Genomic and Functional Uniqueness of the TNF -Associated Factor Family in Amphioxus, the Basal Chordate

This information is current as Shaochun Yuan, Tong Liu, Shengfeng Huang, Tao Wu, Ling of September 26, 2021. Huang, Huiling Liu, Xin Tao, Manyi Yang, Kui Wu, Yanhong Yu, Meiling Dong and Anlong Xu J Immunol 2009; 183:4560-4568; Prepublished online 14 September 2009;

doi: 10.4049/jimmunol.0901537 Downloaded from http://www.jimmunol.org/content/183/7/4560

Supplementary http://www.jimmunol.org/content/suppl/2009/09/14/jimmunol.090153 Material 7.DC1 http://www.jimmunol.org/ References This article cites 38 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/183/7/4560.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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Genomic and Functional Uniqueness of the TNF Receptor-Associated Factor Gene Family in Amphioxus, the Basal Chordate1

Shaochun Yuan, Tong Liu, Shengfeng Huang, Tao Wu, Ling Huang, Huiling Liu, Xin Tao, Manyi Yang, Kui Wu, Yanhong Yu, Meiling Dong, and Anlong Xu2

The TNF-associated factor (TRAF) family, the crucial adaptor group in innate immune signaling, increased to 24 in amphioxus, the oldest lineage of the Chordata. To address how these expanded molecules evolved to adapt to the changing TRAF mediated signaling pathways, here we conducted genomic and functional comparisons of four distinct amphioxus TRAF groups with their human counterparts. We showed that lineage-specific duplication and rearrangement were responsible for the expansion of amphioxus TRAF1/2 and 3 lineages, whereas TRAF4 and 6 maintained a relatively stable genome and structure. Am- Downloaded from phioxus TRAF1/2 and 3 molecules displayed various expression patterns in response to microbial infection, and some of them can attenuate the NF-␬B activation mediated by human TRAF2 and 6. Amphioxus TRAF4 presented two unique functions: activation of the NF-␬B pathway and involvement in somite formation. Although amphioxus TRAF6 was conserved in activating NF-␬B pathway for antibacterial defense, the mechanism was not the same as that observed in humans. In summary, our findings reveal the evolutionary uniqueness of the TRAF family in this basal chordate, and suggest that genomic duplication and functional divergence of the TRAF family are important for the current form of the TRAF-mediated signaling pathways in humans. The http://www.jimmunol.org/ Journal of Immunology, 2009, 183: 4560–4568.

umor necrosis factor receptor-associated factors (TRAFs)3 genitor cells (3). It also binds the ste20 kinase msn and specifically are crucial adaptors linking immune receptors and inter- activates the JNK pathway when triggered by Eiger, a T mediate signal transducers to a variety of immune re- unique TNF homologue in Drosophila (4). However, human sponses and developmental processes. There are six TRAFs in TRAF4 is not associated with classical TNFRs but serves as a mammals, three in Drosophila, and one in Caenorhabditis elegans negative regulator of TRIF (Toll/IL-1R resistance domain contain- (1). The TRAF family can be classified into three major groups, ing adaptor-inducing IFN-␤)-dependent pathways in the vertebrate by guest on September 26, 2021 two ancestral groups, TRAF4 and TRAF6, and the newly evolved TLR system (5). Several studies also implicate its role in human group comprising TRAF1, 2, 3, and 5 (2). All TRAF share diseases and carcinogenesis (6, 7). a similar structure but differ from one another mainly in physio- DTRAF2, the null mutant of which shows strong immune logical function. deficiencies and affects normal dorsal-ventral (D-V) patterning DTRAF1, the ortholog of human TRAF4, is predominantly ex- in Drosophila embryos, physically and functionally interacts pressed during nervous system development and in epithelial pro- with Pelle to mediate the Toll-MyD88-Dorsal pathway (8, 9). Humans TRAF6, the ortholog of DTRAF2, has not only con- served its ancestral role in TLR but also State Key Laboratory of Biocontrol, National Engineering Research Center of South China Sea Marine Biotechnology, Department of Biochemistry, College of Life Sci- adapted to the increasingly complex TNF system, having di- ences, Sun Yat-sen (Zhongshan) University, Guangzhou, People’s Republic of China verse functions from immunity to development processes, such Received for publication May 15, 2009. Accepted for publication July 17, 2009. as the development of lymph nodes, skin, and the CNS (10). In The costs of publication of this article were defrayed in part by the payment of page addition, human TRAF6 can be recruited by the IL-1R subfam- charges. This article must therefore be hereby marked advertisement in accordance ily that binds specific IL-1-related cytokines through their ex- with 18 U.S.C. Section 1734 solely to indicate this fact. tracellular Ig-like structures to participate in multiple immuno- 1 This work was supported by Project 2007CB815800 of the National Basic Research Program (973), Projects 2006AA09Z433 and 2008AA092603 of the State High-Tech logical and inflammatory processes (11). Development Project (863), Project 2007DFA30840 of International S&T Coopera- Although knowledge of the functions of DTRAF3 remains tion Program from the Ministry of Science and Technology of China, a key project (0107) from the Ministry of Education, a key project of Commission of Science and elusive, those of TRAF1, 2, 3, and 5 are well documented in Technology of Guangdong Province and Guangzhou City, a project of Sun Yet-sen humans. Upon differing TNFR engagement, TRAF2 and 5 are University Science Foundation, and the First Degree Supporting Project 20080440119 involved in the activation of JNK and NF-␬B signaling (12, 13), of China Postdoctoral Science Foundation. 2 whereas TRAF3 functions as an inhibitor of TRAF2- or 5-me- Address correspondence and reprint requests to Dr. Anlong Xu, Department of ␬ Biochemistry, College of Life Sciences, Sun Yat-Sen (Zhongshan) University, 135 diated activation of the noncanonical NF- B pathway (14, 15). XinGangXi Road, 510275, Guangzhou, People’s Republic of China. E-mail address: In addition to the cooperation with classical TNFRs, new stud- [email protected]. ies highlight their crucial roles in the induction of type I IFN, 3 Abbreviations used in this paper: TRAF, TNF-associated factor; Amphi, refers to all the critical component of innate defense against viral infections. amphioxus species; Bbt, Branchiostoma belcheri tsingtauense; Bf, Branchiostoma floridae; BC, bacterially challenged; ORF, open reading frame; TIR, Toll/IL-1R re- For example, TRAF2 participates in the antiviral action of IFN sistance domain; TRIF, Toll/IL-1R resistance domain containing adaptor-inducing by rapidly binding to the IFNAR1 subunit (16). TRAF3 directly IFN-␤; UC, unchallenged. and specifically interacts with several components of antiviral Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 pathways, such as TRIF, TBK1, and Cardif, demonstrating its www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901537 The Journal of Immunology 4561 unique role as a positive regulator of type I IFN production (17–19). It is clear that functions of TRAFs vary substantially among groups and between Drosophila and humans, thus it is of par- ticular interest to discover how these conserved adaptors in- creased in number and evolved their protein structures to adapt to the changing functions involved in TRAF-mediated path- ways, progressing from Drosophila to humans via intermediate species. One of the key intermediate species, amphioxus, rep- resents the oldest lineage of the chordata and contains 24 TRAFs, 48 TLRs, and a sophisticated TNF system not observed in other invertebrates (20). Thus, investigation into amphioxus TRAFs is not only of importance in revealing the novel func- tions of this complex defense systems in this primitive species but also will help to shed on the evolution and develop- ment of TRAF-mediated signaling in humans.

Materials and Methods

Adults and embryos, cells, and reagents Downloaded from Adult Chinese amphioxus (Branchiostoma belcheri tsingtauense (Bbt)) were obtained from Qingdao, China. Samples of the blastula, gastrula, neurula, and larva were collected in the breeding season. Human HEK293T or Hela cells were grown in DMEM (Invitrogen) supplemented with 10% FCS and antibiotics. LPS of E. coli 0111:B4 was purchased from Sigma-Aldrich.

FIGURE 1. Sequences analysis and comparison of amphioxus TRAF http://www.jimmunol.org/ Sequence data family. A, Domain topology comparison of 11 amphioxus TRAF1/2 mem- bers, which were arrayed on scaffold_21. B, Domain topology comparison The draft genome sequence of the amphioxus Branchiostoma floridae (Bf) of TRAFs among Drosophila, amphioxus, and human. Gene identification and the corresponding gene predictions can be accessed on the Branchios- was mainly performed with the HMMER2.0 plus SMART data set access- toma floridae version 1.0 website of the Joint Genome Institute, U.S. De- partment of Energy (http://genome.jgi-psf.org/Brafl1/Brafl1.home.html). Se- ing on the Branchiostoma floridae version 1.0 website of the Joint Genome quences of four amphioxus TRAFs were deposited in the National Center for Institute, U.S. Department of Energy (see Materials and Methods). The Biotechnology Information database and assigned as BbtTRAF2a RING finger and the TRAF domains were predicted by SMART main page (EF210343), BbtTRAF3a (EF210344), BbtTRAF4 (EF210345), and of website, whereas the zinc finger domains were manually predicted by BbtTRAF6 (EF210346). sequences alignment. Regarding the scaffold_21, the gray triangles indi- cated the down-regulated after challenge by 454 sequencing, by guest on September 26, 2021 Cloning of BbtTRAFs cDNAs and genomic sequences whereas the black triangles made the contrary meaning. Full-length cDNAs of BbtTRAF2a, 3a, 4, and 6 were obtained by RT-PCR, 5Ј-RACE and 3Ј-RACE using gene specific primers according to the Gene- tag and cloned into pcDNA3.0. The TRAF domain only (2CT, 3CT, 4CT, and RACE Kit (Invitrogen). Genomic sequences of BbtTRAF2a, 3a, 4, and 6 6CT) of four BbtTRAFs were fused with Flag tag and HA tag separately and were amplified by specific primers based on their open reading frame cloned into pcDNA3.0. For functional comparison, the ORFs of human (ORFs) (shown in supplemental Table S1).4 TRAF2, 3, 4, and 6 (h2P, h3P, h4P, and h6P), and segments without the N-terminal RING and zinc fingers of human TRAF2 and 6 (hsTRAF2 DN and Southern blot analysis of genomic DNA hsTRAF6 DN) were fused with Flag tag and cloned into pcDNA3.0. Genomic DNA from two individuals was separately digested by the same Transient transfection and luciferase reporter assay enzyme and then transferred to Hybond Nϩ nylon membranes (Amersham Biosciences). DNA probes for BbtTRAF2a and BbtTRAF3a were ampli- HEK293T cells (2 ϫ 105 cells per well) in 24-well plates were transiently fied by primers shown in Table S1 and labeled according to the DIG DNA transfected with 1 ␮g of DNA plasmids, including the indicated amount of Labeling System Kit (Roche). After overnight hybridization and stringent detected gene plasmids, 200 ng of indicated luciferase reporter plasmid, washing, the nylon membranes were immunodetected according to the 100 ng of ␤-galactosidase gene, and the supplemented empty vectors. At manufacturer’s protocol. 24-h posttransfection, cells were lysed, and luciferase activities were mea- sured using the luciferase reporter assay (Promega). The luciferase activity Section and whole-mount in situ hybridization was normalized to ␤-galactosidase activity and expressed as the fold stim- ulation relative to empty vector-transfected cells. Values were expressed as Digoxigenin-labeled sense and antisense probes for BbtTRAF4 and Bbt- mean relative stimulation for a representative experiment from three sep- TRAF6 were synthesized by primers shown in supplemental Table S1 us- arate experiments each performed in duplicate. ing Dig RNA synthesis (Roche). After a series of disposals, the probes of sense or antisense were added to the sections at a concentration of 1 Coimmunoprecipitation ␮g/ml, and the sections were hybridized overnight at 42°C. After high- stringency washing, hybridization signals were immunodetected. Whole- HEK293T cells in 6-well dishes (5 ϫ 106 cells per well) were transfected mount in situ hybridizations for embryos at different developmental stages with 2 ␮g of DNA plasmids (1 ␮g/each expression vector). At 48-h post- were conducted according to the protocol of Holland (21). transfection, cells were lysed and incubated with primary Abs (4 ␮gof anti-FLAG [M2] (Sigma-Aldrich)) at 4°C overnight, then incubated with Expression plasmids protein G-Sepharose for an additional4hat4°C. Analysis was conducted using SDS-PAGE followed by Western blot using the ECL protocol (Am- For confocal analyses, the ORFs of four BbtTRAFs were inserted into ersham Biosciences), with anti-HA (1/1000) and anti-Flag (1/1000) mAb. pEGFP-N1 vector, respectively. For reporter assays and CO-IP tests, the ORFs of four BbtTRAFs (2P, 3P, 4P, and 6P), the truncated mutant without the RING finger domain (6ZT) of BbtTRAF6, and the truncated mutant Results without the TRAF domain (6RZ) of BbtTRAF6 were all fused with Flag TRAF family in amphioxus A search of the amphioxus genome in our previous study iden- 4 The online version of this article contains supplemental material. tified 24 TRAF gene models, including one corresponding to 4562 GENOMIC AND FUNCTIONAL UNIQUENESS OF AMPHIOXUS TRAF FAMILY

homologues on scaffold_351 showed the similar protein struc- tures, we used the MEGA3.1 software to analyses the simple selections among these sequences by dN/dS ratio test, which is one of the most widely used statistical tests to quantify the selection pressures by comparing the rate of substitutions at synonymous sites (dS) to the rate of substitutions at nonsyn- onymous sites (dN) (22).The overall dN/dS value of the TRAF domain was close to zero, indicating that these regions may be dominated by purifying selection, whereas the dN/dS ratio of the RING and zinc finger region was two folds higher than those of the TRAF domain, indicating that these regions are under more unfixed pressure (supplemental Fig. S2).

Protein structure comparison of four amphioxus TRAFs with their counterparts in Drosophila and human To gain further insight into this uniquely expanded TRAF family, four representatives from each TRAF group were isolated from the intestinal cDNA library of Chinese amphioxus Bbt and designated as BbtTRAF2a, 3a, 4, and 6, respectively. The similarity among Downloaded from four BbtTRAFs and several classical TRAFs ranged from 23 to 66% (supplemental Fig. S3A). Although all four BbtTRAFs showed similar domain topology with their human counterparts, they were different from those of insects (Fig. 1B). First, both BbtTRAF4 and human TRAF4 contained a RING finger whereas DTRAF1 did not. Second, BbtTRAF6 and human TRAF6 had five http://www.jimmunol.org/ FIGURE 2. Genomic structure and rearrangement of amphioxus TRAF zinc fingers, whereas DTRAF2 had only two such domains. In family. A, Southern blot analyses of BbtTRAF2a and 3a with digoxygenin- addition, BbtTRAF6 had a large insertion at the linker region be- labeled cDNA probes indicated that they were heavily duplicated and tween the zinc finger and the N-TRAF region. Similar to the or- highly polymorphic in amphioxus. Genomic DNA in each lane was extracted ganization in humans, RING finger domains of BbtTRAF2a, 3a, 4, from single amphioxus. B, Comparison of intron-exon organizations of four and 6 were organized as CX2CX11,12CX1HX2CX11CX2C, but cer- amphioxus TRAFs between two different species, the Bbt, and Bf. C, Com- tain insertions and deletions were observed in the zinc finger re- parison of intron-exon organizations of two closed TRAF3 members on Bf gions (supplemental Fig. S3, B and C). Collectively, amphioxus genome. Numbers indicate the length in the unit of base pairs. The hollow TRAF family members were more similar to their human coun- by guest on September 26, 2021 rectangles indicated the splicing differences between two sequences. terparts than to those in Drosophila, indicating that the TRAF fam- ily, in this early chordate, has established a basic structure for the TRAF6, one to TRAF4, four to TRAF3, and 16 to TRAF1/2 development of TRAF-mediated network signaling in humans. (20). The TRAF4 and TRAF6 subgroups represented the most ancient lineages, whereas TRAF1, 2, 3, and 5 lineages diverged Genomic rearrangement of amphioxus TRAFs from the common ancestor before the amphioxus-vertebrate Southern blot analyses confirmed that BbtTRAF2a and 3a were separation (supplemental Fig. S1). In this study, our analyses multiple duplicated genes in the amphioxus genome, whereas suggested that 11 amphioxus TRAF1/2 lineage members were the most unexpected finding was that the genomic DNA from arrayed on scaffold_21, and 4 amphioxus TRAF3 homologues two individuals showed different band patterns (Fig. 2A). To on scaffold_251 (Fig. 1A). Although these TRAF1/2 molecules investigate the reason for this observation, we conducted were newly generated by lineage-specific duplication, they dif- genomic comparison of amphioxus TRAF2a, 3a, 4, and 6 in two fered from one another in protein structures (Fig. 1A). Protein species, the Bbt and the Bf. The BbtTRAF2a had intron-exon structures of the N-terminal RING and zinc finger regions were organization similar to that of BfTRAF2a, whereas Bbt- more variable than those of the TRAF domain, indicating that TRAF3a, 4, and 6 contained fewer exons than those from Bf these newly generated TRAFs may be associated with the same (Fig. 2B). The significant difference was observed in the pattern upstream proteins but cooperate with distinct downstream mol- of splice junction in the RING and zinc finger domains. We also ecules to convey diverse immune signals. Since four TRAF3 compared the genomic organization of two newly duplicated

Table I. Distinct expression patterns of amphioxus TRAF molecules after bacterial challengea

TRAF1/2 Lineage_scaffold_21

Gene Model 68986 68989 68990 68991 68992 68993 68994 68995 68997 Transcripts (UC-BC) Ϫ43Ϫ300Ϫ105Ϫ1

TRAF1/2 Lineage TRAF3 Lineage TRAF4 TRAF6

Gene Model 107021 107017 99999 237951 237984 238009 115711 69354 Transcripts (UC-BC) 0 2 19 110133

a Gene model indicated EST sequences matching the corresponding JGI gene models. Transcript difference was obtained by comparing the transcript numbers between the BC library and the UC library. The Journal of Immunology 4563

FIGURE 3. BbtTRAF4 is a novel somite formation-related protein. A and B, Blastopore view and side view of late gastrula showed the expression of BbtTRAF4 in the dorsal presomitic mesoderm on either side. C and D, Side view and dorsal view of late neurula showed BbtTRAF4 expression in the Downloaded from somites. E–G, Side view of 20-h larva, 24-h larva, and 48-h larva showed BbtTRAF4 expression in the posterior somites and tail bud. Scale bar was 100 ␮m for whole embryos.

TRAF3 homologues arrayed on scaffold_251 (models 237951 confirmed that most of the predicted TRAFs were real-tran- and 238009). Results showed that, although they had the same scribed genes and may play nonredundant roles in antimicrobial

protein structure and intron-exon organization, their introns infection. http://www.jimmunol.org/ were altered in size (Fig. 2C). In short, our comparisons implied Similar to their human counterparts, the GFP-tagged BbtTRAF2a that the genomic region of RING and zinc fingers was less and 3a were localized mainly to the cytoplasm. Round patches stable than that of the TRAF domain and that the loci of TRAF2 were also observable when cells expressed high amounts of and TRAF3 were unstable and highly polymorphic, facilitating ectopic BbtTRAF2a- or 3a-GFP proteins in Hela cells (see Fig. the gene expansion when they were initially derived. 5, A and B). We further showed that overexpression of full- length BbtTRAF2a alone did not activate the NF-␬B or IFN-␤ Functional characterizations of amphioxus TRAF1/2 and pathway in 293T cells, whereas human TRAF2 induced Ͼ50- 3 lineages fold activation of NF-␬B and 10-fold induction of IFN-␤ (see Because it was difficult to distinguish among copies of am- Fig. 5, E and F). Neither BbtTRAF3a nor human TRAF3 alone by guest on September 26, 2021 phioxus TRAF1/2 and TRAF3 homologues through in situ hy- showed an effect on NF-␬B activation or IFN-␤ induction (data bridization and real time PCR, we analyzed a total of 400,000 not shown). However, human TRAF3 enhanced IFN-␤ induc- EST sequences from two intestinal cDNA libraries of adult am- tion through human TBK1 whereas BbtTRAF3a did not phioxus Bbt by 454-pyrosequencing, the bacterially challenged (Fig. 5G). (BC) library and the unchallenged (UC) library (S. Huang, un- ␬ published data). First, we identified 11 predicted TRAF1/2 Amphioxus TRAF4 not only activates the NF- B pathway but is genes and all four TRAF3 homologues. We then compared the also involved in the somite formation transcript differences between these two libraries and found that Because TRAF4 is unique among TRAF family in showing a expression of 3 TRAF1/2 (gene model: 68995, 68989, and restricted expression pattern during embryogenesis, we per- 107017) and 2 TRAF3 genes (gene model: 99999 and 237951) formed whole-mount in situ hybridization to detect the temporal were significantly increased in the BC library, whereas tran- expression pattern of this conserved molecule in amphioxus. scripts of two TRAF1/2 homologues (gene model: 68986 and Transcripts of BbtTRAF4 were first detected in the presomitic 68990) were reduced (Table I and Fig. 1). Thus, these findings mesoderm of the late gastrula and reached the highest level in

FIGURE 4. Tissue distribution of BbtTRAF4 and 6. A and B, Section in situ hybridization analyses of BbtTRAF4 antisense probe (A) and BbtTRAF6 antisense probe (B) showed that they were predominantly expressed in amphioxus digestive system. C, Macroscopic view of the hybridization signals of BbtTRAF6 in the intestine and mature oocytes. D, Section in situ hybridization analyses of BbtTRAF6 using sense probe as negative control. n is notochord, I is intestine, and g is gonad. Scale bar for A, B, and D was 500 ␮m, except for C, which was 200 ␮m. 4564 GENOMIC AND FUNCTIONAL UNIQUENESS OF AMPHIOXUS TRAF FAMILY Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 5. Subcellular localization and downstream signaling comparison of BbtTRAFs with their human orthologs. A–D, Subcellular localization of four BbtTRAFs in Hela cells. A presented for BbtTRAF2a, B for BbtTRAF3a, C for BbtTRAF4, and D for BbtTRAF6. Scale bar was 25 ␮m. E and F, Compared with human TRAF2, amphioxus TRAF2 alone in 293T cells was not able to activate either NF-␬B or IFN-␤ responses. G, BbtTRAF3 in 293T cells did not enhance the IFN-␤ induction through human TBK1, whereas human TRAF3 can augment the induction to a higher level. H, Name and structure of fusion proteins used in this study. 2P, 3P, 4P, and 6P indicated the vectors containing ORFs corresponding to BbtTRAF2a, 3a, 4, and 6, whereas 2CT, 3CT, 4CT, and 6CT corresponding to the vectors just containing the TRAF domains. 6ZT and 6RZ indicated the vector without RING finger of BbtTRAF6 and the vector without the TRAF domain of BbtTRAF6, respectively. h2P, h3P, h4P, and h6P indicated the vectors containing the ORFs of human TRAF2, 3, 4, and 6, whereas hsTRAF2 DN and hsTRAF6 DN corresponded to the truncated mutants without the N-terminal RING and zinc fingers of human TRAF2 and 6. I, BbtTRAF4 was able to activate the NF-␬B response in 293T cells whereas human TRAF4 could not. J and K, Compared with human TRAF6, BbtTRAF6 activated NF-␬B response at a lower level but did not participate in IFN-␤ production in 293T cells. the developing and nascent somites in neurula. Subsequently, Section in situ hybridization revealed that transcripts of BbtTRAF4 the expression of BbtTRAF4 was rapidly down-regulated and were concentrated in the digestive system, which is thought to be the maintained in the anterior and posterior somites of the neurula. first line of defense for adult amphioxus (Fig. 4A). Real-time PCR At 48 h, BbtTRAF4 was detected only in the developing tail showed that transcripts of BbtTRAF4 were weakly up-regulated after bud (Fig. 3). Our data demonstrated that BbtTRAF4 may play LPS challenge (supplemental Fig. S4). Moreover, BbtTRAF4 was a key role in somite formation in amphioxus rather than func- capable of activating NF-␬B responses in 293T cells, although its tion in epithelial progenitor cells and nerve system demon- human counterpart alone did not (Fig. 5I). All these observations im- strated for other species. plied a role of BbtTRAF4 in amphioxus antibacterial defense. The Journal of Immunology 4565

The ectopic GFP-tagged human TRAF4 proteins were local- ized to cytoplasm and barely detected in nuclear, whereas the endogenous human TRAF4 proteins were found mainly in the nuclear lysates (23). Parallel confocal microscopy showed that the ectopic GFP-tagged BbtTRAF4 proteins also localized to the cytoplasm in a few and round patches in Hela cells (Fig. 5C). However, we could not determine whether endogenous BbtTRAF4 can stay in nuclear because of the lack of amphioxus cell lines at present. Functional characterization of amphioxus TRAF6 Similar to BbtTRAF4, transcripts of BbtTRAF6 were detected strongly in the epithelial cells of gut villi and midgut diverticuli and in connective tissue and coelomic cells (Fig. 4, B and C). Real-time PCR also showed that transcripts of BbtTRAF6 were up-regulated after LPS challenge (supplemental Fig. S4). How- ever, whole-mount in situ hybridization of BbtTRAF6 revealed ubiquitous expression, with no clear pattern during embryogenesis

(data not shown). Compared with human TRAF6, whose ectopic Downloaded from overexpression resulted in the localization to the general cyto- plasm and punctate cytoplasmic structures (24, 25), the GFP- FIGURE 6. Analysis of BbtTRAF6 activities and its relation with other amphioxus TRAFs. A, Activity analyses of BbtTRAF6-truncated mutants tagged BbtTRAF6 appeared to accumulate to large patches around on NF-␬B activation. B, BbtTRAF2a negatively regulated BbtTRAF6 me- the nuclear in Hela cells based on confocal microscopy (Fig. 5D). diated NF-␬B activation. C, Full-length BbtTRAF6 formed a hetero-mul- ␬ In addition, BbtTRAF6 alone activated NF- B at a lower level but timer with full-length BbtTRAF2a. D, BbtTRAF6 formed homo- or het-

did not induce IFN-␤ production (Fig. 5, J and K). eromultimer with BbtTRAF2a through its TRAF domain. http://www.jimmunol.org/ To investigate which domain was essential for the activation of the NF-␬B pathway by BbtTRAF6, several truncated mutants were constructed for reporter assays (Fig. 5H). The truncated mutant via BbtTRAF6 (Fig. 7, D and E). We also assumed that without the RING finger domain of BbtTRAF6 (6ZT) activated BbtTRAF2a, 3a, and 6 may form heteromultimer with human NF-␬B response at a lower level than the full-length vector, TRAF6 via their TRAF domains (Fig. 7F). whereas the truncated mutant without the TRAF domain (6RZ) or only with the TRAF domain (6CT) did not make the same acti- Discussion vation (Fig. 6A). In addition, activation through full-length Bbt-

Expansion and dynamic structure modulation of amphioxus by guest on September 26, 2021 TRAF6 was down-regulated when cotransfected with vector 6RZ, TRAF family but unexpectedly was enhanced by vector 6CT (Fig. 6A). To determine the relationship between BbtTRAF6 and the other The TRAF family is generally thought to have undergone a routine three amphioxus TRAFs, BbtTRAF2a, 3a, 4, and their TRAF do- evolutionary process increasing from three in Drosophila to six in main only were individually coexpressed with BbtTRAF6. Besides humans. However, this view is challenged by the fact that the BbtTRAF2a and its TRAF domain only constructs (2P and 2CT), TRAF family increased to 24 members in amphioxus, 7 in sea neither BbtTRAF3a nor 4 influenced the effect of BbtTRAF6 on urchin, and 8 in zebra fish. Although these intermediate species all NF-␬B activation (Fig. 6B). Further coimmunoprecipitation tests contain a comparable large number of TRAF genes, the huge ex- confirmed that BbtTRAF6 could form heteromultimer with pansion of the TRAF1/2 and TRAF3 lineages was just observed in BbtTRAF2a via their TRAF domain (Fig. 6, C and D). Coim- amphioxus (20, 26, 27). Usually, a complex genome contains a munoprecipitation test also confirmed that BbtTRAF6 was able greater number of gene family members to increase complexity to form homomultimer via its TRAF domain (Fig. 6D). In sum- and functional specificity, suggesting that expansion of the mary, to activate NF-␬B responses, BbtTRAF6 has to form ho- TRAF1/2 and TRAF3 lineages at the transition stage from inver- momultimer to exert its function. Moreover, these activities tebrates to vertebrates may have been due to new functional re- could be regulated by forming heteromultimer with other TRAF quirements and/or a new body plan (28). Despite lineage specific protein. expansion, genomic structures of amphioxus TRAFs differed among individuals and between two amphioxus species. The most BbtTRAFs negatively regulate the activities of human TRAF2 variable region among various TRAFs appeared in the pattern of and BbtTRAF6 splicing junction of the RING and zinc finger domains, which de- To determine whether BbtTRAFs interact with human TRAF2 and termine the downstream signaling activities for distinct TRAF pro- 6 to regulate their activities, human TRAF2 was coexpressed with teins (29, 30). Thus, the evolutionary development of the TRAF1/2 2CT, 3CT, and 6CT (Fig. 5H). Results showed that the 2CT and and TRAF3 lineages is not just restricted to increase in number but 3CT inhibited the NF-␬B activation by human TRAF2 whereas also includes dynamic structure modulation, which is important for 6CT did not (Fig. 7A). In addition, human TRAF2-DN did not conveying diverse signaling when recruited by different receptors. inhibit NF-␬B activation through BbtTRAF6 (Fig. 7B). To deter- mine the reason for such inhibition, coimmunoprecipitation tests Functional innovation of TRAF1/2 and 3 lineages were performed to demonstrate that BbtTRAF2a and 3a interacted Because transcripts of BbtTRAF2a are up-regulated in a Staphy- with human TRAF2 via their TRAF domains but BbtTRAF6 did lococcus aureus-challenged suppression subtractive hybridization not (Fig. 7C). Likewise, TRAF domain only mutants of library (31), and transcripts of BbtTRAF3a (gene model: 99999) BbtTRAF2a, 3a, and 6 suppressed NF-␬B activation through hu- and the other TRAF3 homologue (model:237951) increase after man TRAF6, and human TRAF6-DN inhibited NF-␬B activation bacterial challenge by 454 sequencing technology, the TRAF1/2 4566 GENOMIC AND FUNCTIONAL UNIQUENESS OF AMPHIOXUS TRAF FAMILY

FIGURE 7. BbtTRAFs regulated the NF-␬B activity induced by human TRAF2 and 6 in a mammalian cell line. A, BbtTRAF2a and 3a negatively reg- ulated the NF-␬B activation induced by human TRAF2. B, Human TRAF2- DN(‚1–272), which contained an de- letion of the RING finger and five zinc fingers, inhibited the NF-␬B activation induced by human TRAF2 but not BbtTRAF6. C, Coimmunoprecipitation confirmed that BbtTRAF2a and 3a formed a heteromultimer with human TRAF2 through their TRAF domain whereas BbtTRAF6 could not. D, BbtTRAF2a, 3a, and 6 negatively reg- ulated the NF-␬B activation induced by human TRAF6. E, Human TRAF6- DN(‚1–282), which contained an de- Downloaded from letion of the RING finger and five zinc fingers, inhibited the NF-␬B activation induced by both human TRAF6 and BbtTRAF6. F, Coimmunoprecipitation confirmed that human TRAF6 strongly interacted with BbtTRAF2a, 3a, and 6. http://www.jimmunol.org/

and 3 lineages should play nonredundant roles in antibacteria de- Changing physiological and cellular roles of TRAF4 fense in amphioxus. Although BbtTRAF2a and BbtTRAF3a alone DTRAF1, most closely related to mammalian TRAF4, is predom- have no effect on NF-␬B activation in human 293T cell lines, they inantly expressed during normal eye development and recruited by are negative regulators of this conserved pathway when mediated Eigen-Wengen pathway (32, 33). Mouse TRAF4 is observed

by human TRAF2 or amphioxus TRAF6, much as human TRAF3 by guest on September 26, 2021 throughout embryogenesis, most notably during ontogeny of the serves as a negative regulator for TRAF2 or 5 in the nonclassical CNS and peripheral nervous system (34). In this study, we found NF-␬B pathway (14, 15). In addition, members of amphioxus TRAF1/2 and 3 lineages are disparate in domain architectures, that BbtTRAF4 is involved in somite formation, especially in the whose tiny differences may affect their activities on downstream posterior somite until the tail bud formed, suggesting that the func- signaling. Therefore we can assume that some of these duplicated tion of TRAF4 in the nervous system is not conserved in molecules may have positive effort on the NF-␬B activation. Since amphioxus. amphioxus has developed a novel innate defense system by ex- DTRAF1 is restricted to utilization by the Eiger-Wengen-JNK panding its immune receptors and intermediate signal transducers pathway, whereas vertebrate TRAF4 is not involved in classical (20), we can also assume that if the pattern recognition receptors TNFR signaling. Only one study has reported that human TRAF4 distinguish among pathogen-associated molecular patterns and use may participate in the TRIF-dependent pathway (5). Our current distinct TRAF adaptors for downstream signaling regulation, this study did not indicate whether amphioxus TRAF4 is directly in- novel defense system should be comparable to vertebrate adaptive volved in TNFR or TLR signaling. What we have found was that immunity. amphioxus TRAF4 alone already has obtained its NF-␬B activity,

FIGURE 8. Genomic and func- tional comparison of the TRAF-me- diated signaling pathways among Drosophila, amphioxus, and human. The Journal of Immunology 4567

which has not been observed in Drosophila. In addition, transcripts thermore, our genomic and functional comparisons suggest that the of BbtTRAF4 were abundant in the amphioxus digestive system increase in members of the TRAF family, with functional differ- and up-regulated after LPS challenge, indicating that BbtTRAF4 entiation, is critical to the current form of vertebrate TRAF-medi- may be a component of amphioxus defense system by activating ated signaling pathways, corresponding to the extension of TNF NF-␬B pathway. In summary, our observations indicated that and TLR systems (Fig. 8). physiological roles of TRAF4 have changed and differed widely among phyla, although it is thought to be the most conserved adap- Acknowledgments tor in the TRAF family. We thank Dr. Hong Tang for his gift of human TBK1, human TRAF2, 3, and 6 plasmids. Vectors related to these genes were reconstructed based on Functional similarities and differences of TRAF6 among these original constructions. We also thank Dr. Hong Tang for his con- Drosophila, amphioxus, and humans structive comments on the functional comparison. Upon stimulation by distinct TLRs, cascades through human Disclosures TRAF6 can result in anti-bacterial (TLR4) and anti-viral defense The authors have no financial conflict of interest. (TLR7/8/9) responses but have no apparent roles in normal devel- opment (10, 35). In Drosophila, the MyD88-Pelle-DTRAF2 cas- References cade specifically relays the signal for anti-bacterial defense and 1. Bradley, J. R., and J. S. Pober. 2001. receptor-associated normal D-V axis patterning but not for antiviral response (8). At factors (TRAFs). Oncogene 20: 6482–6491. 2. Grech, A., R. Quinn, D. Srinivasan, X. Badoux, and R. Brink. 2000. Complete the transition stage, amphioxus TRAF6 was found to be abundant structural characterisation of the mammalian and Drosophila TRAF genes: im-

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