The Mechanism of Tissue-Restricted Antigen GeneExpression by AIREKristina Zumer,* Kalle Saksela,* and B. Matija Peterlin*,†
The autoimmune regulator is a critical transcriptionfactor for generating central tolerance in the thymus.Recent studies have revealed how the autoimmune reg-ulator targets many otherwise tissue-restricted Ag genesto enable negative selection of autoreactive T cells. TheJournal of Immunology, 2013, 190: 2479–2482.
The autoimmune regulator (AIRE ) gene was identifiedby positional cloning of the genetic locus linked to arare autoimmune disease, autoimmune-polyendocri-
nopathy-candidiasis-ectodermal dystrophy (APECED) (1, 2).The encoded AIRE protein is expressed primarily in medul-lary thymic epithelial cells (mTECs) (3). AIRE is also ex-pressed in peripheral lymphoid tissues (4), where its contri-bution to tolerance remains to be investigated. The mousemodel of the human APECED disease, the Aire2/2 mouse,was instrumental in identifying the cellular role of AIRE,which is to regulate promiscuous expression of tissue-restrictedAg (TRA) genes in mTECs (5). The key finding was thatmTECs from Aire2/2 versus wild-type mice expressed fewerTRAs. Although this promiscuous gene expression in mTECshad been recognized previously (6), the underlying mecha-nism remained elusive. To date, AIRE is the only identifiedtranscription factor that regulates this process. Among others,AIRE activates insulin, interphotoreceptor retinoid-bindingprotein A, and mucin 6 genes, all of which had been linkedto autoimmunity in humans or AIRE-deficient mice (7, 8).Expressed TRAs are then processed and presented on thesurface of mTECs or taken up by dendritic cells (9, 10). Ex-posure of maturing T cells to these Ags is critical for negativeselection of T cells in the thymus (11, 12). In the absence ofAIRE, autoreactive T cells mature and escape into the pe-riphery, which can lead to autoimmunity (7, 8, 13).Transcription of protein-coding genes occurs in several
phases, all of which are regulated (14). First, transcriptionfactors and RNA polymerase II (RNAPII) are recruited topromoters. Although RNAPII initiates transcription, it thenpauses because of the action of negative elongation factor(NELF) and 6-dichloro-1-a-D-ribofuranosylbenzimidazole sen-
sitivity inducing factor (DSIF). The release of RNAPII toelongation is mediated by the positive transcription elonga-tion factor b (P-TEFb), composed of a regulatory cyclin sub-unit (CycT1 or CycT2) and the cyclin-dependent kinase 9(15). P-TEFb phosphorylates subunits of DSIF and NELF aswell as serine residues at position 2 (Ser2) in the C-terminaldomain (CTD) repeats of the largest RNAPII subunit (RPB1)(16). DSIF is thus changed to an elongation factor, NELF isreleased from the nascent RNA, and the phosphorylatedRNAPII can now elongate (16). The CTD of human RNAPIIcontains 52 heptapeptide repeats (YSPTSPS). The serines,threonines, and tyrosines in these repeats are phosphorylatedby P-TEFb and other kinases in distinct phases of transcrip-tion, giving rise to diverse set of instructions, which areknown as the CTD code (17). The phosphorylated elongatingRNAPII directs cotranscriptional processing, that is, splicingand polyadenylation of genes (18). Transcription elongationproceeds until termination, upon which RNAPII becomesdephosphorylated, and the cycle begins anew. Recent genome-wide analyses revealed an unexpectedly high abundance ofpaused RNAPII at most promoters, including those of inac-tive genes (19–22).AIRE is a transcription factor that assembles into oligomers
and forms punctate structures that colocalize with CREB-binding protein, P-TEFb, and small nuclear ribonucleopro-teins in the nucleus (23–25). The estimated number of AIRE-regulated genes ranges from several hundred to thousands.They have diverse promoters and are regulated by distincttranscription factors in their corresponding tissues. Thesefindings raise the conundrum of how AIRE can regulate sucha large repertoire of divergent genes. Recent work from othersand us has addressed this question and revealed the mecha-nisms of action of this enigmatic protein (26–31).The AIRE protein has a predicted molecular mass of 58
kDa (Fig. 1A) (3). It forms large oligomers, which are detectedin a .670-kDa fraction by gel filtration (32). The N terminusof AIRE contains the homogeneously staining region (HSR)and the Sp100, AIRE-1, NucP41/75, and DEAF-1 (SAND)domain (Fig. 1A). Mutations in HSR from APECED patients
*Department of Virology, Haartman Institute, Helsinki University Central Hospital,University of Helsinki, FIN-00014 Helsinki, Finland; and †Department of Medicine,The Rosalind Russell Medical Research Center, University of California at San Fran-cisco, San Francisco, CA 94143
Received for publication November 21, 2012. Accepted for publication January 8, 2013.
Address correspondence and reprint requests to Dr. B. Matija Peterlin, University ofCalifornia at San Francisco, 533 Parnassus Avenue, Box 0703, Room U-432, SanFrancisco, CA 94143-0703. E-mail address: [email protected]
Abbreviations used in this article: AIRE, autoimmune regulator; APECED, autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy; CTD, C-terminal domain; DNA-
disrupt multimerization, which is essential for AIRE function(32, 33). The AIRE SAND domain is also involved in thisoligomer formation. It lacks the canonical KDWK motif,which is required for DNA binding of other SAND domains(34). Although DNA binding of the AIRE SAND domainhad been reported in vitro (35, 36), it appears to be non-specific (37) and may be irrelevant for its recruitment to targetgenes in vivo. Indeed, we found that mutating KNKA resi-dues in the AIRE SAND domain, which correspond to theKDWK motif in other members of this family, had no effecton AIRE-induced expression of a plasmid reporter gene (26).In some APECED patients an autosomal-dominant G228Wmutation was found in the SAND domain (Fig. 1A), whichcauses the wild-type AIRE protein to coaccumulate in largerstructures that do not colocalize with sites of active tran-scription (25). Thus, dominant-negative effects of thisG228W mutation could be due to an increased affinity for thewild-type AIRE protein. The N terminus of AIRE is requiredfor nuclear localization (38). The nuclear localization signalwas mapped to basic residues from positions 131 to 133 (Fig.1A) (39).AIRE also contains two plant homeodomains, plant ho-
mology domain (PHD)1 and PHD2 (Fig. 1A). PHDs are zincfingers closely related to RING domains, which are common
in proteins involved in ubiquitylation (40). PHDs are pro-tein–protein interaction modules, which can mediate bindingto nucleosomes (41, 42). Such PHDs bind to N-terminal tailsof histone H3, discriminating between those methylated(H3K4me3) or unmodified (H3K4) at Lys4. AIRE PHD1 ismost closely related to the BHC80 PHD that interacts selectivelywith the unmodified H3K4 (42). Indeed, AIRE PHD1 alsobinds to the unmodified H3K4 (Fig. 1A), and this interaction isrequired for AIRE to activate transcription of genes in chro-matin (26, 30, 37). The AIRE PHD2 sequence is divergent anddoes not interact with nucleosomes, but it contributes struc-turally to the activation of TRA genes by AIRE (27, 31, 43).AIRE PHD1 also binds to the DNA-dependent protein
kinase (DNA-PK) (Fig. 1A) (44). DNA-PK is a nuclear ki-nase, which not only functions to repair DNA double-strandbreaks and mediate V(D)J recombination, but it also supportstranscription and chromatin remodeling (45). DNA-PK alsophosphorylates two sites in the N terminus of AIRE (44).However, pharmacological inhibition of DNA-PK and bring-ing AIRE to a promoter via heterologous DNA tethering incells lacking DNA-PK revealed that the kinase activity ofDNA-PK is dispensable for gene activation by AIRE. Instead,these interactions represent a key mechanism for the recruit-ment of AIRE to its target genes (26). An important aspect of
FIGURE 1. AIRE protein domains, key interacting partners, and mechanism of TRA gene expression. (A) AIRE contains several domains that are related to
those in other transcription factors. From the N terminus they are: HSR (green), which is important for the oligomerization of AIRE and may function as
a caspase recruitment domain (58); SAND (green); PHD1 and PHD2 (violet); proline-rich region (PRR; orange); and the TAD (red). Protein residues cor-
responding to human AIRE are labeled above, and the position of the four LXXLL motifs is marked below the diagram. The location of the nuclear localization
signal is also indicated (KRK). Mutations discussed in this review are marked with an asterisk below the diagram with accompanying labels. Arrows depict
interactions between DNA-PK, H3K4, P-TEFb, and AIRE. (B) Schematic diagram depicting the molecular mechanism of AIRE-regulated TRA gene expression.
Combinatorial interactions between AIRE, unmodified H3K4 (yellow), DNA-PK (red), and RNAPII (blue) recruit AIRE to a TRA promoter. AIRE brings P-
TEFb (red) to phosphorylate the RNAPII CTD, and this results in transcription elongation, mRNA processing, and TRA gene expression. Histone H2AX with
phosphorylated Ser139 (gH2AX), which marks DNA double-strand breaks, is depicted in yellow. (C) The Venn diagram marks sets of genes that contain un-
modified H3K4, engaged RNAPII, and DNA-PK at their promoters. AIRE is targeted to and can regulate expression of genes at the intersection of these sets.
this targeting is that AIRE interacts with DNA-PK, which isassociated with the histone variant H2AX phosphorylated atSer139 (gH2AX) that marks DNA double-strand breaks (26).The C terminus of AIRE does not share obvious homology
with functional domains in other proteins, but it is highlyconserved between human and mouse AIRE proteins. It servesas a transcriptional activation domain (TAD) (Fig. 1A) (27). Itbinds to P-TEFb and brings it to TRA genes (23, 27). Thisleads to the phosphorylation of Ser2 in the RNAPII CTD andproductive elongation with cotranscriptional processing ofnascent mRNA species. The key role of this domain is under-scored by an APECED patient mutation that affects only theextreme C terminus in AIRE (Fig. 1A) (46) but completelyabolishes its function (27).AIRE contains four LXXLL motifs, two in the HSR, one in
a proline-rich region between the two PHDs, and one in theTAD (Fig. 1A). LXXLL motifs are known to mediate pro-tein–protein interactions between nuclear receptors and theircoactivators (47). Their role for AIRE remains to be estab-lished.Armed with all of this knowledge of interacting proteins and
chromatin targeting strategies of AIRE, we can now proposea model for the AIRE-regulated promiscuous TRA gene ex-pression in mTECs. The defining feature of AIRE target genesis that although they are inactive, they have already engagedRNAPII on their promoters (Fig. 1B) (23, 27, 29). Thismeans that the basal transcriptional machinery is already inplace, but RNAPII is stalled or generates only sterile andunprocessed transcripts, which are unstable (48, 49). Becausethis RNAPII is not phosphorylated at its CTD, it also failsto recruit chromatin-modifying machineries. Thus, H3K4remains unmodified, leading to PHD1-mediated recruitmentof AIRE to such inactive genes (Fig. 1B) (30, 37). Initiation oftranscription has been associated with topoisomerase II–cat-alyzed formation of transient double-strand DNA breaks,leading to DNA-PK recruitment to initiating RNAPII (50,51). DNA-PK–associated AIRE is thereby brought into closeproximity of the initiating RNAPII (Fig. 1B). Because thebinding between AIRE PHD1 and unmodified H3K4 is in-sufficient to target AIRE to specific TRA genes (52), only thecombinatorial interactions of AIRE with all its partners, theunmodified H3K4, DNA-PK, and engaged RNAPII, lead tothe recruitment of AIRE with associated P-TEFb to thesetranscription units (Fig. 1C). Levels and distribution of thesefactors at distinct TRA genes will vary from cell to cell,thereby giving rise to the seemingly stochastic nature of TRAgene expression by AIRE.However, several other aspects of AIRE remain to be in-
vestigated. For example, how is its transcription regulated inmature mTECs or in peripheral lymphoid tissues (53, 54)?There exist three splice variants of human AIRE, some ofwhich lack the N-terminal domains required for the forma-tion of oligomers (2). Do they play any role in central tol-erance? Furthermore, several posttranslational modificationsof AIRE have been reported (44, 55, 56). How do they affectthe function of AIRE and who directs these transcriptionaland posttranslational events to direct central tolerance? Fi-nally, P-TEFb, the essential coactivator of AIRE, is itselftightly regulated in cells. Among other stimuli, P-TEFb canalso be activated by cellular stress (57). Thus, it is likely thatincreased numbers of DNA breaks in mTECs also activate P-
TEFb (28), thereby further enhancing the expression of TRAgenes to optimize the establishment of central tolerance.
ConclusionsAIRE is a transcription factor that activates the expression ofTRA genes in mTECs. Their promoters must be occupied byRNAPII, unmodified H3K4, and DNA-PK. Sufficient levelsof these proteins ensure that AIRE is recruited to these sites.AIRE oligomers then bring P-TEFb to RNAPII, which leadsto its extensive phosphorylation. Thus modified, RNAPII iscompetent for elongation and cotranscriptional processing oftarget genes, which leads to the expression of TRAs and theirpresentation to T cells via MHC class II determinants.
DisclosuresThe authors have no financial conflicts of interest.
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