isotype control or PE-conjugated Ab to pan MHC class I. Samples were precipitated material was calculated from the standard curve and nor- analyzed on a LSRII flow cytometer (BD Biosciences) with settings de- malized relative to control immunoprecipitates. The data are represen- termined by nontransfected cells and isotype staining. Viable cells were tative of a minimum of three independent chromatin preparations, and distinguished based on forward scatter and side scatter characteristics and represent average and SD of at least two independent immunoprecipi- propidium iodide staining. Plots were generated using WinMDI software. tations. The following primers were used, with positions shown relative The top 15% of enhanced GFP (EGFP) expressing transfected cells were to reference sequences indicated in Table I: ERAP1–240 F (5Ј-GGATC sorted. CGCGTTCAGAAAGG-3Ј), ERAP1–137 R (5Ј-CCAGGAAGGGAAT Immunofluorescent staining was performed on human tonsil sections TGGTAAATG-3Ј); TAPASIN ϩ 86F(5Ј-CCAGGCACCTTCACCTA after heat-mediated Ag retrieval. The staining protocol was performed es- ACC-3Ј), TAPASIN ϩ 187R(5Ј-CAGCCATGAAGCCTCCTCTT-3Ј); sentially as previously described (35). Sections were viewed using a Zeiss MECL1–135 F (5Ј-GGGCACAGCAAGGGACAT-3Ј), MECL1–46 R AxioPlan2 imaging fluorescence microscope. Images were captured and (5Ј-GTGGCGGTTTTCTGCATCTT-3Ј); LMP7 ϩ 123F(5Ј-GCTCG processed using the ISIS3 image capture system (MetaSystems). The use of GACCCAGGACACTAC-3Ј), LMP7 ϩ 202R(5Ј-TACTGCCCCGAC human tissue was approved by the local research ethics committee. CTGCAT-3Ј); PA28␣-6F(5Ј-ACTACCCAGGAAGGCGGAG-3Ј), PA28␣ϩ77R(5Ј-CGCACAAGGAGTGGAGTGG-3Ј); PA28␤ϩ202 F Results (5Ј-CGCCACTGAATACCCCCTTT-3Ј), PA28␤ϩ285R(5Ј-GGCT TATAGCTAGGGCCAACTG-3Ј); TAP2–245 F (5Ј-CAGATAAAGT An overlapping PRDM1/IRF-E-binding site distinguishes the TGCCCTTGAGACAA-3Ј), TAP2–123 R (5Ј-CACTGTACAGGCCT promoters of MHC class I Ag-processing and -presentation GCAATGA-3Ј); KRT-10 F (5Ј-TGGACACACCCTCTCAGTATATAA machinery genes AGG-3Ј), KRT-10 R (5Ј-AGAGTAGTGCTTGCTTGAGCTGTATC-3Ј). Our laboratory has previously reported that PRDM1 represses Flow cytometry and immunofluorescence IFN-␥-mediated activation of the MHC CIITA (CIITA) promoter ␥ Evaluation of MHC class I surface expression was performed on HeLa IV and competes with the IFN- -induced transcription factors cells 48 h posttransfection. Cells were cultured in the presence or absence IRF-1 and IRF-2 for occupancy of the IRF-E (19). The overlap of IFN-␥ for the final 24 h. HeLa cells were stained with PE-conjugated between the optimum PRDM1 consensus and the IRF-E suggests The Journal of Immunology 7617 Downloaded from http://www.jimmunol.org/

FIGURE 1. PRDM1 binds to the promoters of ERAP1, TAPASIN, MECL1, and LMP7 in vivo. ChIP analysis of PRDM1, IRF-1, and IRF-2 by guest on September 26, 2021 binding to endogenous promoters. Chromatin prepared from U266 my- eloma cells was immunoprecipitated with control rabbit IgG, anti-PRDM1 (A), anti-IRF-1 (B), or anti-IRF-2 (C). The amount of promoter sequence present was then quantified by real-time PCR. The ratio of product recov- ered relative to control IgG is displayed as fold enrichment. These data are FIGURE 2. PRDM1 and IRF occupancy is mutually exclusive. EMSA derived from two to four separate immunoprecipitations and are displayed evaluation of the identified IRF-E sites for hetero-occupancy using nuclear as the mean Ϯ SD. extracts from COS cells transfected with PRDM1, IRF-1, or IRF-2. Initial titrations of PRDM1-, IRF-1-, and IRF-2-containing extracts with each of the oligos were performed to determine equivalent binding activities (data not shown). Comparable amounts of extracts containing IRF-1 or IRF-2 that other target genes may be under similar antagonistic regula- were mixed with extract containing PRDM1 alone, or in the presence of E tion (28). To establish whether IFN-␥-regulated components of the specific Abs. The migration of PRDM1 (arrowhead), IRF-1 ( ), and IRF-2 F MHC class I pathway were likely to be PRDM1 targets, we ex- ( ) is indicated. amined the basal promoter sequences of these genes (4, 30, 36– 41) for evidence of an overlapping PRDM1/IRF-E consensus (28). more positions within the extended core, which include critical Remarkably, we discovered that all eight promoters examined con- residues necessary for efficient PRDM1 binding (12, 19). tain IRF-E sequences which match the PRDM1 consensus-binding site with at most a single base mismatch (Table I). Moreover, only PRDM1 binds to the promoters of MHC class I-processing one of three mismatches lies within the extended core sequence of and -presentation machinery genes in vivo the PRDM1 site, G(T/C)GAAAG(T/C)(G/T), and in each case the The results above demonstrate the presence of a PRDM1/IRF-E presence of an overlapping PRDM1/IRF-E consensus in the basal consensus in the promoters of the genes encoding multiple com- promoter is evolutionarily conserved. ponents of the MHC class I-processing and -presentation machin- This degree of overlap between PRDM1 consensus and IRF- ery. To evaluate promoter occupancy in vivo, we used ChIP to controlled sites is not a common feature of IFN-␥- or IRF-regu- examine the association between PRDM1 and these promoters in lated genes. In a published comparison of IRF-responsive ele- myeloma cell lines U266 and H929. Constitutive PRDM1 binding ments, other than the known PRDM1 site within the IFN␤ to the promoters of the identified target genes ERAP1, TAPASIN, promoter (12) only 1 of the other 32 sites listed displays this de- MECL1, and LMP7 was observed (Fig. 1A and data not shown). In gree of overlap with the PRDM1 consensus (42). Particularly im- contrast, PA28␤ was weakly bound, while the promoter sequences portant examples in this context are the sequences present in the of PA28␣ and TAP2 were enriched Ͻ2-fold relative to control. ␤ promoters of 2-microglobulin and the MHC class I genes them- Therefore, PRDM1 is probably not bound to all available consen- selves (Table I). These differ from the PRDM1 consensus at two or sus sequences. 7618 PRDM1/BLIMP-1 CONTROLS CLASS I Ag-PROCESSING PATHWAY Downloaded from http://www.jimmunol.org/

FIGURE 4. PRDM1 represses IFN-␥ stimulation of ERAP1, TAPASIN, MECL1, and LMP7 promoter activity. A, Luciferase reporter assays of promoter activation. HeLa cells cotransfected with luciferase reporter con- structs containing promoter regions of ERAP1, TAPASIN, MECL1, and by guest on September 26, 2021 LMP7 and either 50 ng of empty expression vector or 50 ng of expression vector encoding PRDM1 were evaluated for luciferase activity after treat- ment with medium alone or containing 200 IU/ml IFN-␥ for6h.B, Func- tional evaluation of mutated IRF-E sites. HeLa cells were cotransfected with PRDM1 expression vector and mutated luciferase reporter constructs, corresponding to the sequences used in EMSA, and evaluated for luciferase activity following6hofIFN-␥ (200 IU/ml) stimulation. These data are FIGURE 3. PRDM1 occupies a precise IRF-E site. Determination of derived from triplicate samples and are displayed as the mean Ϯ SD. IRF-E sequence requirements for PRDM1, IRF-1, and IRF-2 binding. Nu- clear extracts from IRF-1-, IRF-2-, or PRDM1-transfected COS cells were evaluated for binding to wild-type IRF-E sequence in the presence of 10- moters bound by either PRDM1 or the IRFs, as expected in the or 100-fold excess wild-type or mutated competitor probe. The core se- context of dynamic and antagonistic transcriptional regulation. Al- quence of each probe and the mutated version with positions 5 and 11 of ternatively, PRDM1, IRF-1, and IRF-2 might co-occupy the IRF-E the IRF-E changed from G to C are listed below. target sequences. To distinguish between these possibilities, next we examined interactions in vitro by EMSA. Oligonucleotides rep- We previously found that U266 and H929 myeloma cells also resenting the various IRF-E sequences were incubated with COS express IRF-1 and IRF-2 (19). To test for occupancy by these cell extracts containing PRDM1, IRF-1, or IRF-2 alone, or in com- transcription factors, we performed additional ChIP experiments. bination. Binding to the IRF-E sequences was clearly observed A mutually exclusive pattern of promoter occupancy was previ- with the individual factors. The sites were occupied by IRF pro- ously described at CIITA-pIV and the bidirectional TAP1/LMP2 teins either in a single or multimeric fashion (Fig. 2). When ex- promoter (19). In contrast, we observed that ERAP1, TAPASIN, tracts containing PRDM1 and IRF-1 or IRF-2 were used in com- MECL1, and LMP7 promoters all showed substantial binding by bination, no additional complexes were observed containing both IRF-1 and IRF-2, as well as PRDM1 (Fig. 1, B and C). PA28␣, PRDM1 and either of the IRFs (Fig. 2). Thus, PRDM1, IRF-1, and PA28␤, and TAP2 promoters were occupied by IRF-1 and IRF-2 IRF-2 occupy IRF-E sequences separately and do not co-occupy suggesting that PRDM1 is unable to efficiently compete at these the same sites. sites. At the IRF-E of the CIITA promoter IV, positions 5 and 11, are critical in determining dual regulation by IFN-␥ and PRDM1 (19). PRDM1 and IRF occupancy of the IRF-E sequences is mutually However, it is not known whether this is a common feature of exclusive and dependent on a precise sequence other promoters containing similar sites. To investigate this pos- The occupancy of ERAP1, TAPASIN, MECL1, and LMP7 promot- sibility, we used oligonucleotides representative of the ERAP1, ers by all three factors potentially reflects combinations of pro- TAPASIN, MECL1, and LMP7 IRF-E altered at these positions in The Journal of Immunology 7619 Downloaded from http://www.jimmunol.org/

FIGURE 5. Repression of IRF-Es is a highly conserved PRDM1 func- tion. A, Prdm1 from sea urchin, zebrafish, and frog repress TAPASIN pro-

moter activity. HeLa cells cotransfected with the TAPASIN promoter lu- by guest on September 26, 2021 ciferase reporter construct and either 50 ng of empty expression vector or 50 ng of expression vector encoding Prdm1 from the indicated species were evaluated for luciferase activity after treatment with medium alone or containing 200 IU/ml IFN-␥ for6h.B, Prdm1 orthologs require an intact IRF-E to mediate repression. Data were obtained as described in A using the mutated TAPASIN promoter luciferase reporter construct. These data FIGURE 6. PRDM1 modulates MHC class I surface expression. The are derived from triplicate samples and are displayed as the mean Ϯ SD. level of MHC class I expression on HeLa cells transfected with PRDM1 IRES2 EGFP or control IRES2 EGFP vector after treatment with 200 IU/ml IFN-␥ for 24 h was assessed by flow cytometry. Left panel, The level EMSAs. The relative efficiency of PRDM1, IRF-1, and IRF-2 of EGFP expression in the transfected populations. Right panel, The level Ϫ high binding to the divergent sites was assessed in cold competitor as- of PE-MHC class I on cells gated for EGFP (black line) or EGFP (gray filled). These data are representative of at least four separate evalu- says. The altered IRF-E sequences successfully competed with the ations of EGFP and PRDM1 transfectants. wild-type IRF-E sequences for binding to IRF-1 and IRF-2. In contrast, competition for binding to PRDM1 was barely detectable (Fig. 3). Thus, in the context of these IRF-Es, positions 5 and 11, zinc-finger domain was sufficient to repress IFN-␥-dependent pro- determine the capacity to bind PRDM1. Our findings suggest that moter activation (data not shown). the presence of a G rather thanaCatpositions 5 and 11 reflect In EMSA, the mutant oligonucleotides preserve IRF, but not evolutionary selection for dual regulation of these promoters. PRDM1 binding. These same substitutions abolished PRDM1-de- pendent repression in response to IFN-␥ at the ERAP1, TAPASIN, ␥ PRDM1 represses IFN- -dependent promoter activation of and LMP7 promoters and significantly reduced repression at the MHC class I Ag-processing and -loading genes in a fashion MECL1 promoter (Fig. 4B). Together, these data demonstrate that dependent on the IRF-E sequence PRDM1 represses both the basal and IFN-␥-dependent activities of To demonstrate whether PRDM1 binding to the promoters of these promoters via the IRF-E sequence. MHC class I Ag-processing pathway genes causes transcriptional repression, we used luciferase reporter assays. As shown in Fig. 4, Distant orthologs of PRDM1 repress human TAPASIN promoter PRDM1 mediates substantial repression of both basal and IFN-␥- activity with the same strict sequence dependence dependent promoter activation (Fig. 4A). In the presence of PRDM1 orthologs act as important transcriptional regulators PRDM1, promoter activity remains below basal levels even after among both vertebrates and invertebrates (15, 43–46), including IFN-␥ stimulation. ERAP1, TAPASIN, and MECL1 gave similar sea urchins, which lack an anticipatory/adaptive immune system results, while partial inhibition was observed for the LMP7 pro- and MHC class I genes (47). Hence, PRDM1 function has been moter. Furthermore, a vector encoding the PRDM1 DNA-binding co-opted during evolution into regulation of the adaptive immune 7620 PRDM1/BLIMP-1 CONTROLS CLASS I Ag-PROCESSING PATHWAY Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 7. PRDM1 selectively affects expression of MHC class I assembly components. A, PRDM1 does not affect MHC class I transcript levels. mRNA levels of HLA-A, HLA-B, HLA-C,or␤-actin were assessed by RT-PCR in sorted EGFPhigh HeLa cells transfected with PRDM1 IRES2 EGFP or control IRES2 EGFP vector after treatment with 200 IU/ml IFN-␥ for 24 h. B, Transfected PRDM1 represses endogenous ERAP1, TAPASIN, MECL1, and LMP7. Transcript levels were assessed by quantitative RT-PCR in sorted EGFPhigh cells stimulated with 200 IU/ml IFN-␥ for6h.C, Transcript levels of ERAP1, TAPASIN, MECL1, and LMP7 from the samples used in A were assessed by quantitative RT-PCR. The data are derived from three separate transfections and are displayed as the mean Ϯ SD. D, Chromatin prepared from HeLa cells transfected with PRDM1 IRES2 EGFP or control IRES2 EGFP vector was immunoprecipitated with control rabbit IgG or anti-PRDM1 and the fold enrichment of ERAP1, TAPASIN, MECL1, LMP7,orKRT-10 promoter sequence was determined by real-time PCR. The amount of DNA in PRDM1 ChIP samples from EGFP-transfected cells was equivalent to control IgG at all loci. These data were derived from two separate transfection experiments and are displayed as the mean Ϯ SD. E, siRNA knockdown of PRDM1. U266 cells were transfected with control or PRDM1-specific siRNA and evaluated for relative protein expression at 24 h by Western blotting. F, Endogenous PRDM1 represses ERAP1, TAPASIN, MECL1, and LMP7. Transcript levels of the indicated genes were quantified relative to ␤-actin from the triplicate samples in E. The data are displayed as the expression detected in samples transfected with PRDM1-specific siRNA relative to control transfected cells. response. The degree of conservation among PRDM1 orthologs is mediating repression of the TAPASIN promoter in vitro. All three greatest in the zinc-finger DNA-binding domains (15). The IRF- orthologs, including sea urchin Prdm1 (blimp1/krox), clearly have E/PRDM1 sites in the MHC class I Ag-processing pathway genes the ability to repress both basal and IFN-␥-dependent promoter include perfect matches to the optimum human and mouse activation at the TAPASIN promoter (Fig. 5A). As for human PRDM1 consensus binding site (27, 28), and thus provide ideal PRDM1, the ability of the orthologs to repress the TAPASIN pro- targets for testing conservation of PRDM1 function. moter was eliminated by mutation of the IRF-E at position 5 and We therefore examined the ability of frog (48), zebrafish (43), 11 (Fig. 5B). In contrast to frog and zebrafish (2), the sea urchin, and sea urchin (15) Prdm1 to substitute for human PRDM1 in Strongylocentrotus purpuratus, does not possess an anticipatory/ The Journal of Immunology 7621 adaptive immune system or MHC Ag-presentation pathways (47). Thus, the ability of PRDM1 to regulate IRF-E sites is ancient and must have existed in a common ancestor before the evolution of the adaptive immune system. PRDM1 represses IFN-␥-dependent induction of surface MHC class I and expression of the endogenous target genes Repression of multiple target genes would be expected to impact on induction of MHC class I surface expression in response to IFN-␥. To test this, HeLa cells were transfected with PRDM1 IRES2 EGFP or control IRES2 EGFP vector and treated with IFN-␥. MHC class I surface expression was then assessed. EGFPhigh and EGFPϪ untransfected populations were compared (Fig. 6). In controls, we observed indistinguishable levels of either basal or IFN-␥-induced MHC class I expression. The EGFPhigh population in PRDM1-transfected cells showed only a slight de- crease in basal MHC class I expression, but displayed a complete failure to induce surface MHC class I expression in response to

IFN-␥. Thus, PRDM1 expression has a profound effect on the abil- Downloaded from ity of cells to up-regulate MHC class I surface expression in re- FIGURE 8. PRDM1 and TAPASIN show mutually exclusive patterns ␥ sponse to IFN- . of expression in tonsil crypt epithelium. Sections of normal human tonsil To examine whether this inhibition of surface MHC class I ex- were stained by multicolor immunofluorescence with Ab to KERATIN pression might be due to repression of MHC class I mRNA in- (blue), PRDM1 (red), and TAPASIN (green). A representative field using duction, EGFPhigh populations were sorted and MHC class I a ϫ20 objective is shown. The figure displays composite and split color

mRNA induction was assessed by RT-PCR. Control and PRDM1- channels. http://www.jimmunol.org/ transfected EGFPhigh populations fail to display a significant dif- ference in levels of MHC class I mRNA expression (Fig. 7A). In contrast, IFN-␥-dependent induction of ERAP1, TAPASIN, Ag-presentation pathway in this tissue, we focused on TAPASIN MECL1, and LMP7 mRNA was markedly inhibited in EGFPhigh expression, because an available Ab to this protein worked effec- PRDM1-transfected populations relative to control at 6 h post- tively in multicolor immunofluorescence. TAPASIN was ex- stimulation (Fig. 7B). Repression of ERAP1 and TAPASIN mRNA, pressed strongly in the lymphoid compartments of the tonsil, but but not MECL1 or LMP7, remained substantial at 24 h (Fig. 7C). was also expressed in tonsil crypt epithelium identified by staining ChIP assays confirmed that repression of these promoters was ac- with a pan-keratin Ab (Fig. 8). In crypt epithelium, TAPASIN and companied by PRDM1 occupancy (Fig. 7D). PRDM1 therefore PRDM1 displayed inverse expression. TAPASIN was strongest in by guest on September 26, 2021 blocks IFN-␥-dependent induction of MHC class I surface expres- basal and intermediate, but absent in superficial layers. This con- sion due to repression of these components of the Ag-processing trasted with the restricted zone of PRDM1 staining in more super- and peptide-loading pathway, and not MHC class I structural ficial epithelium. This pattern of PRDM1 and TAPASIN expres- genes. sion in the tonsil epithelium strengthens the idea that PRDM1 We have previously shown that the myeloma cell line U266 functions to repress this pathway in vivo. expresses both IRF-1 and IRF-2 in the absence of IFN-␥ stimula- tion. Furthermore, the presence of these factors is sufficient to me- Discussion diate increased expression of an otherwise IFN-␥-dependent gene, The absolute level of MHC class I surface expression and the when PRDM1 levels are reduced (19). To demonstrate that endog- repertoire of peptides presented by MHC class I control CD8 T cell enous PRDM1 mediates repression of ERAP1, TAPASIN, MECL1, activation and target cell recognition. IFN-␥ induces expression of and LMP7, we examined the effect of PRDM1 knockdown in MHC class I genes (53) and key components of the machinery U266 cells on the expression of these genes. PRDM1 protein was responsible for peptide generation, loading, and trimming. In this substantially reduced following transfection with specific siRNA study, we have demonstrated that PRDM1 can exert a dominant (Fig. 7E). Loss of PRDM1 led to an increase in expression of all opposing role and repress the MHC class I Ag-presentation four target genes, ranging from 1.6- to 4.6-fold (Fig. 7F). Collec- pathway. tively, these data establish that the expression of genes on the We have identified a panel of novel PRDM1 target genes on this MHC class I Ag-processing and -loading pathway is directly re- pathway. Our data reveal that four promoters are substantially oc- sponsive to the relative levels of PRDM1 and IRF-1 or IRF-2. cupied by PRDM1 under physiological conditions in a myeloma cell line. When bound to the newly defined target promoters, Mutually exclusive expression of PRDM1 and TAPASIN in PRDM1 is a potent repressor of basal activity and IFN-␥-depen- tonsil crypt epithelium is consistent with transcriptional dent induction. TAP2, PA28␣, and PA28␤ are also potential tar- repression in vivo gets of PRDM1-mediated repression, based on the presence of an The tonsil crypt epithelium generates a specialized niche for Ag overlapping PRDM1/IRF-E consensus site, but gave little evidence sampling (49), and represents an important site for class I presen- of occupancy in this study. At the initial step of peptide generation, tation of viral pathogens such as HIV and EBV (50, 51). The PRDM1 acts to repress the transcription of two, MECL1 and modified squamous epithelium of the crypt is heavily infiltrated by LMP7, of the three catalytic components that distinguish the im- lymphocytes and potentially subject to chronic cytokine stimula- munoproteasome (3). Because these three components are assem- tion. This epithelium displays strong expression of PRDM1 in su- bled in a cooperative fashion, absence of one or more subunits is perficial epithelial cells (52). To evaluate whether PRDM1 expres- sufficient to compromise assembly of the immunoproteasome and sion correlates with repression of components of the MHC class I the enhanced generation of antigenic peptides (54, 55). The next 7622 PRDM1/BLIMP-1 CONTROLS CLASS I Ag-PROCESSING PATHWAY component of the pathway targeted by PRDM1, TAPASIN, acts as tation during cellular stress responses. Under conditions of cellular a chaperone protein tethering the empty MHC class I molecule to stress, Ag presentation may be profoundly altered, and consequent the TAP transporter and facilitating the loading of antigenic pep- T cell responses can contribute to the initiation of autoimmune tide (6, 7). TAPASIN-deficient cell lines and mice have profound disease (11). In addition to its induction during cellular viral in- defects in Ag presentation and the efficiency with which stable, fection (12), we have shown that PRDM1 expression is responsive optimized MHC class I peptide complexes are formed (6, 56, 57). to a range of stressors, in particular those targeting the unfolded Because MHC class I alleles display differences in their relative protein response of the ER (32). Responses of this type are linked dependence on TAPASIN for optimal peptide loading (7), repres- to MHC class I-dependent Ag presentation in a number of ways. sion of TAPASIN by PRDM1 has the potential to alter both the First, the misfolding of the MHC class I allele HLA-B27, partic- overall efficiency with which MHC class I peptide complexes are ularly in the context of IFN-␥ stimulation, can initiate an ER stress formed and the relative level of surface MHC class I alleles ex- response (64). Second, the generation of antigenic peptides is al- pressed. Finally, PRDM1 represses ERAP1 which has recently tered by the phosphorylation of the eIF2␣ subunit of the ribosome been shown to be the primary peptidase responsible for trimming (65). The selection of cryptic ribosomal initiation sites generates antigenic peptides to fit the MHC class I-binding groove (8, 9, 58). alternate immunogenic peptide products, and a general role for Because peptides generated by the immunoproteasome may be defective ribosomal products as a source of antigenic peptides is particularly dependent on N-terminal trimming to fit the MHC well-established (66). Third, chemical modification of proteins and class I peptide-binding groove, repression of ERAP1 has the po- the generation of altered peptides can occur and contribute to au- tential to profoundly alter the generation of MHC peptide com- toimmune disease (67). We propose that PRDM1 induction con- plexes (10, 59, 60). The impact on IFN-␥-dependent induction of tributes to the control of MHC class I Ag presentation during cel- Downloaded from surface MHC class I expression therefore reflects the combined lular stress to help prevent inappropriate and deleterious Ag action of PRDM1 on multiple linked steps in this functional path- presentation. It is notable that lineages which express PRDM1, way. However, we cannot exclude that repression of one or more such as macrophages, squamous epithelia and endothelium, are of these steps mediate a dominant effect or that selective effects particularly prone to exposure to adverse environmental condi- may be evident, dependent on the relative levels of PRDM1 and tions. We conclude that PRDM1 is a unique regulator of Ag

IRF expression. presentation. http://www.jimmunol.org/ A striking feature at the level of promoter sequences is the se- lection for overlapping PRDM1/IRF-E-binding sites in this set of Acknowledgments genes, which act at sequential steps of a single functional pathway. We thank Peter Cockerill for pXPG vector, Philip Ingham for Danio rerio Because the degree of overlap seen at these promoters is not a Prdm1/Blimp-1 expression vector and pCSII, Christoph Niehrs for Xeno- common feature among IRF- or IFN-regulated promoters (42), this pus laevis Prdm1/Blimp-1 expression vector, and Eric Davidson for S. suggests a particular selection for dual regulation. The lack of such purpuratus Prdm1/blimp1/krox. We thank Liz Straszynski for cell sorting. We are indebted to Liz Bikoff for helpful advice on the manuscript. selection in the MHC class I promoters themselves (61) argues against a direct role for PRDM1 in repressing MHC class I gene Disclosures by guest on September 26, 2021 transcription, and we demonstrate that the blocked induction of The authors have no financial conflict of interest. MHC class I surface expression by PRDM1 is not reflected in repression of MHC class I transcription. We have previously References shown that PRDM1 represses the IFN-␥ responsive promoter IV of 1. Litman, G. W., J. P. Cannon, and L. J. Dishaw. 2005. Reconstructing immune CIITA (19). In addition to regulating MHC class II expression, phylogeny: new perspectives. Nat. Rev. Immunol. 5: 866–879. ␥ 2. Danchin, E., V. Vitiello, A. Vienne, O. Richard, P. Gouret, M. F. McDermott, and CIITA has been shown to play a role in the IFN- -responsive P. Pontarotti. 2004. The major histocompatibility complex origin. Immunol. Rev. transcription of MHC class I (62, 63). Repression of CIITA poten- 198: 216–232. tially provides yet another mechanism by which PRDM1 may tar- 3. Strehl, B., U. Seifert, E. Kruger, S. Heink, U. Kuckelkorn, and P. M. Kloetzel. 2005. Interferon-␥, the functional plasticity of the ubiquitin- system, get MHC class I expression. However, the data presented here and MHC class I antigen processing. Immunol. Rev. 207: 19–30. demonstrate that repression of MHC class I surface expression by 4. Wright, K. L., L. C. White, A. Kelly, S. Beck, J. Trowsdale, and J. P. Ting. 1995. PRDM1 is not dependent on transcriptional inhibition of MHC Coordinate regulation of the human TAP1 and LMP2 genes from a shared bidi- rectional promoter. J. Exp. Med. 181: 1459–1471. class I structural genes. 5. White, L. C., K. L. Wright, N. J. Felix, H. Ruffner, L. F. Reis, R. Pine, and The evolutionary conservation of the PRDM1 protein is striking J. P. Ting. 1996. Regulation of LMP2 and TAP1 genes by IRF-1 explains the paucity of CD8ϩ T cells in IRF-1Ϫ/Ϫ mice. Immunity 5: 365–376. and centered in the PR- and DNA-binding domains (15). We dem- 6. Ortmann, B., J. Copeman, P. J. Lehner, B. Sadasivan, J. A. Herberg, onstrate that Prdm1 orthologs from both lower vertebrates and in- A. G. Grandea, S. R. Riddell, R. Tampe, T. Spies, J. Trowsdale, and P. Cresswell. vertebrates have the ability to effectively substitute for human 1997. A critical role for tapasin in the assembly and function of multimeric MHC ␥ class I-TAP complexes. Science 277: 1306–1309. PRDM1 in mediating repression of IFN- -dependent promoter in- 7. Elliott, T., and A. Williams. 2005. The optimization of peptide cargo bound to duction. Moreover, the strict dependence on the precise sequence MHC class I molecules by the peptide-loading complex. Immunol. Rev. 207: of the IRF-E/PRDM1 sites at position 5 and 11 is conserved. The 89–99. 8. Serwold, T., F. Gonzalez, J. Kim, R. Jacob, and N. Shastri. 2002. ERAAP cus- sea urchin and zebrafish Prdm1 orthologs are derived from species tomizes peptides for MHC class I molecules in the endoplasmic reticulum. Na- that diverged before, and soon after, the evolutionary acquisition ture 419: 480–483. 9. Saric, T., S. C. Chang, A. Hattori, I. A. York, S. Markant, K. L. Rock, of the adaptive immune system (1). Our results indicate that the M. Tsujimoto, and A. L. Goldberg. 2002. An IFN-␥-induced aminopeptidase in PRDM1 DNA-binding specificity was fixed before acquisition of the ER, ERAP1, trims precursors to MHC class I-presented peptides. Nat. Im- the adaptive immune system. The presence of an IRF-1/IRF-2 or- munol. 3: 1169–1176. 10. Hammer, G. E., F. Gonzalez, E. James, H. Nolla, and N. Shastri. 2007. In the tholog in the sea urchin immune system (47) suggests that an an- absence of aminopeptidase ERAAP, MHC class I molecules present many un- tagonistic module of PRDM1 and IRF transcription factors was stable and highly immunogenic peptides. Nat. Immunol. 8: 101–108. pre-existent and co-opted as a functional unit into the adaptive/ 11. Gleimer, M., and P. Parham. 2003. Stress management: MHC class I and class I-like molecules as reporters of cellular stress. Immunity 19: 469–477. anticipatory immune system. 12. Keller, A. D., and T. Maniatis. 1991. Identification and characterization of a The wider role for PRDM1-dependent regulation of Ag presen- novel repressor of ␤-interferon . Genes Dev. 5: 868–879. 13. Turner, C. A., Jr., D. H. Mack, and M. M. Davis. 1994. Blimp-1, a novel zinc tation remains to be determined. However, we propose a model in finger-containing protein that can drive the maturation of B lymphocytes into which PRDM1 provides a mechanism for controlling Ag presen- immunoglobulin-secreting cells. Cell 77: 297–306. The Journal of Immunology 7623

14. Tunyaplin, C., M. A. Shapiro, and K. L. Calame. 2000. Characterization of the B relationship to the placental leucine aminopeptidase/oxytocinase gene. J. Bio- lymphocyte-induced maturation protein-1 (Blimp-1) gene, mRNA isoforms and chem. 130: 235–241. basal promoter. Nucleic Acids Res. 28: 4846–4855. 42. Fujii, Y., T. Shimizu, M. Kusumoto, Y. Kyogoku, T. Taniguchi, and 15. Livi, C. B., and E. H. Davidson. 2006. Expression and function of blimp1/krox, T. Hakoshima. 1999. Crystal structure of an IRF-DNA complex reveals novel an alternatively transcribed regulatory gene of the sea urchin endomesoderm DNA recognition and cooperative binding to a tandem repeat of core sequences. network. Dev. Biol. 293: 513–525. EMBO J. 18: 5028–5041. 16. Ren, B., K. J. Chee, T. H. Kim, and T. Maniatis. 1999. PRDI-BF1/Blimp-1 43. Baxendale, S., C. Davison, C. Muxworthy, C. Wolff, P. W. Ingham, and S. Roy. repression is mediated by corepressors of the Groucho family of proteins. Genes 2004. The B-cell maturation factor Blimp-1 specifies vertebrate slow-twitch mus- Dev. 13: 125–137. cle fiber identity in response to Hedgehog signaling. Nat. Genet. 36: 88–93. 17. Yu, J., C. Angelin-Duclos, J. Greenwood, J. Liao, and K. Calame. 2000. Tran- 44. Vincent, S. D., N. R. Dunn, R. Sciammas, M. Shapiro-Shalef, M. M. Davis, scriptional repression by blimp-1 (PRDI-BF1) involves recruitment of histone K. Calame, E. K. Bikoff, and E. J. Robertson. 2005. The zinc finger transcrip- deacetylase. Mol. Cell. Biol. 20: 2592–2603. tional repressor Blimp1/Prdm1 is dispensable for early axis formation but is 18. Gyory, I., J. Wu, G. Fejer, E. Seto, and K. L. Wright. 2004. PRDI-BF1 recruits required for specification of primordial germ cells in the mouse. Development the histone H3 methyltransferase G9a in transcriptional silencing. Nat. Immunol. 132: 1315–1325. 5: 299–308. 45. Ohinata, Y., B. Payer, D. O’Carroll, K. Ancelin, Y. Ono, M. Sano, S. C. Barton, ␥ 19. Tooze, R. M., S. Stephenson, and G. M. Doody. 2006. Repression of IFN- T. Obukhanych, M. Nussenzweig, A. Tarakhovsky, et al. 2005. Blimp1 is a induction of class II transactivator: a role for PRDM1/Blimp-1 in regulation of critical determinant of the germ cell lineage in mice. Nature 436: 207–213. cytokine signaling. J. Immunol. 177: 4584–4593. 46. Ng, T., F. Yu, and S. Roy. 2006. A homologue of the vertebrate SET domain and 20. Ancelin, K., U. C. Lange, P. Hajkova, R. Schneider, A. J. Bannister, zinc finger protein Blimp-1 regulates terminal differentiation of the tracheal sys- T. Kouzarides, and M. A. Surani. 2006. Blimp1 associates with Prmt5 and directs tem in the Drosophila embryo. Dev. Genes Evol. 216: 243–252. histone arginine methylation in mouse germ cells. Nat. Cell. Biol. 8: 623–630. 47. Hibino, T., M. Loza-Coll, C. Messier, A. J. Majeske, A. H. Cohen, 21. Shapiro-Shelef, M., K. I. Lin, L. J. McHeyzer-Williams, J. Liao, D. P. Terwilliger, K. M. Buckley, V. Brockton, S. V. Nair, K. Berney, et al. 2006. M. G. McHeyzer-Williams, and K. Calame. 2003. Blimp-1 is required for the The immune gene repertoire encoded in the purple sea urchin genome. Dev. Biol. formation of immunoglobulin secreting plasma cells and pre-plasma memory B 300: 349–365. cells. Immunity 19: 607–620.

48. de Souza, F. S., V. Gawantka, A. P. Gomez, H. Delius, S. L. Ang, and C. Niehrs. Downloaded from 22. Shapiro-Shelef, M., and K. Calame. 2005. Regulation of plasma-cell develop- 1999. The zinc finger gene Xblimp1 controls anterior endomesodermal cell fate ment. Nat. Rev. Immunol. 5: 230–242. in Spemann’s organizer. EMBO J. 18: 6062–6072. 23. Martins, G. A., L. Cimmino, M. Shapiro-Shelef, M. Szabolcs, A. Herron, E. Magnusdottir, and K. Calame. 2006. Transcriptional repressor Blimp-1 regu- 49. Perry, M., and A. Whyte. 1998. Immunology of the tonsils. Immunol. Today 19: lates T cell homeostasis and function. Nat. Immunol. 7: 457–465. 414–421. 24. Kallies, A., E. D. Hawkins, G. T. Belz, D. Metcalf, M. Hommel, L. M. Corcoran, 50. Frankel, S. S., K. Tenner-Racz, P. Racz, B. M. Wenig, C. H. Hansen, D. Heffner, P. D. Hodgkin, and S. L. Nutt. 2006. Transcriptional repressor Blimp-1 is essen- A. M. Nelson, M. Pope, and R. M. Steinman. 1997. Active replication of HIV-1 tial for T cell homeostasis and self-tolerance. Nat. Immunol. 7: 466–474. at the lymphoepithelial surface of the tonsil. Am. J. Pathol. 151: 89–96. 51. Pegtel, D. M., J. Middeldorp, and D. A. Thorley-Lawson. 2004. Epstein-Barr 25. Chang, D. H., C. Angelin-Duclos, and K. Calame. 2000. BLIMP-1: trigger for http://www.jimmunol.org/ differentiation of myeloid lineage. Nat. Immunol. 1: 169–176. virus infection in ex vivo tonsil epithelial cell cultures of asymptomatic carriers. 26. Horsley, V., D. O’Carroll, R. Tooze, Y. Ohinata, M. Saitou, T. Obukhanych, J. Virol. 78: 12613–12624. M. Nussenzweig, A. Tarakhovsky, and E. Fuchs. 2006. Blimp1 defines a pro- 52. Angelin-Duclos, C., G. Cattoretti, K. I. Lin, and K. Calame. 2000. Commitment genitor population that governs cellular input to the sebaceous gland. Cell 126: of B lymphocytes to a plasma cell fate is associated with Blimp-1 expression in 597–609. vivo. J. Immunol. 165: 5462–5471. 27. Keller, A. D., and T. Maniatis. 1991. Selection of sequences recognized by a 53. van den Elsen, P. J., T. M. Holling, H. F. Kuipers, and N. van der Stoep. 2004. DNA binding protein using a preparative southwestern blot. Nucleic Acids Res. Transcriptional regulation of antigen presentation. Curr. Opin. Immunol. 16: 19: 4675–4680. 67–75. 28. Kuo, T. C., and K. L. Calame. 2004. B lymphocyte-induced maturation protein 54. Groettrup, M., S. Standera, R. Stohwasser, and P. M. Kloetzel. 1997. The sub- (Blimp)-1, IFN regulatory factor (IRF)-1, and IRF-2 can bind to the same regu- units MECL-1 and LMP2 are mutually required for incorporation into the 20S latory sites. J. Immunol. 173: 5556–5563. proteasome. Proc. Natl. Acad. Sci. USA 94: 8970–8975.

29. Bert, A. G., J. Burrows, C. S. Osborne, and P. N. Cockerill. 2000. Generation of 55. Griffin, T. A., D. Nandi, M. Cruz, H. J. Fehling, L. V. Kaer, J. J. Monaco, and by guest on September 26, 2021 an improved luciferase reporter gene plasmid that employs a novel mechanism R. A. Colbert. 1998. Immunoproteasome assembly: cooperative incorporation of for high-copy replication. Plasmid 44: 173–182. interferon ␥ (IFN-␥)-inducible subunits. J. Exp. Med. 187: 97–104. 30. Herberg, J. A., J. Sgouros, T. Jones, J. Copeman, S. J. Humphray, D. Sheer, 56. Garbi, N., P. Tan, A. D. Diehl, B. J. Chambers, H. G. Ljunggren, F. Momburg, P. Cresswell, S. Beck, and J. Trowsdale. 1998. Genomic analysis of the Tapasin and G. J. Hammerling. 2000. Impaired immune responses and altered peptide gene, located close to the TAP loci in the MHC. Eur. J. Immunol. 28: 459–467. repertoire in tapasin-deficient mice. Nat. Immunol. 1: 234–238. 31. Herrmann, F., J. Trowsdale, C. Huber, and B. Seliger. 2003. Cloning and func- 57. Williams, A. P., C. A. Peh, A. W. Purcell, J. McCluskey, and T. Elliott. 2002. tional analyses of the mouse tapasin promoter. Immunogenetics 55: 379–388. Optimization of the MHC class I peptide cargo is dependent on tapasin. Immunity 32. Doody, G. M., S. Stephenson, and R. M. Tooze. 2006. BLIMP-1 is a target of 16: 509–520. cellular stress and downstream of the unfolded protein response. Eur. J. Immunol. 58. York, I. A., S. C. Chang, T. Saric, J. A. Keys, J. M. Favreau, A. L. Goldberg, and 36: 1572–1582. K. L. Rock. 2002. The ER aminopeptidase ERAP1 enhances or limits antigen 33. Schreiber, E., P. Matthias, M. M. Muller, and W. Schaffner. 1989. Rapid detec- presentation by trimming epitopes to 8–9 residues. Nat. Immunol. 3: 1177–1184. tion of octamer binding proteins with “mini-extracts”, prepared from a small 59. Hammer, G. E., F. Gonzalez, M. Champsaur, D. Cado, and N. Shastri. 2006. The number of cells. Nucleic Acids Res. 17: 6419. aminopeptidase ERAAP shapes the peptide repertoire displayed by major histo- 34. Johnson, D. R. 2000. Differential expression of human major histocompatibility compatibility complex class I molecules. Nat. Immunol. 7: 103–112. class I loci: HLA-A, -B, and -C. Hum. Immunol. 61: 389–396. 60. York, I. A., M. A. Brehm, S. Zendzian, C. F. Towne, and K. L. Rock. 2006. 35. Cattoretti, G., C. Angelin-Duclos, R. Shaknovich, H. Zhou, D. Wang, and Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims MHC class I-presented B. Alobeid. 2005. PRDM1/Blimp-1 is expressed in human B-lymphocytes com- peptides in vivo and plays an important role in immunodominance. Proc. Natl. mitted to the plasma cell lineage. J. Pathol. 206: 76–86. Acad. Sci. USA 103: 9202–9207. 36. Hayashi, M., T. Ishibashi, K. Tanaka, and M. Kasahara. 1997. The mouse genes 61. Gobin, S. J., M. van Zutphen, A. M. Woltman, and P. J. van den Elsen. 1999. encoding the third pair of ␤-type proteasome subunits regulated reciprocally by Transactivation of classical and nonclassical HLA class I genes through the IFN- IFN-␥: structural comparison, chromosomal localization, and analysis of the pro- stimulated response element. J. Immunol. 163: 1428–1434. moter. J. Immunol. 159: 2760–2770. 37. Foss, G. S., and H. Prydz. 1999. Interferon regulatory factor 1 mediates the 62. Martin, B. K., K. C. Chin, J. C. Olsen, C. A. Skinner, A. Dey, K. Ozato, and interferon-␥ induction of the human immunoproteasome subunit multicatalytic J. P. Ting. 1997. Induction of MHC class I expression by the MHC class II endopeptidase complex-like 1. J. Biol. Chem. 274: 35196–35202. transactivator CIITA. Immunity 6: 591–600. ␣ 38. Namiki, S., T. Nakamura, S. Oshima, M. Yamazaki, Y. Sekine, K. Tsuchiya, 63. Gobin, S. J., A. Peijnenburg, V. Keijsers, and P. J. van den Elsen. 1997. Site ␥ R. Okamoto, T. Kanai, and M. Watanabe. 2005. IRF-1 mediates upregulation of is crucial for two routes of IFN -induced MHC class I transactivation: the ISRE- LMP7 by IFN-␥ and concerted expression of immunosubunits of the proteasome. mediated route and a novel pathway involving CIITA. Immunity 6: 601–611. FEBS Lett. 579: 2781–2787. 64. Turner, M. J., D. P. Sowders, M. L. DeLay, R. Mohapatra, S. Bai, J. A. Smith, 39. Kohda, K., T. Ishibashi, N. Shimbara, K. Tanaka, Y. Matsuda, and M. Kasahara. J. R. Brandewie, J. D. Taurog, and R. A. Colbert. 2005. HLA-B27 misfolding in 1998. Characterization of the mouse PA28 activator complex gene family: com- transgenic rats is associated with activation of the unfolded protein response. plete organizations of the three member genes and a physical map of the approx- J. Immunol. 175: 2438–2448. imately 150-kb region containing the ␣- and ␤-subunit genes. J. Immunol. 160: 65. Schwab, S. R., J. A. Shugart, T. Horng, S. Malarkannan, and N. Shastri. 2004. 4923–4935. Unanticipated antigens: translation initiation at CUG with leucine. PLoS Biol. 40. Arons, E., V. Kunin, C. Schechter, and R. Ehrlich. 2001. Organization and func- 2: e366. tional analysis of the mouse transporter associated with antigen processing 2 66. Yewdell, J. W., and C. V. Nicchitta. 2006. The DRiP hypothesis decennial: sup- promoter. J. Immunol. 166: 3942–3951. port, controversy, refinement and extension. Trends Immunol. 27: 368–373. 41. Hattori, A., K. Matsumoto, S. Mizutani, and M. Tsujimoto. 2001. Genomic or- 67. Doyle, H. A., and M. J. Mamula. 2001. Post-translational protein modifications ganization of the human adipocyte-derived leucine aminopeptidase gene and its in antigen recognition and autoimmunity. Trends Immunol. 22: 443–449.