The Mechanism of Tissue-Restricted Antigen Gene Expression by AIRE Kristina Zumer, Kalle Saksela and B. Matija Peterlin This information is current as J Immunol 2013; 190:2479-2482; ; of September 29, 2021. doi: 10.4049/jimmunol.1203210 http://www.jimmunol.org/content/190/6/2479 Downloaded from References This article cites 58 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/190/6/2479.full#ref-list-1

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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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Mechanism of Tissue-Restricted Antigen Gene Expression by AIRE Kristina Zumer,*ˇ Kalle Saksela,* and B. Matija Peterlin*,† The autoimmune regulator is a critical transcription sitivity inducing factor (DSIF). The release of RNAPII to factor for generating central tolerance in the thymus. elongation is mediated by the positive transcription elonga- Recent studies have revealed how the autoimmune reg- tion factor b (P-TEFb), composed of a regulatory cyclin sub- ulator targets many otherwise tissue-restricted Ag genes unit (CycT1 or CycT2) and the cyclin-dependent kinase 9 to enable negative selection of autoreactive T cells. The (15). P-TEFb phosphorylates subunits of DSIF and NELF as Journal of Immunology, 2013, 190: 2479–2482. well as serine residues at position 2 (Ser2) in the C-terminal domain (CTD) repeats of the largest RNAPII subunit (RPB1)

(16). DSIF is thus changed to an elongation factor, NELF is Downloaded from AIRE he autoimmune regulator ( ) gene was identified released from the nascent RNA, and the phosphorylated by positional cloning of the genetic locus linked to a RNAPII can now elongate (16). The CTD of human RNAPII T rare autoimmune disease, autoimmune-polyendocri- contains 52 heptapeptide repeats (YSPTSPS). The serines, nopathy-candidiasis-ectodermal dystrophy (APECED) (1, 2). threonines, and tyrosines in these repeats are phosphorylated The encoded AIRE protein is expressed primarily in medul- by P-TEFb and other kinases in distinct phases of transcrip- lary thymic epithelial cells (mTECs) (3). AIRE is also ex- tion, giving rise to diverse set of instructions, which are http://www.jimmunol.org/ pressed in peripheral lymphoid tissues (4), where its contri- known as the CTD code (17). The phosphorylated elongating bution to tolerance remains to be investigated. The mouse 2/2 RNAPII directs cotranscriptional processing, that is, splicing model of the human APECED disease, the Aire mouse, and polyadenylation of genes (18). Transcription elongation was instrumental in identifying the cellular role of AIRE, proceeds until termination, upon which RNAPII becomes which is to regulate promiscuous expression of tissue-restricted dephosphorylated, and the cycle begins anew. Recent genome- Ag(TRA)genesinmTECs(5).Thekeyfindingwasthat 2/2 wide analyses revealed an unexpectedly high abundance of mTECs from Aire versus wild-type mice expressed fewer TRAs. Although this promiscuous gene expression in mTECs paused RNAPII at most promoters, including those of inac- by guest on September 29, 2021 had been recognized previously (6), the underlying mecha- tive genes (19–22). nism remained elusive. To date, AIRE is the only identified AIRE is a transcription factor that assembles into oligomers transcription factor that regulates this process. Among others, and forms punctate structures that colocalize with CREB- AIRE activates insulin, interphotoreceptor retinoid-binding binding protein, P-TEFb, and small nuclear ribonucleopro- protein A, and mucin 6 genes, all of which had been linked teins in the nucleus (23–25). The estimated number of AIRE- to autoimmunity in humans or AIRE-deficient mice (7, 8). regulated genes ranges from several hundred to thousands. Expressed TRAs are then processed and presented on the They have diverse promoters and are regulated by distinct surface of mTECs or taken up by dendritic cells (9, 10). Ex- transcription factors in their corresponding tissues. These posure of maturing T cells to these Ags is critical for negative findings raise the conundrum of how AIRE can regulate such selection of T cells in the thymus (11, 12). In the absence of a large repertoire of divergent genes. Recent work from others AIRE, autoreactive T cells mature and escape into the pe- and us has addressed this question and revealed the mecha- riphery, which can lead to autoimmunity (7, 8, 13). nisms of action of this enigmatic protein (26–31). Transcription of protein-coding genes occurs in several The AIRE protein has a predicted molecular mass of 58 phases, all of which are regulated (14). First, transcription kDa (Fig. 1A) (3). It forms large oligomers, which are detected factors and RNA polymerase II (RNAPII) are recruited to in a .670-kDa fraction by gel filtration (32). The N terminus promoters. Although RNAPII initiates transcription, it then of AIRE contains the homogeneously staining region (HSR) pauses because of the action of negative elongation factor and the Sp100, AIRE-1, NucP41/75, and DEAF-1 (SAND) (NELF) and 6-dichloro-1-a-D-ribofuranosylbenzimidazole sen- domain (Fig. 1A). Mutations in HSR from APECED patients

*Department of Virology, Haartman Institute, Helsinki University Central Hospital, PK, DNA-dependent protein kinase; DSIF, 6-dichloro-1-a-D-ribofuranosylbenzimidazole University of Helsinki, FIN-00014 Helsinki, Finland; and †Department of Medicine, sensitivity inducing factor; H3K4, histone H3 unmodified at Lys4; HSR, homogeneously The Rosalind Russell Medical Research Center, University of California at San Fran- staining region; NELF, negative elongation factor; PHD, plant homology domain; P- cisco, San Francisco, CA 94143 TEFb, positive transcription elongation factor b; RNAPII, RNA polymerase II; SAND, Sp100, AIRE-1, NucP41/75, and DEAF-1; TAD, transcriptional activation domain; Received for publication November 21, 2012. Accepted for publication January 8, 2013. TRA, tissue-restricted Ag. Address correspondence and reprint requests to Dr. B. Matija Peterlin, University of California at San Francisco, 533 Parnassus Avenue, Box 0703, Room U-432, San Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 Francisco, 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- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203210 2480 BRIEF REVIEWS: MECHANISM OF AIRE FUNCTION Downloaded from http://www.jimmunol.org/

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. by guest on September 29, 2021 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. disrupt multimerization, which is essential for AIRE function in proteins involved in ubiquitylation (40). PHDs are pro- (32, 33). The AIRE SAND domain is also involved in this tein–protein interaction modules, which can mediate binding oligomer formation. It lacks the canonical KDWK motif, to nucleosomes (41, 42). Such PHDs bind to N-terminal tails which is required for DNA binding of other SAND domains of histone H3, discriminating between those methylated (34). Although DNA binding of the AIRE SAND domain (H3K4me3) or unmodified (H3K4) at Lys4. AIRE PHD1 is had been reported in vitro (35, 36), it appears to be non- most closely related to the BHC80 PHD that interacts selectively specific (37) and may be irrelevant for its recruitment to target with the unmodified H3K4 (42). Indeed, AIRE PHD1 also genes in vivo. Indeed, we found that mutating KNKA resi- binds to the unmodified H3K4 (Fig. 1A), and this interaction is dues in the AIRE SAND domain, which correspond to the required for AIRE to activate transcription of genes in chro- KDWK motif in other members of this family, had no effect matin (26, 30, 37). The AIRE PHD2 sequence is divergent and on AIRE-induced expression of a plasmid reporter gene (26). does not interact with nucleosomes, but it contributes struc- In some APECED patients an autosomal-dominant G228W turally to the activation of TRA genes by AIRE (27, 31, 43). mutation was found in the SAND domain (Fig. 1A), which AIRE PHD1 also binds to the DNA-dependent protein causes the wild-type AIRE protein to coaccumulate in larger kinase (DNA-PK) (Fig. 1A) (44). DNA-PK is a nuclear ki- structures that do not colocalize with sites of active tran- nase, which not only functions to repair DNA double-strand scription (25). Thus, dominant-negative effects of this breaks and mediate V(D)J recombination, but it also supports G228W mutation could be due to an increased affinity for the transcription and chromatin remodeling (45). DNA-PK also wild-type AIRE protein. The N terminus of AIRE is required phosphorylates two sites in the N terminus of AIRE (44). for nuclear localization (38). The nuclear localization signal However, pharmacological inhibition of DNA-PK and bring- was mapped to basic residues from positions 131 to 133 (Fig. ing AIRE to a promoter via heterologous DNA tethering in 1A) (39). cells lacking DNA-PK revealedthatthekinaseactivityof AIRE also contains two plant homeodomains, plant ho- DNA-PK is dispensable for gene activation by AIRE. Instead, mology domain (PHD)1 and PHD2 (Fig. 1A). PHDs are zinc these interactions represent a key mechanism for the recruit- fingers closely related to RING domains, which are common ment of AIRE to its target genes (26). An important aspect of The Journal of Immunology 2481 this targeting is that AIRE interacts with DNA-PK, which is TEFb (28), thereby further enhancing the expression of TRA associated with the histone variant H2AX phosphorylated at genes to optimize the establishment of central tolerance. Ser139 (gH2AX) that marks DNA double-strand breaks (26). The C terminus of AIRE does not share obvious homology Conclusions with functional domains in other proteins, but it is highly AIRE is a transcription factor that activates the expression of conserved between human and mouse AIRE proteins. It serves TRA genes in mTECs. Their promoters must be occupied by as a transcriptional activation domain (TAD) (Fig. 1A) (27). It RNAPII, unmodified H3K4, and DNA-PK. Sufficient levels binds to P-TEFb and brings it to TRA genes (23, 27). This of these proteins ensure that AIRE is recruited to these sites. leads to the phosphorylation of Ser2 in the RNAPII CTD and AIRE oligomers then bring P-TEFb to RNAPII, which leads productive elongation with cotranscriptional processing of to its extensive phosphorylation. Thus modified, RNAPII is nascent mRNA species. The key role of this domain is under- competent for elongation and cotranscriptional processing of scored by an APECED patient mutation that affects only the target genes, which leads to the expression of TRAs and their extreme C terminus in AIRE (Fig. 1A) (46) but completely presentation to T cells via MHC class II determinants. abolishes 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 the Disclosures The authors have no financial conflicts of interest. TAD (Fig. 1A). LXXLL motifs are known to mediate pro- tein–protein interactions between nuclear receptors and their Downloaded from coactivators (47). Their role for AIRE remains to be estab- References 1. Finnish-German APECED Consortium. 1997. An autoimmune disease, APECED, lished. caused by mutations in a novel gene featuring two PHD-type zinc-finger domains. 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