Increased Apoptosis after Autoimmune Regulator Expression in

Mar 11, 2010 - Aire knockout mice develop various autoimmune disorders affecting different organs, indicating ...... Journal of Autoimmunity (2005), 2...
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Increased Apoptosis after Autoimmune Regulator Expression in Epithelial Cells Revealed by a Combined Quantitative Proteomics Approach Nuria Colome´,‡ Javier Collado,# Joan J. Bech-Serra,‡ Ingrid Liiv,† Luis C. Anto ´ n,§ † ‡ # Pa¨rt Peterson, Francesc Canals, Dolores Jaraquemada, and In ˜ aki Alvarez*,# Immunology Unit and Institute of Biotechnology and Biomedicine, Autonomous University of Barcelona, Bellaterra, 08193 Barcelona, Spain, Proteomics Laboratory. Research Institute Foundation and Vall d’Hebron Institute of Oncology (VHIO). Vall d’Hebron University Hospital, Barcelona, Spain, Molecular Pathology, IGMP, Biomedicum, Ravila 19, University of Tartu, 50414 Tartu, Estonia, and CBMSO (Centro de Biologı´a Molecular ‘Severo Ochoa’), Consejo Superior de Investigaciones Cientı´ficas/Universidad Auto´noma de Madrid, Cantoblanco, 28049 Madrid, Spain Received January 18, 2010

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare autosomal recessive autoimmune disease, affecting many endocrine tissues. APECED is associated to the lack of function of a single gene called AutoImmune REgulator (AIRE). Aire knockout mice develop various autoimmune disorders affecting different organs, indicating that Aire is a key gene in the control of organ-specific autoimmune diseases. AIRE is mainly expressed by medullary thymic epithelial cells (mTECs), and its absence results in the loss of tolerance against tissue restricted antigens (TRAs). Aire induces the transcription of genes encoding for TRAs in mTECs. In this report, the analysis of AIRE’s effect on the cellular proteome was approached by the combination of two quantitative proteomics techniques, 2D-DIGE and ICPL, using an AIRE-transfected and nontransfected epithelial cell line. The results showed increased levels of several chaperones, (HSC70, HSP27 and tubulin-specific chaperone A) in AIRE-expressing cells, while various cytoskeleton interacting proteins, that is, transgelin, caldesmon, tropomyosin alpha-1 chain, myosin regulatory light polypeptide 9, and myosin-9, were decreased. Furthermore, some apoptosis-related proteins were differentially expressed. Data were confirmed by Western blot and flow cytometry analysis. Apoptosis assays with annexin V and etoposide demonstrated that AIRE-positive cells suffer more spontaneous apoptosis and are less resistant to apoptosis induction. Keywords: AIRE • apoptosis • autoimmunity • mass spectrometry • 2D-DIGE • ICPL

Introduction Autoimmune polyendocrine syndrome type 1 (APS1; OMIM 240300), also called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), is a rare autosomal recessive autoimmune disease affecting endocrine tissues, with chronic candidiasis and other ectodermal disorders.1,2 APECED is characterized by at least 2 of the following 3 clinical features: hypoparathyroidism, candidiasis, and adrenal insufficiency. The global prevalence of APECED is low, being more frequent in populations such as Iranian Jews (1:9000),3 Sardinians (1: 14 400),4 and Finns (1:25 000).1,2,5 Autoimmune diseases are * To whom correspondence should be addressed. In ˜ aki Alvarez, Ph.D., Institut de Biotecnologia i Biomedicina Vicent Villar Palası´, Universitat Auto`noma de Barcelona, 08193, Bellaterra, Barcelona, Spain. Phone: 34-93581 2409. Fax: 34-93-581 2011. E-mail: [email protected]. ‡ Vall d’Hebron University Hospital. # Autonomous University of Barcelona. † University of Tartu. § Consejo Superior de Investigaciones Cientı´ficas/Universidad Auto´noma de Madrid.

2600 Journal of Proteome Research 2010, 9, 2600–2609 Published on Web 03/11/2010

usually complex syndromes involving various genetic and environmental factors which result in the loss of tolerance against self-antigens. However, APECED is associated with the lack of function of a single gene identified by positional cloning in 1997, the AutoImmune REgulator (AIRE).6,7 The gene is mapped to chromosome region 21q22.3, and encodes a 55 kDa protein. Many efforts have been made in the past decade to elucidate AIRE’s structure and function and its role in autoimmunity.8,9 AIRE contains several structural domains found in some transcriptional regulators and chromatin-binding proteins, including a caspase-recruitment domain (CARD), also referred as a homogeneously staining region (HSR), a nuclear-localization signal (NLS), a SAND (Sp100, AIRE, NucP41/75 and DEAF1) domain, two plant homeodomains (PHD), a proline-rich region (PRR), and four LXXLL sequences. CARD domains are related to the dimerization of different proteins involved in apoptosis or inflammation,10 and could be the domain responsible of AIRE oligomerization.11,12 SAND domains are present in different transcriptional modifiers that associate to DNA, 10.1021/pr100044d

 2010 American Chemical Society

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AIRE-Induced Changes in the Cellular Proteome although the specificity of AIRE’s SAND domain remains uncertain. PHD-containing molecules compose a family of zinc-finger proteins that includes DNA-binding proteins. PHD domains are also involved in protein-protein interactions13 and in interactions with methylated or unmethylated histones.14,15 AIRE’s PHD1 interacts with unmethylated histone H3K4.16 LXXLL are protein-protein interaction motifs and can act as nuclear-receptor-binding motifs. The AIRE’s LXXLL-interactingproteins remain to be identified. So far, over 60 different APECED-related mutations along the AIRE sequence are known worldwide, most of them clustering in the CARD/HSR and PHD1 domains. AIRE is expressed in lymphoid tissues, mostly in the thymus, although it has been reported to be also expressed in lymph nodes, spleen and fetal liver.17 The thymus is the primary lymphoid organ where thymocytes mature and the immunocompetent T cell repertoire is generated. To be efficient against pathogens and to avoid self-reactivity, the set of mature T cells that exit the thymus must show a high number of different specificities and not react with self-proteins. The generation of this exquisite repertoire requires that thymocytes go through extensive selection processes, which includes positive selection mainly involving interaction with cortical thymic epithelial cells (cTECs), and negative selection, involving medullary thymic epithelial cells (mTECs) and dendritic cells (DCs). In the thymus, AIRE is specifically expressed by mTECs and at lower levels by DCs. To achieve an efficient central tolerance, peptides displayed by presenting cells in the thymus should be a representation of the proteome that T cells will see in the periphery, including tissue-restricted antigens (TRAs). For many years, thymic expression of TRAs remained unclear, and tolerance to TRAs was assigned to peripheral tolerance mechanisms. However, in the last years, TRA transcription has been fully demonstrated in the thymus. The role of Aire in central tolerance and prevention of autoimmunity has been demonstrated in animal models.18-22 Specifically, the lack of Aire’s function in knockout (KO) mice results in the presence of self-reactive T cells to different TRAs in periphery, which escape from negative selection in the thymus. Experiments with RNA arrays comparing gene expression in mTECs from KO versus wild-type mice have demonstrated that Aire promotes the so-called “promiscuous gene expression” of different TRAs in these cells,23,24 indicating that AIRE regulates the expression of many TRAs. It can be considered that mTECs mirror the peripheral gene expression, as shown by studies using different animal models23,25 and human tissue.26 Recent reports show that Aire affects gene transcription in other cell types, modifying the expression of a set of genes partially overlapping those expressed by mTECs.27 The role of AIRE as a transcriptional regulator is well accepted, but the mechanisms involved in its function remain unknown. In addition, other functions have been proposed for AIRE. It has been shown that AIRE interacts with DNA-PK, Ku70 and Ku80, all involved in the DNA repair machinery.8 An in vitro E3 ubiquitin ligase activity for the PHD1 domain was also reported,28 but was not confirmed when the structure of this domain was resolved by nuclear magnetic resonance (NMR).29 It has also been described that Aire induces apoptosis of mTECs,30 which could favor cross-presentation of TRAs by DCs after phagocytosis of apoptotic AIRE+ cells. During apoptosis, specific biochemical events and morphological changes occur

that involve cytoskeleton rearrangement and overexpression of stress chaperones. The transcriptional effect of AIRE on gene expression can happen in different tissues and cells.31 So far, no attempt has been made to study how the AIRE-mediated transcriptional modifications affect the composition of cellular proteomes. To this end, we transfected the thyroid epithelial cell line HT93 with the human AIRE gene and compared the proteomes from AIRE-positive and negative cells. The analysis was performed using a conservative strategy combining two quantitative proteomic techniques: 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) and Isotope Coded Protein Label (ICPL). The expression of AIRE by HT93 cells resulted in changes of the relative abundance of some proteins. Thus, an increase of the level of several cellular chaperones was observed in AIREexpressing cells, together with a decrease of some cytoskeletoninteracting proteins. In addition, the amount of some apoptosisrelated proteins was modified. This was confirmed by apoptosis assays. Thus, the data were compatible with the reported role for AIRE as an inducer of apoptosis.30

Experimental Procedures Cell Lines and Antibodies. HT93 is an epithelial cell line of thyroid origin transformed by SV40 infection.32 SK-Hep-1 is a hepatocellular carcinoma-derived cell line33 obtained from the American Type Culture Collection (ATCC). Cells were grown in Dulbecco’s Modified Eagle’s Medium (D-MEM) (SigmaAldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA) and 200 mM Lglutamine (Invitrogen), in 75 or 175 cm2 flask cultures (Nunc A/S, Roskilde, Denmark) at 37 °C and 5% CO2. The following primary antibodies were used: anti-AIRE mAb 6.1,17 anti-caldesmon mAb (Chemicon, Hampshire, U.K.), antiHSP70 mAb (BD Biosciences, Palo Alto, CA). Secondary antibodies were Alexa 488-labeled goat anti-mouse IgG (Invitrogen) and HRP-labeled goat anti-mouse IgG (GE Healthcare, Uppsala, Sweden). Transfection of HT93 and SK-Hep-1. AIRE gene was cloned into pcDNA3.1 (Invitrogen). Cells were transfected as previously described, with some modifications.34 About 5 × 106 cells were trypsinized and washed twice in PBS. Cells were resuspended in 800 µL of PBS, and, after addition of 10 µg of DNA, electroporated in a 4 mm gap size cuvette (BTX, Holliston, MA) at 960 µF, 250 V, 24 Ω. Cells were incubated on ice for 10 min and plated in a 75 cm2 flask. Cells were then incubated at 37 °C for 24 h after which 1 mg/mL G418 was added. AIRE expression by G418-resistant cells was tested by flow cytometry and immunofluorescence. A control mock transfectant was obtained with the pcDNA3.1 alone using the same conditions. Flow Cytometry Analysis. About 5 × 105 cells were washed in staining buffer (SB: PBS with 2% FBS and 0.5% Triton X-100), and incubated with the mAb 6.1 (1 µg/mL) in SB at room temperature (R.T.) for 1 h. Cells were washed twice in SB and incubated 1 h with an Alexa 488-labeled goat anti-mouse IgG antibody at R.T. Cells were then washed twice in SB, resuspended in 400 µL of PBS and analyzed in a FACSCalibur flow cytometer (BD Biosciences-Immunocytometry Systems, San Jose, CA). Immunofluorescence. About 5 × 104 cells were plated in 24well plates, on which coverslips had previously been placed, and cultured at 37 °C for 24 h. Cells were washed three times, fixed with 3.7% formaldehyde for 20 min at R.T., washed again three times with PBS, and stained immediately or stored at 4 Journal of Proteome Research • Vol. 9, No. 5, 2010 2601

research articles °C in PBS for further processing. For staining, cells adhered to coverslips were permeabilized in SB for 5 min. Then, cells were incubated with mAb 6.1 (1 µg/mL) in SB at R.T. for 45 min. Coverslips were washed in PBS, incubated in SB for 5 min and then with the secondary antibody in SB for 45 min. Cells were then washed in PBS, mounted on slides using Fluomont (Dako Industries, Carpenteria, CA), and analyzed by epifluorescence microscopy. Cell Pellets. Cells were grown to about 80% confluence and trypsynized, washed three times in 20 mM Tris/HCl, pH 7.4, 150 mM NaCl, and counted. Pellets of 1 × 107 cells were made by direct freezing in liquid nitrogen.35 Dry pellets were maintained in liquid nitrogen until further use. 2D-DIGE. Three samples containing dry pellets of 1 × 107 HT93 or HT93-AIRE cells each were used. Samples were lysed in lysis buffer (30 mM Tris/HCl pH 8.5, 7 M urea, 2 M thiourea, 4% w/v CHAPS), sonicated three times, and cell extracts were centrifuged for 10 min at 13 000g. Supernatant was recovered and proteins quantified (RC DC Protein Assay, Bio-Rad, Hercules, CA). Samples from either control or AIRE-transfected cells were labeled with Cy3 or Cy5 cyanine dyes by the addition of 400 pmol of Cy dye in 1 µL of anhydrous N,N-dimethylformamide per 50 µg of protein. An internal standard control, consisting of a pool of the same total protein amount of every sample, was labeled with Cy2 dye, using the same method. After 30 min incubation on ice in the dark, the reaction was quenched by addition of 1 µL of 10 mM lysine and additionally incubated for 10 min. Samples were finally combined according to the experimental design, at 50 µg of protein per Cy dye per gel, and diluted 2-fold with IEF sample buffer (7 M urea, 2 M thiourea, 4% w/v CHAPS, 2% w/v DTT, 2% v/v pharmalytes pH 3-10). The 2-DE was performed using GE-Healthcare reagents and equipment. First-dimension IEF was performed on IPG strips (24 cm; linear gradient pH 3-10) using an Ettan IPGphor system. Samples were applied via anodic cup loading on the strips previously incubated overnight in 450 µL of rehydration buffer (7 M urea, 2 M thiourea, 4% w/v CHAPS, 1% v/v pharmalytes pH 3-10, 100 mM DeStreak). After focusing at 67 kVh, strips were equilibrated first for 15 min in 6 mL of reducing solution (6 M urea, 100 mM Tris-HCl, pH 8, 30% v/v glycerol, 2% w/v SDS, 5 mg/mL DTT) and then in 6 mL of alkylating solution (6 M urea, 100 mM Tris-HCl, pH 8, 30% v/v glycerol, 2% w/v SDS, 22.5 mg/mL iodoacetamide) for 15 min, on a rocking platform. Second-dimension SDS-PAGE was run by overlaying the strips on 12.5% isocratic Laemmli gels (24.6 × 20 cm), casted in low fluorescence glass plates, on an Ettan DALTsix system. Gels were run at 20 °C, at constant power 2.5 W/gel for 30 min followed by 17 W/gel until the bromophenol blue tracking front reached the end of the gel. Fluorescence images of the gels were acquired on a Typhoon 9400 scanner (GE Healthcare). Cy2, Cy3 and Cy5 images were scanned at 488 nm/520 nm, 532 nm/580, and 633 nm/670 nm excitation/emission wavelengths, respectively, at a 100 µm resolution. Image analysis and statistical quantification of relative protein abundances was performed using DeCyder V. 6.0 software (GE Healthcare). Gels were poststained using the noncovalent fluorescent stain Flamingo (BioRad, Hercules, CA). Fluorescence images were then matched to those of the DIGE analysis. Protein spots of interest were excised from the gel using an automated Spot Picker (GE Healthcare). In-gel trypsin digestion was performed 2602

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Colome´ et al. using autolysis stabilized trypsin (Promega). Tryptic digests were purified using ZipTip microtiter plates (Millipore). ICPL. The three HT93 or HT93-AIRE samples used in DIGE analysis were pooled. Protein reduction, carbamydomethylation and labeling were performed using the Serva ICPL Kit following the manufacturer’s recommendations. About 200 µg of protein was reduced for 30 min at 60 °C in the presence of 0.5 µL of tris (2-carboxyethyl) phosphine solution. Samples were cooled to room temperature, alkylated with 0.5 µL of iodacetamide and incubated for 30 min at 25 °C in the dark. Reactions were stopped by addition of 0.5 µL of N-acetyl-cysteine to each sample and incubated for 15 min at 25 °C. After carbamidomethylation, 3 µL of 12C-Nic-reagent solution (L) was added to 100 µg of HT93 sample and 3 µL of 13C-Nic-reagent solution (H) to the same amount of HT93-AIRE sample. Samples were covered with argon, sonicated for 1 min and the reaction was allowed to proceed at room temperature for 2 h. Then, 2 µL of hydroxylamine was added to each sample and incubated for 20 min. Reverse-labeling was performed by labeling 100 µg of HT93 protein extract with 13C-Nic-reagent solution (H) and 100 µg of HT93-AIRE protein extract with 12C-Nic-reagent solution (L). A pair of differentially labeled HT93 and HT93-AIRE samples was combined and pH was adjusted to 11-12 by adding 2 N NaOH. After 20 min, samples were neutralized with 2 N HCl. Proteins were precipitated with the Clean up Kit (GE, healthcare) and were dissolved in sample loading buffer (50 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 0.1% (w/v) bromophenol blue, 10% (v/v) glycerol, 5% (v/v) β-mercaptoethanol). Proteins were separated by 1D-electrophoresis in a 12.5% polyacrylamide 1D-gel. Each gel lane was cut into 20 horizontal slices and each slice was subjected to tryptic digestion with modified porcine trypsin (Promega, Madison, WI). Protein Identification by MS. Tryptic digests from excised 2D gels spots were analyzed by MALDI-TOF MS on an Ultraflex TOF-TOF Instrument (Bruker, Bremen, Germany). Samples were prepared using HCCA as matrix on anchor-chip targets (Bruker). Calibration was performed in the external mode using a peptide calibration standard kit (Bruker Daltonics). The spectra were processed using Flex Analysis 3.0 software (Bruker Daltonics). Peak lists were generated using the signals in the m/z 800-4000 region, with a signal-to-noise threshold of greater than 3. The SNAP algorithm included in the software was used to select the monoisotopic peaks from the isotopic distributions observed. After removing m/z values corresponding to usually observed matrix cluster ions, an internal statistical calibration was applied. Peaks corresponding to frequently seen keratin and trypsin autolysis peptides were then removed. The resulting final peak list was used for identification of the proteins by peptide mass fingerprint. Mascot 2.2 program (Matrix Science Ltd., London, U.K.) was used to search the Swiss-Prot 57.0 database, limiting the search to human proteins (20 403 sequences). Search parameters were as follows: trypsin cleavages excluding N-terminal to P, 1 or 2 missed cleavages allowed, cysteine carbamidomethylation set as fixed modification, methionine oxidation as variable modification, mass tolerance less than 50 ppm, monoisotopic mass values. Criteria for positive identification were a significant Mascot probability score (score >55, p < 0.05). Alternatively, proteins were identified by ion trap mass spectrometry as described.36 ICPL tryptic digest were analyzed on an Esquire HCT ion trap mass spectrometer (Bruker), coupled to a nanoHPLC system (Ultimate, LcPackings, Netherlands). Sample was first concentrated on a 300 µm i.d. 1 mm PepMap nanotrapping

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AIRE-Induced Changes in the Cellular Proteome column and then loaded onto a 75 µm i.d., 15 cm PepMap nanoseparation column (LC Packings, Netherlands). Peptides were then eluted by an acetonitrile gradient (Gradient: 0-60% B in 120 min, B ) 80% acetonitrile (ACN), 0.1% formic acid in water; flow rate ca. 300 nL/min) through a PicoTip emitter nanospray needle (New Objective, Woburn, MA) onto the nanospray ionization source of the ion-trap mass spectrometer. MS/MS fragmentation (1.9 s, 100-2,800 m/z) was performed on two of the most intense ions, as determined from a 1.2 s MS survey scan (310-1,500 m/z), using a dynamic exclusion time of 1.2 min for precursor selection. An automated optimization of MS/MS fragmentation amplitude, starting from of 0.60 V was used. Data processing for protein identification and quantitation was performed using Protein Scape 2.1 and WARPLC 1.2 (Bruker), a software platform integrating LC-MS run data processing, protein identification through database search of MS/MS spectra and protein quantitation based on the integration of the chromatographic peaks of MS extracted ion chromatograms for each precursor. Proteins were identified using Mascot to search the Swiss-Prot 57.0 database. MS/MS spectra were searched with a precursor mass tolerance of 1.5 Da, fragment tolerance of 0.5 Da, trypsin specificity with a maximum of 1 missed cleavage, cysteine carbamidomethylation set as fixed modification and methionine oxidation and the Nterminal and Lys ICPL labels as variable modifications. Positive identification criterion was set as an individual Mascot score for each peptide MS/MS spectrum higher than the corresponding homology threshold score. False positive rate for Mascot protein identification was measured by searching a randomized

37

decoy database, and estimated to be under 4%. For protein quantitation, HT93-AIRE/HT93 ratios were calculated averaging the measured HT93-AIRE/HT93 ratios for the observed peptides, after discarding outliers. For selected proteins of interest, quantitation data obtained from the automated WARP-LC analysis was manually reviewed. Western Blot Analysis. Western blot experiments were performed as described.38 Samples of 20 µg of protein extracts were subjected to SDS-PAGE on 12% acrylamide gels in Trisglycine-SDS buffer. Electrophoretically separated proteins were subjected to semidry electrophoretic transfer onto nitrocellulose membranes at 0.8 mA/cm2 for 1 h. Membranes were blocked for 30 min in T-PBS (PBS with 0.1% Tween 20) with 5% skimmed milk, and then incubated with the corresponding antibodies for 2 h at R.T. Membranes were washed three times in T-PBS and incubated with a HRP-labeled goat anti-mouse IgG antibody at a 1:5000 dilution for 1 h. Specific proteins were detected by ECL (Biological Industries Israel Beit Haemek Ltd., Ashrat, Israel). Apoptosis Assay. For analysis of apoptotic cells, 2 × 105 of HT93 and HT93-AIRE cells were seeded onto 60 mm dishes and 1.5 × 105 cells were stained with 5 µL of Annexin V-PE and 5 µL of 7-AAD (7-amino actinomycin) markers after 24, 48, and 72 h according to Annexin V-PE Apoptosis Detection kit I (BD Pharmingen). The percentage of early (Annexin V-PE positive) or late (with 7-AAD positive) apoptotic cells was measured by flow cytometry (FACSCalibur, BD Biosciences) and analyzed by cytometry software (FlowJo7). To estimate the effect of induced apoptosis, etoposide (100 nM) was added to

Figure 1. Characterization of the HT93-AIRE transfectant. (A) Flow cytometry analysis of AIRE expression in HT93 (thin line) and HT93AIRE (bold line). Cells were fixed, permeabilized, and stained with the AIRE-specific mAb 6.1 followed by Alexa 488-labeled goat antimouse IgG. (B-F) Immunofluorescence analysis of AIRE expression. After 24 h incubation at 37 °C, cells on coverslips were fixed, permeabilized and incubated with mAb 6.1 followed by Alexa 488-labeled goat anti-mouse IgG. AIRE could not be detected in HT93 (B), and it was located in different structures into cells (C-F). Left panels, DAPI staining; right panels, 6.1 staining. Journal of Proteome Research • Vol. 9, No. 5, 2010 2603

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cell cultures at 43 h and incubated for 5 h before cell harvest. Cells were further analyzed by flow cytometry and necrotic cells were excluded before the analysis by gating.

Results Generation of the HT93-AIRE Transfected Cells. The human cell line HT93 was transfected with the human AIRE cDNA cloned into the pcDNA3.1 vector. Around 70% of cells expressed AIRE as confirmed by intracellular flow cytometry and immunofluorescence microscopy (Figure 1A,C). No detectable AIRE expression was observed (Figure 1A,B) in untransfected HT93 cells. As described, AIRE was predominantly detected in the nucleus, either with a diffuse nuclear expression (Figure 1D) or concentrated in nuclear dots (Figure 1E), although cells showing a filamentous cytoplasmic staining, and both nuclear and cytoplasmic patterns were also seen (Figure 1F).17,39,40 The same distribution was observed in the AIRE-transfected hepatocellular carcinoma cell line, SK-Hep-1 (data not shown). Proteomic Analysis of HT93 and HT93-AIRE Cells. To study the changes induced in the cell proteome by the expression of AIRE, two proteomic techniques were used: 2D-DIGE and ICPL. Samples were first analyzed in a 3-gel 2D-DIGE design. To avoid any possible bias due to protein labeling, protein extracts from HT93 were labeled with Cye5 dye and HT93-AIRE extracts with Cye3 dye in two gels, while in another gel, the HT93 extract was labeled with Cye3 dye and the corresponding HT93-AIRE extract with Cye5 dye. The internal standard, consisting of a pool of the same total protein amount of every sample, was labeled with Cye2 dye. One pair of samples and the internal standard were separated by isoelectrofocusing and SDS-PAGE electrophoresis in each 2D-gel. The fluorescence images obtained from all gels were analyzed, changes of intensity of each spot were studied, and a statistical analysis was carried out. Eighty-five significant changes (p < 0.01, t-test) greater than 1.5-fold in abundance ratio were observed, 43 of them increased and 42 decreased in the AIRE-transfected cells compared with the nontransfected cells (Figure 2A). Figure 2B shows a peak with higher intensity and Figure 2C with lower intensity in the transfected cells extract. The corresponding gel spots were excised and in-gel digested with trypsin. Protein digests were analyzed by MALDI-TOF MS for protein identification. From a total of 57 proteins identified, 23 were increased and 34 decreased in AIRE-expressing cells. Table 1 of Supporting Information includes the list of proteins with a modified expression identified by DIGE. MALDI-TOF spectra, peak lists and Mascot reports obtained are shown in Figure 1 of Supporting Information. The second quantitative proteomic analysis was carried out by LC-MS analysis using ICPL. Part of the same cell extracts used for the DIGE analysis was separately pooled, both for HT93 and HT93-AIRE. Two experiments were performed in order to avoid differences in protein labeling. First, HT93 and HT93-AIRE protein samples were labeled with the light and heavy isotopes, respectively (ICPL1), and the opposite labeling was performed in the second experiment (ICPL2). In each experiment, HT93 and HT93-AIRE isotope-labeled extracts were pooled, proteins were separated in a monodimensional SDSPAGE gel, and 20 slices of each gel were trypsin-digested. The resulting peptides were separated by RP-HPLC, and fragmented in an online connected IonTrap mass spectrometer. In the first ICPL experiment, 1588 different peptides derived from 565 proteins were sequenced from HT93/HT93-AIRE (Tables 2 and 3, Supporting Information). From these proteins, 89 were 2604

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Figure 2. 2D-DIGE of HT93 vs HT93-AIRE cells. (A) Silver stained 2D-gel of HT93-AIRE protein extract. The spots differentially expressed between HT93 and HT93-AIRE cells are indicated. (B) Three-dimensional representation of the spot containing the protein increased in AIRE-expressing cells, HSP70 (left, HT93AIRE; right, HT93). Corresponding spots are indicated in the 2Dgel. (C) Three-dimensional representation of the spot containing the protein decreased in AIRE-expressing cells, caldesmon (left, HT93-AIRE; right, HT93). Corresponding spots are indicated in the 2D-gel.

increased and 60 were decreased more than 1.5 times in HT93AIRE. In the second ICPL experiment, 1565 peptides, from 606 proteins, were sequenced from HT93/HT93-AIRE (Tables 4 and 5, Supporting Information). From these, 41 were increased and 57 decreased in HT93-AIRE. Identification of Differentially Expressed Proteins. A conservative approach was followed to select those proteins that were differentially expressed in AIRE-positive versus AIREnegative cells and to eliminate false differences. The proteins selected were those with an abundance ratio of more than 1.5 in at least two of the three experiments (DIGE, ICPL1 and ICPL2) without contradictory data in the third experiment. Following this approach, 27 proteins showed a modified expression: 9 were increased and 18 decreased in HT93-AIRE cells (Table 1). Assignments of some of these proteins from each ICPL experiment were done on the basis of only one spectrum. These MS/MS single spectra are shown in Figure 2 of Supporting Information. Some chaperones were detected, including HSP27, HSC70, and tubulin-specific chaperone A among the proteins increased in AIRE-expressing cells. Other proteins were the transitional endoplasmic reticulum ATPase, superoxide dismutase, Ufm1conjugating enzyme 1, programmed cell death protein 5, RNA-

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AIRE-Induced Changes in the Cellular Proteome a

Table 1. Proteins with Differential Expression in HT93 and HT93-AIRE Cells HT93-AIRE/HT93 AC. number

b

DIGE

ICPL 1

ICPL 2

P49748

0.37

0.28

0.55

Q01995 Q05682 P09493 P24844 P05783 P35579 O00151 P09382 P06744 P46777 Q9NYF8 O00233 P30046 Q9UJZ1 P07741 O75475 P55072 P04792 P00441 P11142 O75347 Q9Y3C8 O14737 Q14498 O75083 Q9H773

0.38 0.57 0.59 0.59 0.62 0.62 1.53 1.60 2.28 -

0.37 0.48 0.62 0.45 0.63 0.60 0.49 0.59 0.39 0.67 0.47 0.66 0.59 0.62 0.54 0.41 0.58 1.93 1.61 1.30 1.51 1.62 1.71 2.25 2.65 2.66

0.45 0.30 0.66 0.86 0.48 0.71 0.36 0.51 0.54 0.55 0.55 0.57 0.59 0.61 0.61 0.64 0.64 1.82 1.94 1.60 1.58 2.88 1.83 1.54 2.23 2.12

protein

Very long-chain specific acyl-CoA dehydrogenase, mitochondrial Transgelin Caldesmon Tropomyosin alpha-1 chain Myosin regulatory light polypeptide 9 Keratin, type I cytoskeletal 18 Myosin-9 PDZ and LIM domain protein 1 Galectin-1 Glucose-6-phosphate isomerase 60S ribosomal protein L5 Bcl-2-associated transcription factor 1 26S proteasome non-ATPase regulatory subunit 9 D-dopachrome decarboxylase Stomatin-like protein 2 Adenine phosphoribosyltransferase PC4 and SFRS1-interacting protein Transitional endoplasmic reticulum ATPase Heat shock protein beta-1 Superoxide dismutase [Cu-Zn] Heat shock cognate 71 kDa protein Tubulin-specific chaperone A Ufm1-conjugating enzyme 1 Programmed cell death protein 5 RNA-binding protein 39 WD repeat-containing protein 1 dCTP pyrophosphatase 1

a Only proteins which were increased or decreased 1.5-fold in at least two experiments are included. DIGE and each of the ICPL experiments were considered as individual experiments. b Swiss-Prot accession number.

binding protein 39, WD repeat-containing protein 1 and XTP3transactivated gene A protein. Proteins that were less abundant in AIRE-expressing cells included the mitochondrial very long-chain specific acyl-CoA dehydrogenase, PDZ and LIM domain protein 1, galectin-1, glucose-6-phosphate isomerase, 60S ribosomal protein L5, 26S proteasome non-ATPase regulatory subunit 9, D-dopachrome decarboxylase, adenine phosphoribosyltransferase, PC4 and SFRS1-interacting protein, transitional endoplasmic reticulum ATPase and keratin, type I cytoskeletal 18. The Bcl-2-associated transcription factor 1 was also decreased in AIRE-expressing cells. Interestingly, some proteins involved in the actin-myosin cytoskeleton were decreased in the AIRE positive cells compared to control cells. Thus, transgelin, caldesmon, tropomyosin alpha-1 chain, myosin regulatory light polypeptide 9, myosin-9 and stomatin-like protein 2 were less abundant in AIRE-expressing cells. To identify the pathways affected after AIRE transfection, a search with the Ingenuity software (Ingenuity Pathway Analysis) was done with the genes that encode for the proteins identified in DIGE and ICPL analysis. A network centered in the growth factor receptor-bound protein 2 (GRB2) gene was obtained in which several proteins involved in the action and stabilization of actin filaments were decreased after AIRE expression (Figure 3). Validation of the Proteomic Results. To confirm the results obtained by the 2D-DIGE and ICPL analysis, we studied the abundance of two differentially regulated proteins by Western blot and flow cytometry: caldesmon, a protein with a lower expression in AIRE-positive cells, and HSP70, more abundant

in AIRE-expressing cells as detected by the 2D-DIGE comparison. The different content of these proteins in both cell extracts was confirmed by Western blot (Figure 4A, lanes 1 and 2). Extracts from HT93 transfected with the pcDNA3.1 vector alone were also analyzed to discard that differences in protein expression could be caused by the transfection process, (Figure 4A, lane 3). Furthermore, the data were confirmed using the AIRE-transfected SK-Hep-1 cell, indicating that AIRE expression causes the differences observed in protein abundance (Figure 4A, lanes 4 and 5). The data were further confirmed by flow cytometry (Figure 4B). Increased Apoptosis in HT93-AIRE Cells. To confirm that AIRE positive cells have increased apoptosis, we analyzed HT93 and HT93-AIRE cells for Annexin V-PE and 7-AAD markers to detect early and late apoptosis, respectively, by flow cytometry. Annexin V-PE binds to the phosphatidylserine on cell membranes at early stage, whereas 7-AAD intercalates between DNA double strands upon DNA fragmentation at late stage of apoptotic process. We found that the number of Annexin V-PE and 7-AAD negative cells, indicating early apoptosis, was approximately 2- to 3-fold increased in HT93-AIRE cells when compared to HT93 line (Figure 5A). The enhanced apoptosis in AIRE positive cells was evident at 24 and 48 h time points and started earlier as the difference was slightly less at 72 h, when negative cells started to enter early apoptosis most likely due to prolonged cell culture conditions. To further confirm the increased apoptosis among AIRE positive cells, we studied their sensitivity to genotoxic stress induced by etoposide, a wellknown inhibitor of topoisomerase that causes DNA double stranded breaks.41 Again, we found the increased early apopJournal of Proteome Research • Vol. 9, No. 5, 2010 2605

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Figure 3. Pathway analysis of proteins differentially expressed in HT93-AIRE and HT93 cells analyzed by 2D-DIGE and ICPL. Proteins differentially expressed in HT93 and HT93-AIRE cells were input into Ingenuity pathway analysis and a network with GRB2 and actin in the main nodes was obtained. Information about the analysis of biological functions and pathways as well as network interactions is available at the Ingenuity pathway analysis Web site (Ingenuity Systems 2008). Color shading corresponds to the type of changes, red for increased and green for decreased genes. White open nodes are from proteins outside the lists of proteins identified by 2D-DIGE or ICPL but are associated with the regulation of some of them. A line denotes binding of proteins, whereas a line with an arrow denotes ‘acts on’.

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Figure 4. Validation of proteomic results. (A) Western blot analysis of caldesmon and HSP70. Samples of 20 µg of cell lysates were loaded on a 12% SDS-PAGE and separated by electrophoresis. Proteins were transferred to nitrocellulose membranes, incubated with anti-caldesmon or anti-HSP70 mAbs followed by HRP-labeled goat anti-mouse IgG antibody incubation and revealed by ECL. Lane 1, HT93; lane 2, HT93-AIRE; lane 3, HT93-pcDNA3.1; lane 4, SK-Hep-1; lane 5, SK-Hep-1-AIRE. (B) Flow cytometry analysis of caldesmon and HSP70 expression in HT93 (thin line) and HT93-AIRE (bold line). Cells were fixed, permeabilized, and stained with the caldemon- or HSP-specific mAbs followed by Alexa 488-labeled goat anti-mouse IgG. Negative controls are shown as short-dotted line (HT93) or longdotted line (HT93-AIRE).

tosis in AIRE positive cells using relatively low (100 nM) concentration of etoposide (Figure 5B). Taken together, these experiments show that AIRE enhances apoptotic cell death and are in agreement with increased expression of pro-apoptotic (PCD5) in HT93-AIRE cells.

Discussion This study reports a first analysis of the impact of AIRE expression on the proteome of cultured epithelial cells, by comparing AIRE-transfected and nontransfected HT93 cells with a combination of two quantitative proteomics techniques. AIRE protein is predominantly detected in the nucleus, both in tissue sections and in cultured cells. However, at least in AIRE-expressing transfectants, the protein is also detected in cytoplasmic filaments, which requires the HSR domain.12 The presence of AIRE in filamentous structures has been reported in different cell lines, and in our case, AIRE-positive tubular structures were seen in transfected HT93 and SK-Hep-1 cell lines, confirming previous findings. This extra-nuclear localization strongly suggested cellular functions for AIRE in addition to that established as a transcriptional regulator. The approach used in this study was a combination of two different quantitative proteomic techniques: 2D-DIGE, based in differential fluorescent protein labeling, 2D-gel separation, and fingerprinting identification of proteins by MALDI-TOF MS after trypsin digestion; and ICPL, based in a differential isotope protein labeling, 1D-gel separation and tryptic digestion of gel slices followed by RP-HPLC and LC-MS/MS sequencing with 2606

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Figure 5. Increased apoptosis in AIRE positive cells. (A) A percentage of Annexin V-PE+ 7AAD- cells analyzed by flow cytometry at three time-points (24, 48, and 72 h) from HT93 and HT93-AIRE cultures. (B) Annexin V-PE+ 7AAD- apoptotic cells analyzed from HT93 and HT93-AIRE cultures after treatment with 100 nM etoposide or DMSO for 5 h. Both panels represent data and standard deviation from three independent experiments.

an IonTrap mass spectrometer. Both methods are complementary, and although no biological replicas were analyzed by ICPL, each sample was labeled with both heavy and light isotopes. Therefore, the use of two quantitative methods based on different labeling, protein separation and peptide sequencing techniques, and the reverse labeling in both analyses should minimize false quantitative differences. Furthermore, selected results were confirmed by Western blot and flow cytometry. Criteria to select proteins as differentially expressed were conservative so some information may have been lost. Thus, some proteins related with the actin-myosin cytoskeleton were not included in Table 1 as they did not fulfill all the require-

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AIRE-Induced Changes in the Cellular Proteome ments, although they were detected with lower expression in AIRE-expressing cells in some experiments. The increase of HSP70 in DIGE was only detected by ICPL with an increment of 1.48 (instead of 1.5 required to be considered as differentially expressed), but was confirmed by Western blot and flow cytometry (Figure 4). Despite these data, changes due to AIRE expression were consistently observed. Different chaperones, including HSC70, HSP27 and a tubulin-specific chaperone, were increased in AIRE-expressing cells, whereas proteins related with the actin-myosin cytoskeleton were found among those decreased in the transfectants. Chaperones are stress proteins that can be induced as a response to apoptosis. Specifically, HSP27 and HSP70 exert their functions inhibiting some key effectors of the apoptotic cell machinery.42 It is also known that cytoskeleton rearrangement occurs during apoptosis. Some major cytoskeletal proteins were decreased in the transfected cells. A component of intermediate filaments, cytokeratin 18, that is caspase-cleavaged during apoptosis,43 was also found decreased in the HT93AIRE cell proteome. Finally, differential expression of other apoptosis-related factors was detected, that is, Bcl-2-associated transcription factor 1 and programmed cell death protein 5. These data pointed to an increase of apoptosis and the corresponding survival cell response in AIRE-transfected cells. A role of Aire as an apoptosis inducer was reported in the mouse model. Thus, in mouse mTECs, Aire expressing cells showed postmitotic features and a construct codifying for AireGFP induced apoptosis in a transfected cell line.30 In addition, Aire is expressed in mouse testis, where an early wave of apoptosis occurs during spermatogenesis that is reduced in Aire KO mice.44 This putative role of AIRE as an apoptosis inducer was further demonstrated by annexin V and 7-AAD staining. The CARD domain, that has been related to caspase recruitment and generation of apoptotic signals,45 could be the protein domain of AIRE involved in apoptosis induction, although additional work must be done to confirm it. Recently, a description of AIRE’s interactome has been reported in which four functional groups of AIRE-interacting proteins were identified and a model by which AIRE can exert its function is proposed.46 The proteins that compose AIRE’s inteactome are different from the differentially expressed proteins that we described. The methods and objectives were clearly different. Our aim was to identify proteins whose expression was modified by AIRE’s expression, not the proteins that interacted with AIRE. Some of them could coincide, but the expression of AIRE-interacting proteins should not necessarily change quantitatively after AIRE expression. The protocol to identify AIRE-interacting proteins exclusively used nuclear extracts, whereas we analyzed total cell extracts. This makes the comparison of both sets of proteins very difficult, since most of the differentially expressed proteins were located in the cytosol. DNA-PK and Ku80 were identified as AIRE-interacting proteins by Abramson et al.46 AIRE interacts with DNA-PK, Ku70 and Ku80 proteins, involved in DNA repair machinery, as demonstrated by pull-down experiments.8 By 2D-DIGE, Ku80 was decreased in AIRE-expressing cells. The interaction of AIRE with these proteins may thus affect DNA repair and result in increased apoptosis. Indeed, HT93-AIRE cells were more sensitive than untransfected cells to the genotoxic agent etoposide (Figure 5B). The effect of etoposide is increased in Ku70- or Ku80-deficient cells, which can become resistant after transfection with Ku70 or Ku80 genes.47

AIRE-induced stress response and cell death could be a result of the simultaneous transcription of a high number of genes, including TRAs. Our analysis did not allow the detection of TRAs after AIRE expression. There are several explanations to this. First, AIRE may need other cell lineage-specific factors to control TRA gene expression. Thus, in the pancreas, Aire controls the expression of a set of genes that overlaps but is different to those controlled in the thymus.27 On the other hand, TRA expression by mTECs is very low, so the presence of highly expressed proteins could interfere with the detection of low-abundance proteins. A high number of genes are upregulated in mTECs from wild-type in comparison with KO mice. If the ectopic expression of these genes is produced simultaneously in the same cell,48 the expression should be low to allow cell viability. In addition, AIRE can induce gene transcription but complete protein translation may not occur, and incomplete proteins or defective ribosomal products (DRiPs) could be degraded by the proteasome.49 Unfolded or incomplete proteins are very unstable in the cytosol and are rapidly degraded by the proteasome or other proteolytic systems. Finally, both 2D-DIGE and ICPL require one SDSPAGE step, so all the peptides from which a sequence or relative mass have been obtained in this work must have been derived from a polypeptide of large enough size to be retained in the gel.

Conclusions This is the first proteomic analysis studying how AIRE expression influences the composition of human epithelial cell proteome. Results showed a protein profile that indicated a higher level of apoptosis in AIRE-expressing cells. This agrees with previous data obtained by other techniques in mouse thymic epithelial cells. Thus, the combined proteomics approach used here was useful to reveal that AIRE can play roles in the control of autoimmunity different from that of transcriptional regulation. Abbreviations: APS-1, autoimmune polyendocrine syndrome type 1; APECED, autoimmune polyendocrinopathy-candidiasisectodermal dystrophy; AIRE, autoimmune regulator; CARD, caspase-recruitment domain; HSR, homogeneously staining region; NLS, nuclear-localization signal; SAND, Sp100, AIRE, NucP 41/75 and DEAF-1; PHD, plant homeodomain; PRR, proline-rich region; cTECs, cortical thymic epithelial cells; mTECs, medullary thymic epithelial cells; DCs, dendritic cells; TRAs, tissue-restricted antigens; 2D-DIGE, 2-D fluorescence difference gel electrophoresis; ICPL, isotope coded protein label; ATCC, American Type Culture Collection; D-MEM, Dulbecco’s Modified Eagle’s Medium; FBS, fetal bovine serum; AAD, 7-amino actinomycin.

Acknowledgment. This study was supported by the Universitat Auto`noma de Barcelona (UAB) Grant EME2006-26, to I.A., and the Eurothymaide CE Intregrated Project LSHB-CT-2003-503410 and the Spanish Ministry of Education Grant SAF2006-08928, to D.J. The authors thank Manuela Costa of the Flow Cytometry Service of the UAB and Dr. Martti Laan and Dr. Kai Kisand from Biomedicum, University of Tartu for their help with flow cytometry analysis. Thanks also to Dr. Carme Roura for critical reading of the manuscript. The Proteomics Laboratory of Hospital Vall d’Hebron is a member of the Spanish National Institute for Proteomics (PROTEORED) funded by Fundacio´n Genoma Espan ˜ a. Journal of Proteome Research • Vol. 9, No. 5, 2010 2607

research articles Supporting Information Available: List of proteins with a modified expression identified by DIGE. MALDI-TOF spectra, peak lists and Mascot reports obtained in the DIGE analysis. List of peptides identified in ICPL1. List of proteins identified in ICPL2. List of peptides identified in ICPL2. List of proteins identified in ICPL2. Annotated MS/MS single spectra. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Peterson, P.; Peltonen, L. Autoimmune polyendocrinopathy syndrome type 1 (APS1) and AIRE gene: new views on molecular basis of autoimmunity. J. Autoimmun. 2005, 25 Suppl., 49–55. (2) Vogel, A.; Strassburg, C. P.; Obermayer-Straub, P.; Brabant, G.; Manns, M. P. The genetic background of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy and its autoimmune disease components. J. Mol. Med. 2002, 80 (4), 201–11. (3) Zlotogora, J.; Shapiro, M. S. Polyglandular autoimmune syndrome type I among Iranian Jews. J. Med. Genet. 1992, 29 (11), 824–6. (4) Rosatelli, M. C.; Meloni, A.; Devoto, M.; Cao, A.; Scott, H. S.; Peterson, P.; Heino, M.; Krohn, K. J.; Nagamine, K.; Kudoh, J.; Shimizu, N.; Antonarakis, S. E. A common mutation in Sardinian autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients. Hum. Genet. 1998, 103 (4), 428–34. (5) Aaltonen, J.; Bjorses, P.; Sandkuijl, L.; Perheentupa, J.; Peltonen, L. An autosomal locus causing autoimmune disease: autoimmune polyglandular disease type I assigned to chromosome 21. Nat. Genet. 1994, 8 (1), 83–7. (6) Finnish-German APECED Consortium. An autoimmune disease, APECED, caused by mutations in a novel gene featuring two PHDtype zinc-finger domains. Nat. Genet. 1997, 17 (4), 399–403. (7) Nagamine, K.; Peterson, P.; Scott, H. S.; Kudoh, J.; Minoshima, S.; Heino, M.; Krohn, K. J.; Lalioti, M. D.; Mullis, P. E.; Antonarakis, S. E.; Kawasaki, K.; Asakawa, S.; Ito, F.; Shimizu, N. Positional cloning of the APECED gene. Nat. Genet. 1997, 17 (4), 393–8. (8) Liiv, I.; Rebane, A.; Org, T.; Saare, M.; Maslovskaja, J.; Kisand, K.; Juronen, E.; Valmu, L.; Bottomley, M. J.; Kalkkinen, N.; Peterson, P. DNA-PK contributes to the phosphorylation of AIRE: importance in transcriptional activity. Biochim. Biophys. Acta 2008, 1783 (1), 74–83. (9) Mathis, D.; Benoist, C. Aire. Annu. Rev. Immunol. 2009, 27, 287– 312. (10) Park, H. H.; Lo, Y. C.; Lin, S. C.; Wang, L.; Yang, J. K.; Wu, H. The death domain superfamily in intracellular signaling of apoptosis and inflammation. Annu. Rev. Immunol. 2007, 25, 561–86. (11) Ramsey, C.; Bukrinsky, A.; Peltonen, L. Systematic mutagenesis of the functional domains of AIRE reveals their role in intracellular targeting. Hum. Mol. Genet. 2002, 11 (26), 3299–308. (12) Pitkanen, J.; Vahamurto, P.; Krohn, K.; Peterson, P. Subcellular localization of the autoimmune regulator protein. characterization of nuclear targeting and transcriptional activation domain. J. Biol. Chem. 2001, 276 (22), 19597–602. (13) Bienz, M. The PHD finger, a nuclear protein-interaction domain. Trends Biochem. Sci. 2006, 31 (1), 35–40. (14) Adams-Cioaba, M. A.; Min, J. Structure and function of histone methylation binding proteins. Biochem. Cell Biol. 2009, 87 (1), 93– 105. (15) Mellor, J. It takes a PHD to read the histone code. Cell 2006, 126 (1), 22–4. (16) Org, T.; Chignola, F.; Hetenyi, C.; Gaetani, M.; Rebane, A.; Liiv, I.; Maran, U.; Mollica, L.; Bottomley, M. J.; Musco, G.; Peterson, P. The autoimmune regulator PHD finger binds to non-methylated histone H3K4 to activate gene expression. EMBO Rep. 2008, 9 (4), 370–6. (17) Heino, M.; Peterson, P.; Kudoh, J.; Nagamine, K.; Lagerstedt, A.; Ovod, V.; Ranki, A.; Rantala, I.; Nieminen, M.; Tuukkanen, J.; Scott, H. S.; Antonarakis, S. E.; Shimizu, N.; Krohn, K. Autoimmune regulator is expressed in the cells regulating immune tolerance in thymus medulla. Biochem. Biophys. Res. Commun. 1999, 257 (3), 821–5. (18) Liston, A.; Lesage, S.; Wilson, J.; Peltonen, L.; Goodnow, C. C. Aire regulates negative selection of organ-specific T cells. Nat. Immunol. 2003, 4 (4), 350–4. (19) Gavanescu, I.; Kessler, B.; Ploegh, H.; Benoist, C.; Mathis, D. Loss of Aire-dependent thymic expression of a peripheral tissue antigen renders it a target of autoimmunity. Proc. Natl. Acad. Sci. U.S.A. 2007, 104 (11), 4583–7.

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(45) Hofmann, K.; Bucher, P.; Tschopp, J. The CARD domain: a new apoptotic signalling motif. Trends Biochem. Sci. 1997, 22 (5), 155– 6. (46) Abramson, J.; Giraud, M.; Benoist, C.; Mathis, D., Aire’s partners in the molecular control of immunological tolerance. Cell 140, (1), 123-35. (47) Jin, S.; Inoue, S.; Weaver, D. T. Differential etoposide sensitivity of cells deficient in the Ku and DNA-PKcs components of the DNAdependent protein kinase. Carcinogenesis 1998, 19 (6), 965–71. (48) Derbinski, J.; Pinto, S.; Rosch, S.; Hexel, K.; Kyewski, B. Promiscuous gene expression patterns in single medullary thymic epithelial cells argue for a stochastic mechanism. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (2), 657–62. (49) Yewdell, J. W.; Anton, L. C.; Bennink, J. R. Defective ribosomal products (DRiPs): a major source of antigenic peptides for MHC class I molecules. J. Immunol. 1996, 157 (5), 1823–6.

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