Aire’s plant homeodomain(PHD)-2 is critical for induction of immunological tolerance

Abstract

Aire impacts immunological tolerance by regulating the expression of a large set of genes in thymic medullary epithelial cells, thereby controlling the repertoire of self-antigens encountered by differentiating thymocytes. Both humans and mice lacking Aire develop multiorgan autoimmunity. Currently, there are few molecular details on how Aire performs this crucial function. The more amino-terminal of its two plant homeodomains (PHDs), PHD1, helps Aire target poorly transcribed loci by “reading” the methylation status of a particular lysine residue of histone-3, a process that does not depend on the more carboxyl-terminal PHD-2. This study addresses the role of PHD2 in Aire function by comparing the behavior of wild-type and PHD2-deleted Aire in both transfected cells and transgenic mice. PHD2 was required for Aire to interact with sets of protein partners involved in chromatin structure/binding or transcription but not with those implicated in pre-mRNA processing; it also was not required for Aire’s nuclear translocation or regional distribution. PHD2 strongly influenced the ability of Aire to regulate the medullary epithelial cell transcriptome and so was crucial for effective central tolerance induction. Thus, Aire’s two PHDs seem to play distinct roles in the scenario by which it assures immunological tolerance.

An important facet of immunological tolerance is the elimination of potentially self-reactive T cells during their differentiation in the thymus (reviewed in ref. 1). The transcription factor, Aire, a member of the zinc-finger family, is responsible for purging the T-cell repertoire of a broad swath of self-reactive specificities (reviewed in refs. 2 and 3). Consequently, both humans and mice with a mutation in the gene encoding Aire develop multiorgan autoimmune disease. Aire is expressed in a subset of thymic MECs, where it regulates the transcription of a large set of genes, in particular loci encoding peripheral-tissue antigens (PTAs). Peptides derived from these PTAs are loaded onto major histocompatibility complex (MHC) molecules and displayed at the medullary epithelial cell (MEC) surface, where they are encountered by differentiating T cells percolating through the thymus. Those thymocytes whose T-cell receptors avidly recognize MEC MHC:peptide complexes undergo clonal deletion, thereby preserving immunological tolerance (4–8).

The molecular mechanisms underlying Aire’s control of PTA gene expression are only vaguely understood at present (2, 3). It seems not to operate like a traditional transcription factor, binding to a promoter and/or enhancer element and inducing transcript initiation, but instead to somehow release RNA polymerase (RNA-Pol)II pausing and promote transcript elongation (9, 10). Aire targets and regulates genes as a component of one or more large multiprotein complexes (11–13). Its numerous associates include sets of proteins involved in nuclear transport, chromatin binding/structure, transcription, and pre-mRNA processing (12). The geographical features of these Aire-containing complexes remain to be elucidated—i.e., which protein(s) Aire binds directly to, which regions of Aire are responsible for these associations, and which of the interactions have what functional consequences.

Among Aire’s structural domains are two plant homeodomains (PHDs) (Fig. 1A), which generally function in protein:protein interactions, notably as histone “readers” or as docking domains involved in stabilization of multiprotein complexes in various stages of transcription (14). The more amino-terminal PHD1 is a functionally critical region of Aire, acting as a reader of histone-3 molecules unmethylated at the lysine-4 residue (H3K4me0) and thereby promoting targeting to transcriptionally dormant genes (15–20). PHD1 has also been reported to be a vital link in Aire’s interaction with DNA–PKcs (20) and to harbor E3 ubiquitin–ligase activity essential for transcriptional induction (21), although the NMR solution structure and subsequent functional assessments did not support this latter role (22). The function of Aire’s more carboxyl-terminal PHD2 remains an enigma, with arguments both for and against a critical role in transcriptional transactivation (11, 13, 16, 21, 23–25).

Fig. 1.

Aire’s PHD2 is required for interaction with some, but not all, of its partners. (A) Domain structure of mouse Aire. Numbers represent the amino acid residues, and boxes represent the functional domains. HSR, homogenously staining region; NLS, nuclear localization signal; SAND, Sp100, Aire, NucP41/75, DEAF1 domain; PRR, proline-rich region (3). (B) Representative microscopic images of HEK-293 cells expressing Aire-WT (Left) or Aire-ΔPHD2 (Right). Nuclei were counterstained with DAPI. (C) Classification of Aire partners into functional groups, as per ref. 12. (D) Co-IP of known Aire partners. HEK-293 cells, transfected with vector alone (C) or with the Aire-WT or -ΔPHD2 construct were lysed, and the nuclear extracts were immunoprecipitated with anti-FLAG Ab, followed by immunoblotting for the indicated proteins. (E) Summary data from four experiments. Quantitative analysis of band intensities was performed by using Fujifilm Multi Gauge (Version 2).

The focus of this report is the function of Aire–PHD2. Does removal of this domain compromise Aire’s access to and localization within the nucleus? Its interaction with other proteins? Its regulation of PTA gene expression in thymic MECs? Its control over autoimmunity? These questions are addressed through analyses of cells transfected with and mice transgenic for an Aire gene devoid of PHD2.

Results

Without Its PHD2, Aire No Longer Interacts with a Subset of Its Partners.

As a first approach to elucidating the function of the PHD2 of Aire, we investigated whether it is required for Aire’s interactions with its known structural associates. Thus, we generated an expression construct encoding a FLAG-tagged, PHD2-deleted form of Aire (Aire-ΔPHD2) and an analogous construct specifying FLAG-tagged, wild-type Aire (Aire-WT), and we compared the outcome of transfecting each into 293T cells, which are widely used for analyses of protein:protein (including Aire) interactions (e.g., refs. 12 and 13). Deletion of PHD2 had no evident effect on Aire’s localization in the nucleus or on its speckled disposition therein (Fig. 1B).

Experiments entailing Aire-targeted coimmunoprecipitation (co-IP) followed by mass spectrometry, as well as multiple RNA interference-mediated knockdown approaches, have identified a large set of proteins that associate with Aire (e.g., refs. 12 and 13). These proteins were classified into four major functional classes: nuclear transport, chromatin binding/structure, transcription, and pre-mRNA processing (12) (Fig. 1C). We chose representative members of each class (except for the nuclear transport group, given that nuclear localization was unaffected by deletion of PHD2), and assayed their interaction with Aire by co-IP experiments on 293T cells transfected with either the Aire-WT or Aire-ΔPHD2 construct. Interestingly, tested members of the pre-mRNA processing group (SFRS3 and DDX5) still coimmunoprecipitated with Aire lacking PHD2, whereas all members of the chromatin binding/structure and transcription groups (DNA–PKcs, Ku80, PARP1, RNA-PolII, TOP2A, DSIF, P-TEFb, HEXIM1, and MCM6) coimmunoprecipitated poorly or not at all (Fig. 1 D and E). These results suggest that PHD2 has a specific function in particular aspects of Aire’s role in regulating transcription.

Construction of Transgenic Mice Expressing Aire with the ΔPHD2 Mutation.

To address the function of Aire’s PHD2 in vivo, we used a described system (26) to generate lines of transgenic mice expressing either WT Aire (iA-WT) or PHD2-deleted Aire (iA-ΔPHD2) quasi-specifically in thymic MECs, on an Aire-knockout (Aire-KO) nonobese diabetic (NOD) background. Mice generated by using this system faithfully recapitulate the critical features of Aire control of immunological tolerance and have been used previously to examine the role of PHD1 in Aire’s activities (19), as well as temporal aspects of Aire’s impact on tolerance (27).

We first compared thymic epithelial cell compartments in the transgenic mice. Aire-expressing cells are a component of the so-called MEC^hi subset, which displays low levels of the Ly51 marker and high levels of MHC class II molecules. As has been reported (28), Aire-KO mice had a significantly higher fraction and number of MEC^hi cells than did Aire-WT animals; similarly, iA-ΔPHD2 mice had more MEC^hi cells than did iA-WT animals (Fig. 2 A–C). Both transgenic lines had a lower fractional representation of Aire⁺ cells in the MEC^hi compartment than did Aire-WT mice, although levels of Aire expression were indistinguishable in the three lines. However, most importantly, the expression of Aire was very similar in the two transgenic lines, whether assessed as % Aire⁺ of MEC^hi (Fig. 2D), as the mean fluorescence intensity (MFI) of Aire in Aire⁺MEC^hi (Fig. 2E), or by the distribution of Aire on thymus sections (Fig. 2F). All in all, the thymus of iA-ΔPHD2 mice appeared grossly normal (including normal-looking thymocyte populations), but their increase in MEC^hi, similar to what is routinely seen in Aire-KO mice, portended a possible deficiency in Aire function.

Fig. 2.

Aire-expressing MEC^hi compartments in the different mouse lines. (A) Representative flow-cytometric analyses of CD45⁻ thymic stromal cells from three independent experiments. MEC^hi was gated as Ly51^-/loMEC classII^hi; numbers indicate the percentage of MEC^hi cells. (B) Summary data for MEC^hi as a fraction of CD45⁻ cells (n = 3). P values are from Student’s t test. (C) Summary data for total numbers of MEC^hi per thymus. (D) Summary data for fraction of Aire⁺ cells within the MEC^hi fraction. (E) Summary data for Aire MFI in Aire⁺MEC^hi cells. (F) Aire staining on 5-μm frozen sections of thymi from 5-wk-old iA-WT (Left) or iA-ΔPHD2 (Right) mice, counterstained with DAPI. Magnification: 100×.

Aire’s PHD2 Is Required for Most of Its Effects on MEC Gene Expression.

Aire controls immunological tolerance in great part by regulating transcription of a large set of genes in thymic MECs, above all loci encoding PTAs. To investigate the role of PHD2 in this function, we compared the transcriptomes of MEC^hi isolated from various mouse lines. Fig. 3A presents a direct comparison of the impact of the WT vs. ΔPHD2 versions of Aire on MEC transcription in the absence of Aire, i.e., a fold change/fold change (FC/FC) plot for iA-WT vs. Aire-KO vis-à-vis iA-ΔPHD2 vs. Aire-KO. Clearly, deletion of PHD2 greatly compromised Aire function because most of the transcripts induced by Aire-WT were only slightly up-regulated by Aire-PHD2. However, the fact that the cloud of Aire-induced transcripts was tilted away from the x-axis suggested that the mutation was less dramatic then the knockout of Aire. This finding was particularly true for a small subset of highly induced transcripts highlighted in Fig. 3B. In brief, then, PHD2 is required for most of Aire’s control over gene expression in MECs.

Fig. 3.

Aire-PHD2 is essential for most Aire-induced gene expression in MECs. (A) Comparing Aire-WTs with Aire-ΔPHD2’s impact on the Aire-less transcriptome. FC/FC plots of triplicate data comparing iA-WT with Aire-KO (x axis) and iA-ΔPHD2 with Aire-KO (y axis). Red dots indicate some of the transcripts highlighted in B. (B) List of genes impacted less differentially (more than threefold less) by the ΔPHD2 than the KO mutation.

Aire-PHD2 Is Necessary for Imprinting Immunological Tolerance.

Two assays were used to evaluate the importance of PHD2 for Aire’s influence on central tolerance, directly comparing cohorts of Aire-WT, Aire-KO, iA-WT, and iA-ΔPHD2 mice. First, at 14 wk of age (or when 15–20% of body weight had been lost), mice were killed, selected organs were removed, and hematoxylin and eosin (H+E) histology was performed. In general, both for females and males, iA-ΔPHD2 and Aire-KO mice presented a very similar picture, whether the criterion evaluated was the number and type of tissues infiltrated (Fig. 4A), average tissue infiltration score (Fig. 4B), or histological presentation (Fig. 4C and Fig. S1A). Second, we assayed the range of autoantibody (autoAb) specificities by Western blotting whole-tissue extracts from organs known to be autoAb targets in Aire-KO mice on the NOD background (eye, lung, pancreas, and stomach) and probing them with sera from the different lines of mice (Fig. 4D and Fig. S1B). Profiles of serum autoAbs were very similar in iA-ΔPHD2 and Aire-KO individuals, whether male or female. In short, then, Aire requires its PHD2 domain to effectively impose immunological tolerance.

Fig. 4.

Critical role of Aire-PHD2 in imparting immunological tolerance. (A) Range of infiltrates. Histological analysis of WT, KO, iA-WT, and iA-ΔPHD2 mice via H+E staining of fixed tissues. Mice were taken at 14 wk of age or at 15–20% of body-weight loss. i, insulitis typical of Aire-positive NOD mice. (B) Each organ (n = 10) was scored as described in Materials and Methods. (C) Representative infiltrates for female mice. White arrows indicate areas of infiltration. Magnification: 20×, except for the stomach (5×). (D) AutoAbs in female mouse serum. Each lane of the multiscreen immunoblot shows autoAbs for an individual mouse. W, Aire-WT; i, iA-WT; Δ, iA-ΔPHD2; K, Aire-KO. Dilutions, 1:25,000.

Discussion

Aire has two PHDs. Several lines of evidence argue that PHD1 binds preferentially to unmethylated H3K4 residues—a mark of poorly transcribed chromatin—and is thereby an element of the mechanism by which Aire chooses its gene targets (15–17, 19, 29). PHD2’s function has been less studied and thus remains in question. Early results on Aire-transfected cell lines suggested a role in transcriptional transactivation (11, 21, 25), although this result proved not to be a general observation (16). In fact, Xenopus Aire lacks a PHD2, and chicken Aire has a substantially truncated version, casting doubt on a required function (30). Here we demonstrated that the PHD2 of Aire does indeed play a critical role in its control of thymic MEC gene transcription and, as a consequence, in immunological tolerance.

Three major points emerge from our studies. First, PHD2 is not required for Aire to localize to the nucleus or for it to concentrate within punctate structures therein. This finding is somewhat surprising given a recent report that a point mutation within PHD2 (human Aire C446G) results in multiorgan autoimmune disease because it leads to aberrant Aire localization confined to the cytoplasm and, consequently, a reduction in Aire-dependent gene transcripts (13). It is likely that the point mutation provokes abnormal folding and aggregation that does not occur when the entire domain is absent.

Second, deletion of PHD2 greatly compromised Aire’s interaction with certain of its partners (members of the chromatin structure/binding and transcription groups) but not with others (members of the pre-mRNA processing and likely nuclear transport groups). This disjunction is reminiscent of our previous finding that shRNA-driven knockdown of TOP2A (a member of the transcription group) did not destroy the interaction between Aire and SFRS2 (a member of the pre-mRNA processing group) (12). Such observations suggest that Aire might participate in multiple multiprotein complexes, differentially dependent on its individual domains. This notion is consistent with recent 3D structural data on a tandem Aire PHD1–PHD2 fragment, which revealed the two domains to be independent and noninteracting (13). This study also showed that point mutations in PHD1 that have a major impact on PTA gene expression had relatively minor effects (less than two-fold) on the interaction of Aire with protein partners involved in chromatin binding/structure or transcription (e.g., RuvBL2, SMC1A, DNA-PKcs, and Msh6).

Third, removal of PHD2 greatly dampened Aire’s impact on the MEC transcriptome. This effect is similar to that reported for a debilitating point mutation in PHD1 (19), and both alterations led to a multiorgan autoimmune disease essentially indistinguishable from that of Aire-KO mice. At present, we have no explanation for why a small subset of genes was notably less affected by the ΔPHD2 than the Aire-null mutation. However, it may be relevant that several of the genes highlighted as being less responsive to the former than the latter are members of multigene families (e.g., Mup) whose members are located together on the chromosome. Interestingly, some of the same loci were previously found to be less affected by a debilitating PHD1 point mutation than the Aire-null mutation (19).

On the basis of these findings (and others), a model worth considering is that PHD1 helps target Aire to poorly transcribed genes by preferentially binding to H₃K₄Me0 residues (or by preferentially avoiding H₃K₄Me3 residues) and that PHD2 serves as a docking site or adaptor for one or more multiprotein complexes that act to release RNA–PolII pausing and promote transcriptional elongation. Once these processes have been set in motion, a different Aire-containing complex—one that does not depend on PHD2—may come into play to regulate mRNA processing.

However, given this potentially central role for Aire-PHD2, how can Xenopus and chickens be immunologically tolerant in its absence or truncation? One possibility is that in these two species Aire-PHD1 and/or the typical binding partners of Aire-PHD2 have evolved to permit PHD1 to take over PHD2’s critical function(s). Another possibility is that these two species are indeed compromised in their ability to purge the thymus of potentially autoreactive T cells, but that other tolerance mechanisms can come into play effectively enough to dampen autoimmunity. Indeed, we have only a very limited view of the relative degree to which Xenopus and chickens succumb to autoimmunity vis-à-vis other vertebrate species.

Materials and Methods

Cell Culture and Transfection.

HEK-293 cells were cultured in DMEM supplemented with 10% (vol/vol) FBS, l-glutamate and pen/strep antibiotics and maintained in a humidified atmosphere at 37 °C with 5% CO₂. For transfection, the cells were counted and seeded in six-well or 10-cm tissue culture plates and transfected with the specified plasmids using TransIT reagent (Mirus) according to the manufacturer’s instructions.

Abs and Plasmids.

Abs recognizing the following protein or protein-tags were purchased: FLAG-tag (Sigma Aldrich); DNA-PKcs and SFRS3 (Abnova); PARP1 (Cell Signaling Technology); Ku80, TOP2A, and HEXIM1 (Abcam); RNA-PolII, CDK9, SPT5, DDX5, and MCM6 (Santa Cruz Biotechnology); and Aire (eBioscience). Anti-mouse and anti-rabbit immunoglobulin(Ig)G secondary Abs conjugated with horseradish peroxide or fluorescein isothiocyanate (FITC) were purchased from Jackson Immunoresearch. Mammalian expression plasmids carrying FLAG-tagged Aire-WT or Aire-∆PHD2 cDNAs were constructed by in-frame insertion of each between the BglII and SalI sites of the pCMV-tag1 vector (Clontech).

Immunofluorescence.

HEK-293 cells, seeded on coverslips and transiently transfected with Aire-WT or Aire-ΔPHD2 constructs, were fixed with 4% (vol/vol) paraformaldehyde for 10 min followed by permeabilization with 0.5% Triton X-100 [in phosphate-buffered saline (PBS), pH 7.4] for 10 min. Cells were blocked with 1% BSA in Tween buffer (PBST; 0.05% Tween 20 in PBS, pH 7.4) for 1 h and stained with anti-FLAG Abs for 2 h, followed by incubation with FITC-conjugated anti-mouse IgG secondary Abs in the dark for 1 h at room temperature. Cells were counterstained with DAPI for visualization of nuclei. Coverslips with cells were mounted on a slide with fluoromount G, and immunofluorescent images were acquired by a fluorescence microscope (Zeiss Axio Imager M1) equipped with filters matching the spectral excitation and emission characteristics of DAPI and FITC. For frozen sections, thymi from 5-wk-old mice were washed and fixed in 4% (vol/vol) paraformaldehyde. Sections were stained by using anti-mouse Aire (5H12 clone) and counterstained with DAPI. Immunofluorescence was visualized by laser scanning confocal microscopy (Olympus).

Immunoprecipitation.

HEK-293 cells, transfected with empty vector (C) or with an Aire-WT or Aire-ΔPHD2 construct for 48 h, were harvested and lysed in a hypotonic lysis buffer [0.05% Nonidet P-40, 10 mM Hepes, 1.5 mM MgCl₂, 10 mM KCl, 5 mM EDTA (30 × 10⁶ cells per mL)] plus complete protease inhibitor mixture (Roche), pH 7.4, and incubated on ice for 15 min. Cell nuclei were separated from the cytosolic fraction and incubated on ice for 1 h in a native nuclear extraction buffer [50 mM Bis-Tris, 750 mM 6-aminocaproic acid, 3 mM CaCl₂, 10% Glycerol, EDTA-free complete protease inhibitor mixture (Roche) and micrococcal nuclease (Nuclease S7; Roche), pH 7.4, (60 × 10⁶ cells per mL)]. Nuclear extracts were incubated with 20 μL of Protein G Sepharose beads (GE Healthcare) conjugated to anti-FLAG Abs (Sigma) for 3 h with rotation at 4 °C. Beads were washed three times with ice-cold PBS containing 0.05% Nonidet P-40, and once with pure ice-cold PBS. Immunoprecipitated proteins were eluted by boiling in sample buffer for 15 min and separated on 10% SDS/PAGE followed by transfer of proteins to polyvinylidene difluoride membranes (Millipore). Membranes were blocked in PBST buffer containing 5% (wt/vol) nonfat dried milk and probed with primary Abs overnight at 4 °C. After a wash with PBST, membranes were incubated with secondary Abs linked to horseradish peroxidase. The blots were then developed with an enhanced chemiluminescence detection system (Thermo Scientific) as per the manufacturer’s instructions.

Mice.

NOD/Lt J (Aire-WT) mice were originally purchased from the Jackson Laboratory. Aire-KO and iA-WT mice on the NOD genetic background have been described (19, 27). An Aire cDNA carrying PHD2-domain deletion (amino acids 434–475) was constructed by QuikChangeII XL (Stratagene) and introduced into the ClaI site of the TOA construct (19). A fragment containing the Aire-ΔPHD2 cDNA intron and polyadenylation site from the rabbit β-globin gene were AatII and AfeI digested for the TOP construct, purified, and microinjected into NOD embryos as described (27). Progeny were screened by a PCR assay specific for the TOA and PHD2 transgene. All mice were bred and housed under specific-pathogen-free conditions at the Harvard Medical School Center for Animal Resources and Comparative Medicine following Institutional Animal Care and Use Committee Protocol 2954.

Isolation of Thymic Epithelial Cells and Sorting of MECs.

Thymi of 5-wk-old individual Aire WT, Aire-KO, iA-WT, or iA-ΔPHD2 mice were routinely used in a four-way comparison. Thymic lobes underwent a small cut and were agitated in RPMI to release thymocytes. The fragments were digested with collagenase (Roche) and DNase (Sigma) for 30 min and then with collagenase/dispase (Roche) for 30 min, as described (10). The released cells were stained for flow-cytometric sorting or analysis, by using a MoFlo (Dako) or LSRII (Becton Dickinson), respectively, as described (31). Data were analyzed with FlowJo software (Tree Star).

Microarray Analysis.

Total RNA was prepared from MECs of individual 5-wk-old Aire-WT, Aire-KO, iA-WT, or iA-ΔPHD2 mice. RNA amplification and microarray hybridization were performed as described (32). Briefly, RNA was amplified using a T7 polymerase-based method, and cDNA was hybridized to random primers. The cDNA was purified, fragmented, and terminally labeled by using the Affymetrix terminal labeling kit. Labeled DNA was hybridized to 1.0 ST Affymetrix arrays, washed, stained, and scanned. Microarray data were normalized by robust multiarray average (33) and analyzed by the multiplot module of GenePattern 3.4.

Autoimmune Disease Monitoring.

Individuals were killed at 14 wk of age or when they had lost 15–20% body weight relative to that of littermates, as described (19, 34). Designated tissues were removed, fixed in 10% formalin, and embedded in paraffin. Tissue sections were stained with H+E, and infiltration of various organs was scored. In general, scores of 0, 0.5, 1, 2, 3, and 4 indicated no, trace, mild, moderate, or severe lymphocytic infiltration, and complete destruction, respectively. For retinal degeneration, scores were as follows: 0, lesion present without any photoreceptor layer lost; 1, lesion present, but less than half of the photoreceptor layer lost; 2, more than half of the photoreceptor layer lost; 3, entire photoreceptor layer lost without or with mild outer nuclear layer attack; and 4, the entire photoreceptor layer and most of the outer nuclear layer destroyed. All of the infiltrated samples were scored blindly and independently by two investigators.

The appearance of autoAbs was monitored as described (34). Briefly, the indicated organs were removed from a 4-wk-old WT NOD mouse and homogenized and centrifuged at 15,700 × g for 10 min. Supernatants were mixed with protein sample buffer, extracts were size-fractionated by 10% SDS/PAGE gel electrophoresis, gel-separated protein were transferred to a poly(vinylidene fluoride) membrane, and the membrane was blocked and incubated with a 1:25,000 dilution of serum from individual mice. After washing with PBST, the bound Abs were reacted with horseradish-peroxidase-conjugated anti-mouse IgG for 1 h and revealed with an enhanced chemiluminescence reagent (Thermo Scientific) and autoradiography.