Tài liệu Báo cáo khoa học: Identification and characterization of the transcription factors involved in T-cell development, t-bet, stat6 and foxp3, within the zebrafish, Danio rerio docx

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Identification and characterization of the transcriptionfactors involved in T-cell development, t-bet, stat6 andfoxp3, within the zebrafish, Danio rerioSuman Mitra, Ayham Alnabulsi, Chris J. Secombes and Steve BirdScottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, UKIntroductionNaive CD4+T-cells, on antigenic stimulation, becomeactivated, expand and differentiate into different effec-tor subsets called T-helper (Th) cells. The differentia-tion of naive T-cells into Th effector cells depends ona variety of stimuli, such as antigen nature, antigendose and the strength and duration of signals throughthe T-cell receptor (TCR)–CD3 complex, as well as thecytokine microenvironment which activates the cellularsignalling pathways [1]. These Th cell subsets are cru-cial for the induction of the most appropriate immuneresponse towards a particular pathogen. In mammals,three types of CD4+Th effector cell populations exist,Th1, Th2 and Th17, characterized by their cytokinerepertoire and how they regulate B-cell and T-cellKeywordsadaptive immunity; fish immunology; T-cells;transcription factors; zebrafishCorrespondenceS. Bird, Scottish Fish Immunology ResearchCentre, School of Biological Sciences,Zoology Building, University of Aberdeen,Aberdeen AB24 2TZ, UKFax: +44 1224 272396Tel: +44 1224 272881E-mail: s.bird@abdn.ac.uk(Received 25 August 2009, revised16 October 2009, accepted 27 October2009)doi:10.1111/j.1742-4658.2009.07460.xThe discovery of cytokines expressed by T-helper 1 (Th1), Th2, Th17 andT-regulatory (Treg) cells has prompted speculation that these types ofresponses may exist in fish, arising early in vertebrate evolution. In thisinvestigation, we cloned three zebrafish transcription factors, T-boxexpressed in T cells (t-bet), signal transducer and activator of transcription6(stat6) and fork-head box p3 (foxp3), in which two transcripts are pres-ent, that are important in the development of a number of these cell types.They were found within the zebrafish genome, using a synteny approach inthe case of t-bet and foxp3. Multiple alignments of zebrafish t-bet, stat6and foxp3 amino acids with known vertebrate homologues revealed regionsof high conservation, subsequently identified to be protein domains impor-tant in the functioning of these transcription factors. The gene organiza-tions of zebrafish t-bet and foxp3 were identical to those of the humangenes, with the second foxp3 transcript lacking exons 5, 6, 7 and 8. Zebra-fish stat6 (21 exons and 20 introns) was slightly different from the humangene, which contained 22 exons and 21 introns. Immunostimulation ofzebrafish head kidney and spleen cells with phytohaemagglutinin, lipo-polysaccharide or Poly I:C, showed a correlation between the expression oft-bet, stat6 and foxp3 with other genes involved in Th and Tregresponsesusing quantitative PCR. These transcription factors, together with many ofthe cytokines that are expressed by different T-cell subtypes, will aid futureinvestigations into the Th and Tregcell types that exist in teleosts.Abbreviationsfoxp3 ⁄ Foxp3, fork-head box p3; IFN-c, interferon-c; IL, interleukin; LPS, lipopolysaccharide; OSBPL7, oxysterol-binding protein-like 7; PHA,phytohaemagglutinin; PPP1R3F, protein phosphatase 1, regulatory (inhibitor) subunit 3F; RACE, rapid amplification of cDNA ends;stat6 ⁄ STAT6, signal transducer and activator of transcription 6; t-bet ⁄ T-bet, T-box expressed in T cells; TCR, T-cell receptor; TGF-b,transforming growth factor-b; Th, T-helper; Treg, T-regulatory.128 FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBSresponses [2]. Th1 cells produce interferon-c (IFN-c)and lymphotoxin, activating cell-mediated immunityand providing protection against intracellular patho-gens and viruses. Th2 cells secrete interleukin-4 (IL-4),IL-13 and IL-25 (also known as IL-17E), which areimportant in the generation of the correct class ofantibodies by B-cells, and for the elimination ofextracellular pathogens, such as helminths and otherextracellular parasites [2]. Th17 is the most recentlyidentified Th cell subset and secretes pro-inflammatorycytokines, such as IL-17A, IL-17F, IL-21 and IL-22[3,4]. Th17 cells play an important role in host defenceagainst extracellular pathogens, in particular extra-cellular bacteria, which are not efficiently cleared byTh1- and Th2-type immunity [5]. Finally, in additionto Th cells, there is a population of CD4+T-cells thatis involved in the regulation of Th responses via thesecretion of cytokines, called T-regulatory (Treg) cells,which help to inhibit harmful immunopathologicalresponses directed against self- or foreign antigens[6,7]. The majority of these cells constitutively expressthe CD25 cell surface marker and secrete two powerfulanti-inflammatory cytokines: IL-10 and transforminggrowth factor-b (TGF-b).Whether a naive T-cell becomes a Th1, Th2, Th17or Tregcell is influenced by the cytokines that are pro-duced within the microenvironment, which, in turn,influence transcription factors and key signalling path-ways [8]. Th1 differentiation is initiated by coordinatesignalling through the TCR and cytokine receptors,for cytokines such as type I and II IFNs or IL-27,which are associated with STAT1 [9,10]. Activation ofSTAT1 induces the transcription factor, T-boxexpressed in T cells (T-bet), which is a master regula-tor of Th1 differentiation [11]. T-bet potentiates theexpression of IFN-c, which, in turn, upregulates theinducible chain of the IL-12 receptor (IL-12Rb2).Binding of IL-12 to its receptor induces signallingthrough STAT4, which further enhances IFN-c pro-duction and induces the expression of IL-18Ra, allow-ing the responsiveness of these now mature Th1 cellsto IL-18 [12]. Th2 differentiation is initiated by TCRsignalling, together with IL-4 receptor signalling viasignal transducer and activator of transcription 6(STAT6). This, in turn, up-regulates the low-levelexpression of GATA3, the master regulator of Th2 dif-ferentiation [13]. GATA3 autoactivates its own expres-sion, eventually enabling mature Th2 cells to expressthe Th2 cytokine cluster, IL-4, IL-5 and IL-13, as aresult of epigenetic changes [14]. Th1 and Th2 cellsnegatively regulate each other’s development. GATA3suppresses STAT4 and the IL-12Rb2 chain expression,factors which are critical to the Th1 pathway [15],whereas IL-27 suppresses Th2 development [16].Th17 differentiation is slightly more complexbecause of differences between mice and humans [17].In mice, Th17 differentiation is initiated by TCRsignalling, together with TGF-b1 and IL-6 receptorsignalling, which activates STAT3 and induces theexpression of the transcription factor retinoic acid-related orphan receptor ct. IL-23 also activates STAT3but, in addition, serves to maintain Th17 cells in vivo.In contrast, human cells do not require TGF-b1, andit is IL-1, IL-6 and IL-23 that promote human Th17differentiation [17]. Lastly, Tregcells are crucial playersin the regulation⁄ suppression of each of the Thresponses and self-reactive T-cells. It is now knownthat there is more than one subtype of Tregcells,although the most important appear to beCD4+CD25+Foxp3+Treg[18]. These cells are affectedby the transcription factor fork-head box p3 (Foxp3),whose induction is initiated by TCR signalling,together with TGF-b1 receptor signalling [19]. Tregsuppressive activity is via IL-10 and TGF-b, althoughit remains unclear whether these cytokines areproduced by CD4+CD25+Foxp3+Tregor whetherthey induce the production of these cytokines fromanother population of cells [20].To date, our knowledge about the different types ofTh and Tregresponses relates to studies performed inmammals, especially mice and humans [12]. In fish,there has been a considerable amount of work under-taken on immunity over the last few decades, and alarge number of genes involved in immune responseshave been discovered. However, although we know alot about the innate and inflammatory immuneresponses of fish [21], relatively little is known aboutthe lymphocyte subpopulations involved in the adap-tive immune responses in fish, and whether Th subsetsexist. Speculation that Th1, Th2, Th17 and Tregresponses may exist in fish, and arose early in verte-brate evolution, has been prompted by the discoveryof many of the cytokines that are expressed by thesecell types [22,23]. However, it is important to note thatnot all the cytokines known in mammals have beenfound in fish, and it remains to be determined whetherthe regulation of adaptive immunity in fish is similarto that found in mammals, and if it is equally complex.In addition, the key transcription factors involved indriving the differentiation of the naive T-cell to Th1,Th2, Th17 or Tregcells may exist in fish. In this inves-tigation, we have identified, for the first time, t-bet andstat6 in zebrafish and, for the first time in any fish spe-cies, foxp3. Lastly, we carried out some preliminaryS. Mitra et al. Zebrafish T-cell transcription factors t-bet, stat6 and foxp3FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS 129expression analyses to investigate their role in theimmune responses of fish.ResultsCloning and sequencingFor t-bet, stat6 and foxp3, three overlapping productswere obtained using PCR and specific primers, whichcontained the complete cDNA sequence for each gene.The zebrafish t-bet cDNA (EMBL accession no.AM942761) consisted of a 36 bp 5¢-UTR, a 419 bp3¢-UTR and a single open reading frame of 1830 bp,giving a predicted 609 amino acid t-bet molecule. Inthe 3¢-UTR, no obvious polyadenylation signal waspresent. The stat6 cDNA transcript (EMBL accessionno. AM941850) consisted of a 135 bp 5¢-UTR, an809 bp 3¢-UTR and a single open reading frame of2277 bp, which translated into a predicted 758 aminoacid stat6 molecule. In the 3¢-UTR, two mRNA insta-bility motifs (attta) were present, and again no obviouspolyadenylation signal was found. The foxp3 cDNAtranscript (EMBL accession no. FM881778) consistedof a 100 bp 5¢-UTR, a 410 bp 3¢-UTR and a singleopen reading frame of 1260 bp, which translated intoa predicted 419 amino acid foxp3 molecule. In the3¢-UTR, four mRNA instability motifs (attta) werepresent upstream of the polyadenylation signal. Analternative transcript of foxp3 (Fig. 1) was also foundand was shown to be missing the region containing thezinc-finger and leucine-zipper domain.Multiple alignment of zebrafish t-bet, stat6 and foxp3with other known T-bet, STAT6 and Foxp3 amino acidsequences (Figs 2–4, respectively) revealed areas ofamino acid conservation throughout the vertebrates.Significant homology was seen in the putative T-boxDNA-binding domain of t-bet, the STAT protein inter-action domain, STAT protein all-alpha domain, STATprotein DNA-binding domain and SH2 domain ofstat6, and the zinc-finger domain, leucine-zipperdomain and fork-head domain of foxp3. In addition,for stat6 and foxp3, there were a few other conservedfeatures. Within the zebrafish stat6 sequence is animportant tyrosine residue (Tyr664), which was con-served in all sequences. Within the foxp3 molecule,some homology was found within the predictedtranscriptional repressor domains, with domain 2containing a large number of proline residues. As withother t-bet, stat6 and foxp3 molecules sequenced todate, the zebrafish t-bet, stat6 and foxp3 peptides didnot possess a signal peptide, as predicted by SignalPv1.1 (data not shown). Zebrafish t-bet had the highestamino acid identity and similarity (Table 1) to Ginbunacrucian carp t-bet (91.0% and 95.4%, respectively),zebrafish stat6 to Tetraodon stat6 (52.9% and 71.5%,respectively) and zebrafish foxp3 to mouse foxp3(31.6% and 49.0%, respectively). Phylogenetic analysisof t-bet, stat6 and foxp3 (Figs 5–7, respectively)grouped t-bet, stat6 and foxp3 with their mammalianhomologues, all of which were strongly supportedstatistically, when all known vertebrate T-box, STATfamily and Foxp family members were compared.Fig. 1. Pairwise alignment of the full-lengthDanio rerio foxp3 (ZFfoxp3) and an obtainedalternative transcript (ZFfoxp3b). The puta-tive transcriptional repressor domains 1 and2, fork-head (FKH), leucine-zipper and zinc-finger domains are highlighted. The EMBLaccession number of the foxp3b alternativetranscript gene is FM881779.Zebrafish T-cell transcription factors t-bet, stat6 and foxp3 S. Mitra et al.130 FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBSFig. 2. Multiple alignment of the predicted Danio rerio t-bet (T-box21) with selected known vertebrate T-bet molecules. Identical (*) and sim-ilar (: or.) residues identified by theCLUSTALX program are indicated. The putative T-bet DNA-binding domain is highlighted. The EMBL acces-sion numbers of the T-box21 genes are as follows: human, Q9UL17; mouse, Q9JKD8; Ginbuna crucian carp, AB290187; zebrafish,AM942761.S. Mitra et al. Zebrafish T-cell transcription factors t-bet, stat6 and foxp3FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS 131t-bet, stat6 and foxp3 gene organization andchromosome syntenyUsing the zebrafish t-bet, stat6 and foxp3 cDNAsequences elucidated by PCR and the regions of thezebrafish genome that contained these sequences,chromosomes 8, 12 and 23, the gene organizationswere obtained (Fig. 8; t-bet GenBank accession no.FN435332, stat6 GenBank accession no. FN435334,foxp3 GenBank accession no. FN435333). t-bet wasFig. 3. Multiple alignment of the predictedDanio rerio stat6 with selected knownvertebrate STAT6 molecules. Identical (*)and similar (: or.) residues identified by theCLUSTALX program are indicated. The putativeSTAT interaction, STAT all-alpha, STAT DNA-binding and SH2 domains are highlighted.Boxed is an important tyrosine residue(Tyr664 in zebrafish). The EMBL accessionnumbers of the STAT6 genes are asfollows: human, P42226; mouse, P52633;zebrafish, AM941850.Zebrafish T-cell transcription factors t-bet, stat6 and foxp3 S. Mitra et al.132 FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBSfound to have six exons and five introns, stat6 wasfound to have 21 exons and 20 introns, and foxp3was found to have 13 exons and 12 introns. In thegenomic sequence, the intron splicing consensus(GT ⁄ AG) is conserved at theand 3¢ ends of the in-trons. The gene organization was found to be similarto that of human t-bet and foxp3 genes (Fig. 8), withhuman stat6 having a slightly different gene organiza-tion of 22 exons and 21 introns. Generally, the sizesof the zebrafish t-bet, stat6 and foxp3 coding exonsmatched well with the corresponding mammalianexons (Fig. 8). Using the Genscan [24], fasta [25]and blast [26] suite of programs, other genes werediscovered on zebrafish chromosomes 8, 12 and 23around the discovered zebrafish t-bet, stat6 and foxp3genes (Fig. 9). On comparison with the human gen-ome, some degree of synteny was found between thetwo organisms for the regions containing the t-betand foxp3 genes. Around t-bet, the genes TBK1-bind-ing protein 1, oxysterol-binding protein-like 7 (OS-BPL7) and mitochondrial ribosomal protein L10 werefound in the same order on zebrafish chromosome 12and human chromosome 17 and, around foxp3, thegene protein phosphatase 1, regulatory (inhibitor)subunit 3F (PPP1R3F) was found in the same orderon zebrafish chromosome 8 and human chromosomeFig. 4. Multiple alignment of the predictedDanio rerio foxp3 with known Foxp3 mole-cules. Identical (*) and similar (: or.) residuesidentified by theCLUSTALX program are indi-cated. The putative transcriptional repressordomains 1 and 2, fork-head (FKH),leucine-zipper and zinc-finger domains arehighlighted. Proline residues within thetranscriptional repressor domains areunderlined. The EMBL accession numbersof the Foxp3 genes are as follows: human,Q9BZS1; mouse, Q99JB6; crab-eatingmacaque, Q6U8D7; zebrafish, FM881778.S. Mitra et al. Zebrafish T-cell transcription factors t-bet, stat6 and foxp3FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS 133X. For stat6, no synteny was found between thislocus on zebrafish chromosome 23 with the stat6locus on human chromosome 12.Quantification of expressed stat6, t-bet andfoxp3 genes in spleen or head kidney tissuesstimulated with immunostimulants (quantitativereal-time PCR)Using RT-PCR, the constitutive expression of t-bet,stat6 and foxp3 was observed in the spleen, kidney,gill, gut, liver and skin tissue of healthy fish (data notshown). After stimulation of kidney cells with a varietyof immunostimulants, the expression of t-bet, stat6and foxp3, together with other selected zebrafish tran-scription factors and cytokines, was compared usingquantitative PCR (Fig. 10). Stimulation of kidney cellswith phytohaemagglutinin (PHA) led to a significantincrease in il-4 and gata3 expression, stimulation withlipopolysaccharide (LPS) led to a significant increasein il-10, and stimulation with Poly I:C led to a signifi-cant increase in ifn-c, mx and t-bet. Stimulation ofspleen cells with PHA led to a significant increase inifn-c, whereas stimulation with LPS led to a significantincrease in il-10 and foxp3, and stimulation with PolyI:C led to a significant increase in mx and t-bet.Up-regulation was observed for a number of othergenes investigated, but expression was not statisticallysignificant.DiscussionThis paper reports the isolation and sequencing ofthree zebrafish transcription factors, which are knownto be important in T-cell subtype differentiation inmammals. T-bet has already been sequenced withinbony fish, in the Ginbuna crucian carp [27], andSTAT6 in mandarin fish [28], whereas Foxp3 has beencharacterized for the first time in fish. The availabilityof sequenced fish genomes has allowed the discoveryof a number of immune relevant genes using the synte-ny (conservation of gene order) found between thehuman and fish genomes [29–32] and, in some cases,has helped determine whether the gene is a true homo-logue of a mammalian gene. To begin with, we used asynteny approach to identify the chromosomal locationcontaining the zebrafish t-bet, stat6 and foxp3 tran-scription factors. We used this approach for t-bet as,at the time of discovery, the Ginbuna crucian carpsequence was unknown. This approach enabled t-betand foxp3 to be obtained quickly, as a major difficultyin the identification of transcription factors is thatTable 1. Amino acid identity ⁄ similarity of zebrafish t-bet, stat6 and foxp3 with other vertebrate T-bet, STAT6 and Foxp3 molecules.HumanT-betMouseT-betZebrafisht-betGinbunaT-betHumanSTAT6MouseSTAT6Zebrafishstat6TetraodonSTAT6HumanFoxp3MouseFoxp3Zebrafishfoxp3HumanT-bet86.90 43.40 42.50 17.50 16.20 15.60 16.40 19.50 18.10 17.60MouseT-bet91.80 43.90 43.80 17.20 17.50 16.60 15.60 19.20 18.80 16.80Zebrafisht-bet57.00 58.00 91.00 17.30 15.80 16.70 17.00 16.90 17.20 16.30GinbunaT-bet57.20 58.70 95.40 17.50 16.40 16.20 26.30 16.80 18.10 16.90HumanSTAT629.20 28.20 28.70 28.90 84.20 34.20 35.40 14.40 14.00 14.10MouseSTAT628.90 29.20 31.20 30.60 90.00 34.40 35.30 15.30 15.80 12.10Zebrafishstat628.60 30.10 32.70 32.80 53.40 54.50 52.90 15.10 14.50 15.20TetraodonSTAT628.50 28.40 30.60 29.90 55.50 55.20 71.50 14.40 14.80 13.50HumanFoxp333.50 32.50 28.90 30.60 24.30 23.80 26.40 25.20 86.10 31.50MouseFoxp331.00 33.20 28.70 29.60 24.10 24.50 24.40 25.40 91.40 31.60Zebrafishfoxp331.40 29.10 27.60 29.10 24.10 23.80 25.20 26.80 48.00 49.00Above diagonal, identity; below diagonal, similarity.Zebrafish T-cell transcription factors t-bet, stat6 and foxp3 S. Mitra et al.134 FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBSmany of them belong to gene families, with membershaving high sequence identity, making it hard to findthe correct sequence in the zebrafish genome. Thisapproach was not used for stat6 as the region in whichthis gene was found in the zebrafish genome shared nosynteny with the human genome. The zebrafish gen-ome was searched using the human stat6 amino acidsequence directly for identification.The zebrafish t-bet homologue is predicted to contain609 amino acids, the stat6 homologue 758 amino acidsand the foxp3 homologue 419 amino acids. None ofthese molecules was found to contain a signal peptide(data not shown), indicating that the molecules are notsecreted through the classical pathway and will remaincytosolic. Also found in the 3¢-UTR of zebrafish stat6and foxp3 were numerous copies of an mRNA instabil-ity motif (attta) which plays a role in mRNA degrada-tion [33], typical of genes coding for inflammatorymediators [34], and suggesting that these genes are tran-siently transcribed. It is unknown whether theseHUMAN TBX2HUMAN TBX2DOG TBX2DOG TBX2MOUSE TBX2MOUSE TBX2ZEBRAFISH TBX2ZEBRAFISH TBX2XENOPUSTR TBX2XENOPUSTR TBX2MOUSE TBX3MOUSE TBX3HUMANTBX3HUMANTBX3CHICKEN TBX3CHICKEN TBX3HUMAN TBX6HUMAN TBX6MOUSE TBX6MOUSE TBX6XENOPUSTR TBX6XENOPUSTR TBX6ZEBRAFISH TBX6ZEBRAFISH TBX6HUMAN THUMAN T--BETBETMOUSE TMOUSE T--BETBETZEBRAFISH TZEBRAFISH T--BETBETGINBUNACARP TGINBUNACARP T--BETBET5858MOUSE TBX20MOUSE TBX20HUMAN TBX20HUMAN TBX20CHICKEN TBX20CHICKEN TBX20XENOPUSTR TBX20XENOPUSTR TBX20ZEBRAFISH TBX20ZEBRAFISH TBX20MOUSE TBX15MOUSE TBX15HUMAN TBX15HUMAN TBX15MOUSE TBX18MOUSE TBX18HUMAN TBX18HUMAN TBX18MOUSE TBX1MOUSE TBX1HUMAN TBX1HUMAN TBX1XENOPUSTR TBX1XENOPUSTR TBX1MOUSE TBX10MOUSE TBX10HUMAN TBX10HUMAN TBX106060MOUSE TBX5MOUSE TBX5RAT TBX5RAT TBX5HUMAN TBX5HUMAN TBX5CHICKEN TBX5CHICKEN TBX5XENOPUSLA TBX5XENOPUSLA TBX5ZEBRAFISH TBX5ZEBRAFISH TBX5DOG TBX4DOG TBX4HUMAN TBX4HUMAN TBX40.10.1TBOXTBOX--2/2/--33TBOXTBOX--6/6/--2121TBOXTBOX--2020TBOXTBOX--15/15/--1818TBOXTBOX--1/1/--1010TBOXTBOX--4/4/--55HUMAN TBX2HUMAN TBX2DOG TBX2DOG TBX2MOUSE TBX2MOUSE TBX2ZEBRAFISH TBX2ZEBRAFISH TBX2XENOPUSTR TBX2XENOPUSTR TBX2MOUSE TBX3MOUSE TBX3HUMANTBX3HUMANTBX3CHICKEN TBX3CHICKEN TBX3HUMAN TBX6HUMAN TBX6MOUSE TBX6MOUSE TBX6XENOPUSTR TBX6XENOPUSTR TBX6ZEBRAFISH TBX6ZEBRAFISH TBX6HUMAN THUMAN T--BETBETMOUSE TMOUSE T--BETBETZEBRAFISH TZEBRAFISH T--BETBETGINBUNACARP TGINBUNACARP T--BETBET5858MOUSE TBX20MOUSE TBX20HUMAN TBX20HUMAN TBX20CHICKEN TBX20CHICKEN TBX20XENOPUSTR TBX20XENOPUSTR TBX20ZEBRAFISH TBX20ZEBRAFISH TBX20MOUSE TBX15MOUSE TBX15HUMAN TBX15HUMAN TBX15MOUSE TBX18MOUSE TBX18HUMAN TBX18HUMAN TBX18MOUSE TBX1MOUSE TBX1HUMAN TBX1HUMAN TBX1XENOPUSTR TBX1XENOPUSTR TBX1MOUSE TBX10MOUSE TBX10HUMAN TBX10HUMAN TBX106060MOUSE TBX5MOUSE TBX5RAT TBX5RAT TBX5HUMAN TBX5HUMAN TBX5CHICKEN TBX5CHICKEN TBX5XENOPUSLA TBX5XENOPUSLA TBX5ZEBRAFISH TBX5ZEBRAFISH TBX5DOG TBX4DOG TBX4HUMAN TBX4HUMAN TBX40.10.1TBOXTBOX--2/2/--33TBOXTBOX--6/6/--2121TBOXTBOX--2020TBOXTBOX--15/15/--1818TBOXTBOX--1/1/--1010TBOXTBOX--4/4/--55Fig. 5. Unrooted phylogenetic tree showing the relationship between the Danio rerio t-bet amino acid sequence for the full-length moleculewith other known vertebrate T-box (TBX) family member sequences. This tree was constructed by the neighbour-joining method using theCLUSTALX and TREEVIEW packages, and was bootstrapped 10 000 times. All bootstrap values less than 75% are shown. The EMBL accessionnumbers of the TBX-1 amino acid sequences are as follows: human, O43435; mouse, P70323; Xenopus tropicalis, Q3SA49. The accessionnumbers of the TBX-2 amino acid sequences are as follows: human, Q13207; mouse, Q60707; dog, Q863A2; X. tropicalis, Q3SA48; zebra-fish, Q7ZTU9. The accession numbers of the TBX-3 amino acid sequences are as follows: human, O15119; mouse, P70324; chicken,O73718. The accession numbers of the TBX-4 amino acid sequences are as follows: human, P57082; dog, Q861Q9. The accession numbersof the TBX-5 amino acid sequences are as follows: human, Q99593; mouse, P70326; rat, Q5I2P1; chicken, Q9PWE8; Xenopus laevis,Q9W7C2; zebrafish, Q9IAK8. The accession numbers of the TBX-6 amino acid sequences are as follows: human, O95947; mouse, P70327,X. tropicalis, Q66JL1; zebrafish, P79742. The accession numbers of the TBX-10 amino acid sequences are as follows: human, O75333;mouse, Q810F8. The accession numbers of the TBX-15 amino acid sequences are as follows: human, Q96SF7; mouse, O70306. The acces-sion numbers of the TBX-18 amino acid sequences are as follows: human, O95935; mouse, Q9EPZ6. The accession numbers of the TBX-20amino acid sequences are as follows: human, Q9UMR3; mouse, Q9ES03; chicken, Q8UW76; X. tropicalis, Q3SA46; zebrafish, Q9I9K7. Theaccession numbers of the TBX-21 (T-BET) amino acid sequences are as follows: human, Q9UL17; mouse, Q9JKD8; Ginbuna crucian carp,AB290187; zebrafish, AM942761.S. Mitra et al. Zebrafish T-cell transcription factors t-bet, stat6 and foxp3FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS 135instability motifs will be found within the t-bet 3¢-UTRas it remains to be fully sequenced. Phylogenetic analy-sis was carried out using the amino acid sequences ofzebrafish t-bet, stat6 and foxp3 plus those of all knownvertebrate T-box, STAT family and Foxp familymembers. The zebrafish genes grouped well with theirvertebrate T-bet, STAT6 and Foxp3 homologues,which was supported by bootstrap values greater than75%, providing further evidence of their identity.Multiple alignments of the zebrafish t-bet, stat6 andfoxp3 amino acids with their vertebrate homologuesrevealed regions of high conservation. These regions0.10.1XENOPUSLA STAT1XENOPUSLA STAT1CHICKEN STAT1CHICKEN STAT1MOUSE STAT1MOUSE STAT1PIG STAT1PIG STAT1HUMAN STAT1HUMAN STAT1SALMON STAT1SALMON STAT1TETRAODON STAT1TETRAODON STAT1HALIBUT STAT1HALIBUT STAT1SNAKEHEAD STAT1SNAKEHEAD STAT1CHICKEN STAT4CHICKEN STAT4MOUSE STAT4MOUSE STAT4HUMAN STAT4HUMAN STAT4ZEBRAFISH STAT4ZEBRAFISH STAT4FUGU STAT4FUGU STAT4TETRAODON STAT4TETRAODON STAT4MOUSE STAT2MOUSE STAT2PIG STAT2PIG STAT2HUMAN STAT2HUMAN STAT2HUMAN STAT6HUMAN STAT6MOUSE STAT6MOUSE STAT6ZEBRAFISH STAT6ZEBRAFISH STAT6TETRAODON STAT6TETRAODON STAT6HUMAN STAT5HUMAN STAT5COW STAT5COW STAT5PIG STAT5PIG STAT55858MOUSE STAT5MOUSE STAT5RAT STAT5RAT STAT5TROUT STAT5TROUT STAT5ZEBRAFISH STAT5ZEBRAFISH STAT56363FUGU STAT5FUGU STAT5TETRAODON STAT5TETRAODON STAT5TROUT STAT3TROUT STAT3ZEBRAFISH STAT3ZEBRAFISH STAT3TETRAODON STAT3TETRAODON STAT3MEDAKA STAT3MEDAKA STAT35555CHICKEN STAT3CHICKEN STAT3MOUSE STAT3MOUSE STAT3RAT STAT3RAT STAT37171PIG STAT3PIG STAT3HUMAN STAT3HUMAN STAT349490.10.10.10.1XENOPUSLA STAT1XENOPUSLA STAT1CHICKEN STAT1CHICKEN STAT1MOUSE STAT1MOUSE STAT1PIG STAT1PIG STAT1HUMAN STAT1HUMAN STAT1SALMON STAT1SALMON STAT1TETRAODON STAT1TETRAODON STAT1HALIBUT STAT1HALIBUT STAT1SNAKEHEAD STAT1SNAKEHEAD STAT1CHICKEN STAT4CHICKEN STAT4MOUSE STAT4MOUSE STAT4HUMAN STAT4HUMAN STAT4ZEBRAFISH STAT4ZEBRAFISH STAT4FUGU STAT4FUGU STAT4TETRAODON STAT4TETRAODON STAT4MOUSE STAT2MOUSE STAT2PIG STAT2PIG STAT2HUMAN STAT2HUMAN STAT2HUMAN STAT6HUMAN STAT6MOUSE STAT6MOUSE STAT6ZEBRAFISH STAT6ZEBRAFISH STAT6TETRAODON STAT6TETRAODON STAT6HUMAN STAT5HUMAN STAT5COW STAT5COW STAT5PIG STAT5PIG STAT55858MOUSE STAT5MOUSE STAT5RAT STAT5RAT STAT5TROUT STAT5TROUT STAT5ZEBRAFISH STAT5ZEBRAFISH STAT56363FUGU STAT5FUGU STAT5TETRAODON STAT5TETRAODON STAT5TROUT STAT3TROUT STAT3ZEBRAFISH STAT3ZEBRAFISH STAT3TETRAODON STAT3TETRAODON STAT3MEDAKA STAT3MEDAKA STAT35555CHICKEN STAT3CHICKEN STAT3MOUSE STAT3MOUSE STAT3RAT STAT3RAT STAT37171PIG STAT3PIG STAT3HUMAN STAT3HUMAN STAT34949STAT-1STAT-1STAT-4STAT-4STAT-2STAT-2STAT-6STAT-6STAT-5STAT-5STAT-3STAT-3Fig. 6. Unrooted phylogenetic tree showing the relationship between the Danio rerio stat6 amino acid sequence for the full-length moleculewith other known vertebrate STAT family member sequences. This tree was constructed by the neighbour-joining method using theCLUSTALX and TREEVIEW packages, and was bootstrapped 10 000 times. All bootstrap values less than 75% are shown. The EMBL accessionnumbers of the STAT-1 amino acid sequences are as follows: human, P42224; mouse, P42225; pig, Q764M5; chicken, CAG32090; Xeno-pus tropicalis, AAM51552; salmon, ACI33829; Tetraodon, AAL09414; halibut, ABS19629; snakehead, ABK60089. The accession numbers ofthe STAT-2 amino acid sequences are as follows: human, P52630; mouse, Q9WVL2; pig, O02799. The accession numbers of the STAT-3amino acid sequences are as follows: human, P40763; mouse, P42227; rat, P52631; pig, Q19S50; chicken, Q6DV79; trout, AAB60926;zebrafish, AAH68320; Tetraodon, AAL09415; medaka, AAT64912. The accession numbers of the STAT-4 amino acid sequences are asfollows: human, Q14765; mouse, P42228; chicken, BAF34318; zebrafish, CAD52132; Fugu, AAS10464; Tetraodon, AAL09416. Theaccession numbers of the STAT-5 amino acid sequences are as follows: human, P51692; mouse, P42232; rat, P52632; pig, Q9TUZ0; cow,Q9TUM3; trout, AAG14946; Tetraodon, AAL09417; Fugu, AAS80167; zebrafish, AAT95391. The accession numbers of the STAT-6 aminoacid sequences are as follows: human, P42226; mouse, P52633; Tetraodon, AAO22057; zebrafish, AM941850.Zebrafish T-cell transcription factors t-bet, stat6 and foxp3 S. Mitra et al.136 FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBSwere subsequently identified to be protein domainsimportant in the functioning of these transcriptionfactors. T-bet (also known as Tbox-21) belongs to theT-box family of genes, consisting of over 20 memberscharacterized in mammals [35]. They contain a con-served sequence, around 200 amino acids in length,called the ‘T-box’, which, in T-bet, is centrally located,whereas, in other members, it is located at the amino-terminus [36]. This region is known to be aDNA-binding domain and is quite clearly conserved inzebrafish, as the sequence, when compared with humanand mouse T-bet [11,37], shows almost completeidentity in this region.STAT6 (also known as IL-4-induced transcriptionfactor) belongs to the STAT family of proteins [38].STAT proteins share structurally and functionally0.10.1XENOPUS FOXP4XENOPUS FOXP4MOUSE FOXP4MOUSE FOXP4HUMAN FOXP4HUMAN FOXP4ZEBRAFISH FOXP3ZEBRAFISH FOXP3MOUSE FOXP3MOUSE FOXP3HUMAN FOXP3HUMAN FOXP3MACAQUE FOXP3MACAQUE FOXP3XENOPUS FOXP2XENOPUS FOXP2HUMAN FOXP2HUMAN FOXP2MACAQUE FOXP2MACAQUE FOXP2MOUSE FOXP2MOUSE FOXP2ZEBRAFISH FOXP1ZEBRAFISH FOXP1XENOPUS FOXP1XENOPUS FOXP1CHICKEN FOXP1CHICKEN FOXP1RAT FOXP1RAT FOXP1MOUSE FOXP1MOUSE FOXP1COW FOXP1COW FOXP1HUMAN FOXP1HUMAN FOXP150500.10.10.10.1XENOPUS FOXP4XENOPUS FOXP4MOUSE FOXP4MOUSE FOXP4HUMAN FOXP4HUMAN FOXP4ZEBRAFISH FOXP3ZEBRAFISH FOXP3MOUSE FOXP3MOUSE FOXP3HUMAN FOXP3HUMAN FOXP3MACAQUE FOXP3MACAQUE FOXP3XENOPUS FOXP2XENOPUS FOXP2HUMAN FOXP2HUMAN FOXP2MACAQUE FOXP2MACAQUE FOXP2MOUSE FOXP2MOUSE FOXP2ZEBRAFISH FOXP1ZEBRAFISH FOXP1XENOPUS FOXP1XENOPUS FOXP1CHICKEN FOXP1CHICKEN FOXP1RAT FOXP1RAT FOXP1MOUSE FOXP1MOUSE FOXP1COW FOXP1COW FOXP1HUMAN FOXP1HUMAN FOXP15050FOXP4FOXP4FOXP3FOXP3FOXP2FOXP2FOXP1FOXP1Fig. 7. Unrooted phylogenetic tree showing the relationship between the Danio rerio foxp3 amino acid sequence for the full-length moleculewith other known vertebrate Foxp family member sequences. This tree was constructed by the neighbour-joining method using theCLUSTALXand TREEVIEW packages, and was bootstrapped 10 000 times. All bootstrap values less than 75% are shown. The EMBL accession numbersof the Foxp1 amino acid sequences are as follows: human, Q9H334; rat, Q498D1; mouse, P58462; cow, A4IFD2; chicken, Q58NQ4; Xeno-pus laevis, Q5W1J5; zebrafish, Q2LE08. The accession numbers of the Foxp2 amino acid sequences are as follows: human, O15409;mouse, P58463; crab-eating macaque, Q8MJ97; Xenopus laevis, Q4VYS1. The accession numbers of the Foxp3 amino acid sequences areas follows: human, Q9BZS1; mouse, Q99JB6, crab-eating macaque, Q6U8D7; zebrafish, FM881778. The accession numbers of the Foxp4amino acid sequences are as follows: human, Q8IVH2; mouse, Q9DBY0; X. laevis, Q4VYR7.S. Mitra et al. Zebrafish T-cell transcription factors t-bet, stat6 and foxp3FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS 137[...]... CH471224; zebrafish, FN435333 conserved domains, including an N-terminal STAT protein interaction domain, which strengthens interactions between STAT dimers on adjacent DNA-binding sites, a coiled-coil STAT protein all-alpha domain, which is implicated in other protein–protein interactions, a STAT protein DNA-binding domain and an SH2 domain, which binds phosphorylated tyrosine residues in the context of a... thought to help mediate DNA binding and may be involved in the induction of dimerization [43] Each of these three regions appears to be present within zebrafish foxp3, as there is very good conservation of protein sequence in these regions when compared with mammalian Foxp3 In addition, within human Foxp3, there are two other domains, not found in other Foxp subfamily members and possibly specific for Treg... analysis of zebrafish t-bet, stat6 and foxp3 detected constitutive expression in the spleen, kidney, gill, gut, liver and skin tissue of healthy fish (data not shown) Previous studies on the expression of these genes within humans and mice, of T-bet within Ginbuna crucian carp [27] and STAT6 in mandarin fish [28] have shown similar widespread expression In the case of STAT6, highest expression in mammals... STAT6, indicating that this gene is expressed in haematopoietic cells and more variably in other lineages [41] The expression of T-bet has been seen within a wide variety of tissues and cells in the Ginbuna crucian carp [27] and, in mammals, in the lung tissue of mouse and spleen and thymus of human and mouse [11,37] Foxp3 is also highly expressed in lymphoid organs, such as the thymus and spleen [49], and. .. be involved in the differentiation of T-cell subsets Our preliminary expression data suggest that these genes are involved in the fish immune response, and that there is a relationship between the expression of these transcription factors and the cytokine genes known to be produced by different T-cell subtypes in mammals These transcription factors, together with many of the important cytokines that are... to their human homologues Both the human and zebrafish T-bet and Foxp3 genes contained the same number of exons and introns t-bet contained six exons and five introns, whereas foxp3 contained 13 exons and 12 introns However, zebrafish stat6 showed some differences from the human homologue, having 21 exons and 20 introns rather than 22 exons and 21 introns This difference was found at the 3¢ end of the. .. contain transcriptional repressor domain 2, the zinc-finger domain and the leucine-zipper domain In humans, a similar scenario exists, with two alternatively spliced isoforms of Foxp3 also being expressed, but here only exon 2 is lacking in the short form [45] This splicing variant has not been reported in mice [44] The gene organizations of zebrafish t-bet, stat6 and foxp3 were also determined and found... different T-cell subtypes [22,23], will aid future investigations into the types of Th and Treg cells that exist in teleost fish Materials and methods Sequence retrieval The zebrafish t-bet, stat6 and foxp3 sequences were found initially using the zebrafish genome (http://www.ensembl.org /Danio_ rerio/ ) assembly version 7 (Zv7) and, in the case of t-bet and foxp3, by exploiting the conservation of synteny... for stat6, zftbet-F1 and zftbet-R1 for t-bet, and zffoxp3-F1, zffoxp3-R1, zffoxp3-F2 and zffoxp3-R2 for foxp3 These primers amplified part of the initial predicted sequence which contained the majority of the open reading frames of zebrafish stat6, t-bet and foxp3 to check they were correct Having isolated these partial sequences, the complete zebrafish t-bet, stat6 and foxp3 cDNA sequences were obtained... transcriptional repressor domains 1 and 2 [44] In mammals, these regions contain fairly high numbers of proline residues [44] Similar domains may exist in zebrafish foxp3, as there is some homology within this region, with a relatively large number of proline residues present in the potential second transcriptional repressor domain, but not in the first Inter- FEBS Journal 277 (2010) 128–147 ª 2009 The . Identification and characterization of the transcription factors involved in T-cell development, t-bet, stat6 and foxp3, within the zebrafish, Danio rerio Suman. all-alpha domain, STATprotein DNA-binding domain and SH2 domain of stat6, and the zinc-finger domain, leucine-zipperdomain and fork-head domain of foxp3. In addition,for
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