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Contents lists available at ScienceDirect

Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi

Short communication

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Molecular cloning and expression analysis of an apoptosis-associated gene Daxx from zebrafish, Danio rerio

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Lin Qi a, b, Zhiming Xiang b, * a

Railway Police College, 31 Agricultural Road, Zhengzhou 450053, China Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 February 2015 Received in revised form 30 March 2015 Accepted 30 March 2015 Available online xxx

The death domain-associated protein Daxx exerts many functions including the induction and inhibition of apoptosis, regulation of chromatin remodeling and gene transcription. In this report, we have cloned and characterized a Daxx ortholog from the zebrafish, Danio rerio. The bioinformatics analysis results indicated that the open reading frame (ORF) of zebrafish Daxx is 2,151bp long and encodes a putative protein of 716 amino acids containing Daxx domain. Though quantitative PCR analyses, Daxx mRNA was detected in embryonic development from 6 h to 120 h and in all 11 selected zebrafish tissues, and the expression of Daxx was increased first and then decreased during megalocytivirus infectious spleen and kidney necrosis virus (ISKNV) infection. Fluorescence microscopy indicated that the full-length protein was located in the nuclei of the tested Hela cells uniformly but punctiform distribution in HEK293T. In the luciferase report assays, the GAL4-Daxx fusion protein inhibited the transcriptional activity of L8G5Luc reporter gene showed that Daxx might act as a transcriptional repressor, following the overexpression in HEK293T, the activation of NF-kB-Luc and p53/p21-Luc reporter genes were repressed by the protein. These results suggested that Daxx might play definite role in apoptosis and innate immunity in zebrafish. © 2015 Published by Elsevier Ltd.

Keywords: Zebrafish Apoptosis Innate immunity ISKNV Daxx

1. Introduction Daxx plays a crucial role in transcriptional regulation, cell apoptosis and chromatin remodeling [1]. It was first described as a pro-apoptotic protein associated with the death receptor Fas [2],

Abbreviations: Daxx, death domain-associated protein; Fas, tumor necrosis factor receptor superfamily member 6; BLM, bloom syndrome; MDM2, mouse double minute 2; Bax, BCL2-associated X protein; Puma, BCL2 binding component 3; Bid, BH3 interacting-domain death agonist; Pax, paired box homeotic protein; ETS1, v-ets avian erythroblastosis virus E26 oncogene homolog 1; EGFP, enhanced green fluorescent protein; RFP, red fluorescent protein; SUMO, small ubiquitin-like modifier protein; SIMs, SUMO-interaction motifs; PML-NB, promyelocytic leukemia (PML) nuclear body; ATRX, associated with alpha-thalassemia retardation syndrome X-linked protein; NDP55, 55 kD nuclear dot protein; NLS, nuclear localization signals; UTR, untranslated regions; EST, expressed sequence tag; ORF, open reading frame; Ads, adenoviruses; HSV-1, herpes simplex virus 1; HCMV, human cytomegalovirus; ISKNV, infectious spleen and kidney necrosis virus; FCS, fetal calf serum; hpi, hour post infection; IB, Immune blot; DAPI, 40 , 6-diamidino-2phenylindole hydrochloride; ANOVA, Analysis of variance. * Corresponding author. Tel./fax: þ86 20 8910 2507. E-mail address: [email protected] (Z. Xiang).

but as more studies have been completed, increasing evidence suggested that Daxx could play an anti-apoptotic role in response to many stresses. For example, Daxx might inhibit Fas and stressmediated apoptosis by suppressing pro-apoptotic gene expression [3]. The detailed mechanisms for this action remain unknown, but the role of the gene in the viral immune defense has recently been proposed. Daxx is a scaffold protein with a complicated structure and diverse roles. Daxx localizes to the nucleus in un-stimulated cells and relocalizes to other cellular regions following the stimulation by different stresses via NLS1 and NLS2, which are two separate nuclear localizing signals (NLS) [4]. NLS1 contributes partially to nuclear localization, whereas NLS2 plays a major role. Both signals are important for the nuclear and promyelocytic leukemia (PML) nuclear body (PML-NB) localization of the full-length protein. PML-NBs are multi-protein complexes that are composed of Daxx, PML, SP100, BLM, SUMO1, NDP55 and other proteins [5,6]. The pleomorphic locations of Daxx indicate that the protein plays multiple roles in the regulation of cell growth, apoptosis and immune defense. PML-NBs protein complexes are involved in the regulation of key nuclear pathways, but their biochemical function in nuclear

http://dx.doi.org/10.1016/j.fsi.2015.03.040 1050-4648/© 2015 Published by Elsevier Ltd.

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metabolism is unknown [7]. PML-NBs are the nuclear localization sites of many DNA viruses. Both of them were found to be viral gene expression and DNA replication centers, which regulate the expression of viral genes and prevent the replication of invading viruses [8]. Many DNA viruses were found to damage PML-NBs for apoptosis prevention and immune escape, such as the adenoviruses (Ads), the herpes simplex virus 1 (HSV-1), human EpsteineBarr virus (EBV), and human cytomegalovirus (HCMV) [9]. Iridoviruses are large double-stranded DNA viruses which infect vertebrates are collectively called piscine iridoviruses, which include lymphocytivirus, ranavirus, and megalocytivirus [10,11]. Recently, a new DNA virus named infectious spleen and kidney necrosis virus (ISKNV) was found belonged to the genus Megalocytivirus. There is fatal threat for some fish aquaculture for this contagious and highly lethal virus which needs to be researched. Its pathogenicity was examined in many host species, such as zebrafish and many cultured teleosts [12]. It is clear that Daxx plays important roles in many signaling pathways to regulate apoptosis, which is triggered by a series of stress signals including irradiation, hydrogen peroxide treatment and infection by pathogens [2,9]. Daxx is a negative regulator of p53, and p53 inhibition induces apoptosis in response to different genotoxic and other stresses [13,14]. As a target protein of Daxx, p53 exerts its functions by regulating the expression of proapoptosis genes, such as Bax, Puma and Bid, or it can induce apoptosis directly by binding to Bcl-2 family members to trigger cytochrome c release [15,16]. Mouse double minute 2 (MDM2) is a ubiquitin ligase (E3) that can enhance the ubiquitination and proteasomal degradation of p53 [14]. Daxx is required for Mdm2 stability, and the down regulation of Daxx decreases Mdm2 levels. When DNA is damaged, the Daxx dissociates from Mdm2 and triggers its self-degradation. p53 is released and activated and then induces apoptosis [17]. In addition, Daxx was also found to modulate NF-kB transcriptional activity [18]. In this paper, we cloned and characterized a cDNA encoding Daxx protein from the zebrafish, Danio rerio. The expression level of zebrafish Daxx was changed following ISKNV infection. Furthermore, fluorescence microscopy results show that zebrafish Daxx located in the nuclei of Hela cells uniformly but punctiform distribution in EK293T. In addition, zebrafish Daxx inhibited the NF-kB and p53/p21 luciferase reporter genes. Altogether, these results indicated that Daxx is involved in the viral immunity in zebrafish. 2. Materials and methods 2.1. Cloning and bioinformatics analysis The cDNA sequence including the 5'- and 3'-untranslated regions (UTRs) of the zebrafish Daxx were amplified with the nested PCR primer named Daxx ou F/R and Daxx in F/R, using zebrafish cDNA as amplification templates according to NCBI zebrafish Daxx (NM_001044949). The PCR products were cloned into a pGEM-T easy vector (Promega, USA) for sequencing using an ABI 3770 sequencer (Applied Biosystems, USA). The following plasmids were constructed and used for mammalian cell transfections. The primers used for the construction of the various expression vectors are listed in Table 1. The cDNA fragment encoding the zebrafish Daxx-myc peptide was amplified using Promega Taq polymerase with the specific primers Daxx-myc F and Daxx-myc R. EcoRⅠand KpnⅠare the restriction enzyme sites of the 50 ends of the primers. The PCR fragment was digested with the appropriate restriction enzymes and subsequently cloned into the EcoR/KpnⅠsites of the pCMV-Myc expression vector (Clontech# 631604, USA). pCMV-BD-Daxx vector (empty vector code: CAT#211342, Stratagene) was constructed for the zebrafish Daxx

Table 1 Sequences of designed primers used in this study. Primer

Sequence (50 to 30 )

Comment

Daxx ou F: Daxx ou R: Daxx in F: Daxx in R: Daxx-myc F: Daxx-myc R: Daxx-RFP-F: Daxx-RFP-R: Daxx-BD-F: Daxx-BD-R: qPCR Daxx F: qPCR Daxx R: qPCR b-actin F qPCR b-actin R

GCATGTTGAGCTGTACAAAGATCCA GAGATCGTTTATTGATTGAGCAGGG TAAGTTCGTGTTTTTGTAAATGCTG TCTTGGTAGAAATGAGGATTGCGT TTTGAATTCTGGCCGGAGCAGGGATGGA CGGGGTACCCTCGGAATCAGACAGA AAAGAATTCATGGCCGGAGCA TTTGGTACCCACTCGGAATCAGACA AAAGAATTCATGGCCGGAGCAGGGA CCCGTCGACTCACTCGGAATCAGACA TGAGTGCAGGGTCTAATG AACGAACGCTACAATCTC ATGCCCCTCGTGCTGTTTTC GCCTCATCTCCCACATAGGA

Nest PCR Nest PCR pCMV-Myc-Daxx pDsRed-N1-Daxx pCMV-BD-Daxx qPCR of Daxx qPCR of b-actin

“F” indicates forward primer and “R” indicates reverse primer.

transcriptional activity analysis. To study the subcellular localization of zebrafish Daxx, the full-length zebrafish Daxx was cloned into the pDsRed1-N1 vector (Clontech# 6921-1, USA) with the same restriction enzyme cutting sites as above in order to express zebrafish Daxx-RFP. All of the constructed recombinant plasmids were cleaved with enzymes, and the inserted fragments of each clone were sequenced. The cDNA and deduced amino acid sequence of zebrafish Daxx were analyzed using the BLAST program at the National Center for Biotechnology Information (NCBI) website (http://blast.ncbi.nlm. nih.gov), and the pI/Mw were computed to predict the theoretical isoelectric point and molecular weight (http://web.expasy.org). The protein motif features were predicted with online software (http:// smart.cmbl-heidelberg.de/ and http://swissmodel.expasy.org). A multiple sequence alignment of the deduced amino acids was performed using ClustalX 1.81, and a neighbor-joining (NJ) phylogenetic tree was constructed using the MEGA5.0 package. The Clustal method was used to correct the distances for multiple substitutions at a single site. The reliability of branching was tested using bootstrap re-sampling (with 1000 pseudo-replicates). 2.2. Animals, virus challenge, and tissue collection Wild-type zebrafish were reared in re-circulating systems at 28  C. The adult fish (3 months old, female:male ¼ 1:1) used in the infection experiments were transferred to the isolated flowthrough system and allowed to acclimate for 7 days prior to infection. ISKNV filtrates were prepared as previously described [12]. The samples were stored in liquid nitrogen until further use. Total RNA samples were extracted using TRIzol according to the manufacturer's instructions. 2.3. RNA isolation and quantitative PCR analysis of zebrafish Daxx Total RNA was isolated according to M&M2.2 using TRIzol reagent and DNase I treatment (Promega, USA). The quantitative PCR methods were prepared as previously described [12]. 2.4. Cell culture, transient transfection and subcellular localization The Hela cells or HEK293T cells used in all studies were maintained according to standard methods in DMEM (Dulbecco's modified Eagle's medium; GIBCO, BRL) supplemented with 10% fetal calf serum (FCS) in a humidified atmosphere of 95% air and 5% CO2 at 37  C. pRFP-Daxx was constructed to generate zebrafish Daxx-RFP fusion protein. Transient transfection was conducted with

Please cite this article in press as: L. Qi, Z. Xiang, Molecular cloning and expression analysis of an apoptosis-associated gene Daxx from zebrafish, Danio rerio, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.03.040

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Lipofectamine 2000 (Invitrogen, USA). At 48 h after transfection, Hela or HEK293T cells were washed with PBS buffer for 5 min, fixed with 4.0% paraformaldehyde for 10 min, and stained with 40 , 6diamidino-2-phenylindole hydrochloride (DAPI). Cells transfected with fluorescent vectors were directly observed under fluorescent microscopy. The transcriptional activation by zebrafish Daxx was tested. Cells were co-transfected with 100 ng/well each of pRL-TK (Promega, USA) and luciferase reporter plasmids; the total amount of transfected DNA was kept constant by adding control vector. pCMV-BD-Daxx or pCMV-BD (empty bait vector) were used as controls. To test transcriptional activation, 100 ng/well of plasmid was transiently co-transfected into HEK293T cells along with the pL8G5-Luc reporter gene [19]. Furthermore, luciferase activity was measured over a range of plasmid concentrations from 50 ng to the maximal transfection of 200 ng of the target gene. Because the Daxx protein can bind DNA elements other transcriptional factors and regulate the expression of target genes, NF-kB(Promega), p53- (Stratagene), or p21-Luc (Addgene) luciferase reporter plasmids, and pCMV-Myc-Daxx or pCMV-Myc vectors were used to investigate the role of zebrafish Daxx on the transcriptional activity of each of these reporter genes. Forty-eight hours later, the luciferase activity of the total cell lysates was measured using Dual-Luciferase Reporter Assay System (Promega, USA) following the manufacturer's instructions and normalized for transfection efficiency by the control vector pRL-TK. Renilla luciferase activity was expressed as the fold-stimulation relative to the cells transfected with empty vector. Values were expressed as the mean relative stimulation from three separate experiments with each performed in duplicate. All data were calculated as the means and standard deviations (±S.D.). 2.5. Western blot assay In the luciferase reporter assays, western blot analysis was applied to the total cell lysates to verify the expression of zebrafish Daxx. The western blot methods were prepared as previously described [12]. 5  106 cells were lysed using lysis buffer (50 mM TriseHCl pH7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X100, 1 mM PMSF). The cell lysateswere separated on 12% SDS-PAGE and transferred to a nitrocellulose membrane. Following blocked in Tris-buffer saline containing 0.05% Tween-20 and 5% defatted milk powder for 1 h at room temperature, the membrane was incubated with mouse anti-Myc or anti-actin monoclonal antibody (1:3000, Cell Signaling Technology, USA) over night at 4  C. Immunoreactive proteins were detected using HRP-conjugated secondary antibody (1: 500, Jackson Immuno Research Laboratories, USA) and enhanced Chemiluminescence reagent (Amersham Pharmacia Biotech, USA). In this experiment, actin was used as an internal control. 2.6. Statistical analysis All results are shown as the mean ± S.D. of the different biological samples. The significance of data was examined using oneway ANOVA, and the differences were considered statistically significant when p < 0.05. 3. Results 3.1. Cloning and sequence analysis of zebrafish Daxx The zebrafish Daxx (GenBank# NM_001044949) cDNA was identified using a homologous search of the NCBI database using the NCBI BLAST program. The cloned zebrafish Daxx cDNA has a

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2151-bp open reading frame (ORF) that encodes 716 amino acids. The deduced amino acid sequences from the ORF are predicted to comprise a protein with a molecular weight of 80 kDa and an isoelectric point (PI) of 4.9. Motif analysis indicated that zebrafish Daxx contained a Daxx domain (Fig. 1A). Multiple sequence alignment was performed using the deduced amino acid sequences of zebrafish Daxx and other known Daxx proteins using BLAST. Only the amino acid sequence of the N-terminal helical domain (196-402 aa) was found to be very well conserved, and with identity values of 75% of HsDaxx (Fig. 1B). Phylogenetic tree analysis was carried out using zebrafish Daxx and members of the Daxx superfamily that were selected from vertebrates and invertebrates (Fig. 1C). The phylogenetic tree suggests an obvious difference between the invertebrate and vertebrate proteins. Zebrafish Daxx is closely related to Astyanax mexicanus AmDaxx, with identity values of 56%. In contrast, zebrafish Daxx, which was located in the fish branches, was distant from HsDaxx, with only 24% amino acid identity. These characteristics suggest that zebrafish Daxx represents a novel member of the Daxx family. 3.2. Expression pattern variation of Daxx in zebrafish To show the expression pattern variation of Daxx in zebrafish, mRNAs from various embryonic stages and different adult zebrafish tissues were assayed by real-time qPCR. According to our results, the expression of zebrafish Daxx mRNA reached its peak level at 6 hpf, decreased to its minimum at 24 hpf, and then showed persistently low expression from 24 hpf to 120 hpf. The expression levels remained stable after this time (Fig. 2A). In addition, zebrafish Daxx could be detected in all 11 selected tissue samples, and zebrafish Daxx was relatively high expressed in the muscle, ovary and testis (Fig. 2B). 3.3. Expression under virus challenge The goal of this study is to obtain more details regarding Daxx and its molecular immune mechanisms in response to ISKNV stimulation in zebrafish. The expression of Daxx mRNA in zebrafish spleens was increased in the first 24 h after infection, and then the expression of the zebrafish Daxx notably and gradually decreased after 12 h post-infection (hpi). The expression dropped approximately 2000-fold at 10 days post-infection (dpi) (Fig. 2C). 3.4. Subcellular localization To examine the subcellular localization of zebrafish Daxx, pDsRed1-N1-Daxx was transfected into Hela and HEK293T cells, respectively. 48 h after transfection, the localization of the RFPDaxx was visualized in the nucleus, as indicated by its overlap with DAPI signals. The overlaid image shows that the zebrafish Daxx proteins were predominantly and uniformly located in the nuclei of the Hela cells and punctate distribution in the nuclei of the HEK293T cells (Fig. 3A, B). 3.5. Zebrafish Daxx is a transcriptional repressor Previous studies have indicated that the homologous proteins of Daxx function as transcription repressors. The pL8G5-Luc system was used to confirm the potential function of zebrafish Daxx in transcriptional regulation. In this system, a pL8G5-Luc reporter contains five copies of the GAL4 DNA binding site upstream of the luciferase reporter transcription startsite, and LexA-VP16 fusion protein is a positive control, act as a transactivator. To examine the potential function of zebrafish Daxx in transcriptional regulation,

Please cite this article in press as: L. Qi, Z. Xiang, Molecular cloning and expression analysis of an apoptosis-associated gene Daxx from zebrafish, Danio rerio, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.03.040

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Fig. 1. Bioinformatics analysis of zebrafish Daxx protein. (A) Domain organization of zebrafish Daxx. (B) Multiple sequence alignment of Daxx with Daxx proteins from other species. Partial amino acid sequences including the helical domain were extracted, and the helical domain is marked between the lines and arrows. (C) Phylogenetic tree of the Daxx proteins. The tree was constructed based on the alignment of full amino acid sequences, and the node values represent the percent bootstrap confidence level derived from 1000 replicates. The bar indicates the genetic distance (0.2). The mus zinc finger E-box-binding homeobox 1(ZEB, XP_006525830.1) was used as the out group. GenBank accession numbers of the available model species of the Daxx sequences used in the analysis are indicated as [NP_001135442.1] Homo sapiens, [XP_003950848.1] Pan troglodytes, [XP_005979825.1] Pantholops hodgsonii, [XP_007460896.1] Lipotes vexillifer, [XP_005665942.1] Sus scrofa, [XP_004711805.1] Echinops telfairi, [XP_006536576.1] Mus musculus, [XP_005313226.1] Chrysemys picta bellii, [XP_006271784.1] Alligator mississippiensis, [XP_007442393.1] Python bivittatus, [XP_006000988.1] Latimeria chalumnae, [XP_002938713.1 ] Xenopus tropicalis, [NP_001038414.1] Danio rerio, [XP_007253141.1] Astyanax mexicanus, [ABO13872.1] Salmo salar, [XP_007564352.1] Poecilia formosa, [XP_003969133.1] Takifugu rubripes, [GAA30833] Clonorchis sinensis, [EKC37012.1] Crassostrea gigas, [EHJ76041.1] Danaus plexippus, [XP_003427694.1] Nasonia vitripennis, [EFN74124.1] Camponotus floridanus, [XP_006525830.1] Mus musculus, based on neighbor-joining analyses of Daxx.

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Fig. 2. Quantitative PCR analysis of zebrafish Daxx mRNA expression. (A) Zebrafish Daxx mRNA expression in different embryonic stages at 6, 12, 24, 36, 48, 72, 96, and 120 hpf. (B) Zebrafish Daxx mRNA expression in various adult tissues: intestines, eye, spleen, gill, liver, brain, kidney, heart, ovary, muscle and testis. The values are shown as the means ± S.D. (N ¼ 3). The same lowercase letter indicates that there is no significant difference (P < 0.01). (C) Zebrafish Daxx mRNA expression patterns in the zebrafish spleen after stimulation by ISKNV. The experiment was repeated thrice, and the values are shown as the means ± S.D. (N ¼ 3). Significant differences compared to the control is indicated with an asterisk (P < 0.05) or two asterisks (P < 0.01).

we constructed a fusion protein of zebrafish Daxx with the DNAbinding domain (DBD) of the yeast transcription factor GAL4 driven under a CMV promoter, termed pCMV-BD-Daxx, and then co-transfected HEK293T cells with pCMV-BD-Daxx and either the pL8G5-Lucor pLexA-VP-16 reporter (Fig. 3C). Relative to the activation of the BD protein, Daxx-BD repressed the luciferase activity to 0.6-fold. VP-16, a transcription activating factor, was used as a positive control to increase the activation of the luciferase approximately 170-fold relative to the BD control, but zebrafish Daxx could inhibit the activation of VP-16 approximately 19.7-fold. These results suggest that zebrafish Daxx is a potent transcriptional repressor. 3.6. Reporter gene repression of zebrafish Daxx To investigate the function of zebrafish Daxx in apoptosis and/or cell cycle arrest pathways, we performed pathway-specific reporter assays to examine the roles of zebrafish Daxx in the regulation of the NF-kB- and p53/p21-Luc reporter genes in HEK293T cells. As shown in Fig. 4, the expression of zebrafish Daxx significantly suppressed the luciferase activity of these reporter genes in a dosedependent manner. As shown in Fig. 4A, zebrafish Daxx down-regulated NF-kBluciferase expression following the increasing concentrations of zebrafish Daxx. To further examine the effect of zebrafish Daxx on the downstream target of the p53 pathway, we found that zebrafish

Daxx also inhibited the transcriptional activity of p53/p21-Luc (Fig. 4B, C). The transcriptional activity of p21 was suppressed by the expression of zebrafish Daxx in a dosage-dependent manner similar to the results from the p53 reporter assay. These results suggested that zebrafish Daxx may play an important role in cell cycle arrest and/or apoptosis pathways. 4. Discussion In our study, we cloned the Daxx cDNA sequence from zebrafish. It contains a 2151-bp ORF encoding a protein of 716 amino acids. Homology analysis revealed that the amino acid sequence of zebrafish Daxx was highly similar to those homologous proteins of other species at the helical domain. Phylogenetic analysis shows that zebrafish Daxx is close to the AmDaxx, all of which belong to the fish branch. All of these results suggest that zebrafish Daxx is a novel member of the Daxx family. Daxx plays a key role in development and tissue metabolism, and it either directly or indirectly suppresses apoptosis in the early embryo. Daxx-deficient mice exhibit extensive apoptosis and embryonic lethality [20]. In adult tissues, HsDaxx is ubiquitously expressed throughout the body, with particularly high expression in the testis [2], which was found to be involved in tumorigenesis and inhibition in these tissues. For example, during ovarian cancer cell proliferation, metastasis, and radio/chemoresistance, the depletion of Daxx inhibited tumor development [21]. In this study,

Please cite this article in press as: L. Qi, Z. Xiang, Molecular cloning and expression analysis of an apoptosis-associated gene Daxx from zebrafish, Danio rerio, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.03.040

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Fig. 3. The Subcellular localization and transcriptional activity analysis of zebrafish Daxx. (A) Zebrafish Daxx was localized in the nuclei of Hela and (B) HEK293T cells. (C) pCMV-BDDaxx, pLexA-VP16 or pCMV-BD along with the pL8G5-Luc reporter were transiently transfected into HEK293T cells. The values are shown as the means ± S.D. (N ¼ 3). The same lowercase letter indicates that there was no significant difference (P < 0.01).

we found that zebrafish Daxx was expressed during embryonic development and was relatively highly expressed in the muscle, ovary and testis of the adult zebrafish. These results according to the expression patterns of the known Daxx in other species, and they provide a new model by which to study the role of Daxx during development and tumorigenesis in zebrafish. When zebrafish were challenged with the ISKNV, and we subsequently observed slightly increased zebrafish Daxx expression in the spleen following virus infection. In addition, the expression of zebrafish Daxx was shown to be down-regulated after 1 day postinfection and decreased about two thousand-fold at 10 dpi. In the early phase of the infection, the host cell secretes IFNs and other

inflammation-associated cytokines to defend against the invasion of the virus. The increased expression of zebrafish Daxx may be used to inhibit apoptosis at this time, while later in the infection, zebrafish Daxx was found to decrease in order to induce apoptosis. Many studies have indicated that the nuclear localization of Daxx is controlled by two independent signals, the accurate subcellular localization mechanism of Daxx is unknown [4]. Zebrafish Daxx contains two NLSs, which may be responsible for the localization of the zebrafish Daxx. Following the expression of the DaxxRFP, the nuclear localization of zebrafish Daxx was observed under a fluorescence microscope in Hela cells. DNA virus proteins such as the BNRF1, an EBV tegument protein, could stimulate Daxx to

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Fig. 4. Transcriptional activation analysis of zebrafish Daxx: (A) Inhibition of NF-kB, (B) p53 and (C) p21-Luc transcriptional activity by the expression of zebrafish Daxx. The control plasmid is pCMV-Myc. The values are shown as the means ± S.D. (N ¼ 3). Lowercase letters indicate that there is no significant difference.

localize to the nuclear bodies [22]. In the HEK293T cells, the location of zebrafish Daxx was changed by the stably expressed adenoviruses proteins. Daxx may indirectly interact with and regulate the activation of other transcription factors to regulate the expression of the genes, such as Pax-3, Pax-5 and ETS1 [23e25]. In addition, Daxx has been found to participate in large multiprotein complexes that include core histones and histone deacetylases II (HDACs) to regulate the transcription of the genes [26]. All of these studies suggested that Daxx possesses the ability to regulate transcription, but the exact mechanism for the transcriptional regulation by Daxx is still unknown. In this report, our results show that zebrafish Daxx is a transcription repressor that can strongly inhibit the activation of the L8G5-luc reporter system. To investigate the functional properties of zebrafish Daxx in the NF-kB signaling pathway, a pathway analysis was performed with the HEK293T cell line. Effective repression of NF-kB reporter gene was observed when cells were transfected with zebrafish Daxx. These results indicated that zebrafish Daxx has a role in regulating the transcriptional activity of NF-kB, and the protein is involved in various cellular events and plays critical roles (Fig. 4A). In zebrafish, Daxx was found to inhibit the p53-Luc reporter gene in a dose-dependent manner (Fig. 4B). As a proliferation inhibitor, p21 plays an important role in preventing tumor development, and it is regulated by p53 through the p53-binding sequences in the p21 promoter. In this paper, zebrafish Daxx was found to repress the expression of a p21 luciferase reporter gene (Fig. 4C).

In conclusion, the transcription profile analysis showed that Daxx is expressed during embryonic development and in selected adult tissues of the zebrafish. In the spleen of the zebrafish, the expression of Daxx mRNA level was dramatically changed when challenged with ISKNV. Fluorescence localization assays indicated that zebrafish Daxx was located in the cell nucleus uniformly in Hela cells but different in HEK293T cells. As a transcription repressor, zebrafish Daxx repressed the activation of the L8G5-Luc reporter gene. In addition, the over-expression of zebrafish Daxx could inhibit the NF-kB- and p53/p21-Luc reporter genes. All of these results indicated that Daxx might play a key role in the regulation of cell apoptosis and the defense against viral infection. Acknowledgments This research was supported by the National Science Foundation of China (No. 31202021), Program of the Administration of Ocean and Fisheries of Guangdong Province, China (A201301B08), Sun Yat-sen University State Key Laboratory of Biocontrol Open Program (SKLBC2010K06). References [1] P. Salomoni, A.F. Khelifi, Daxx: death or survival protein? Trends Cell Biol. 16 (2006) 97e104. [2] X. Yang, R. Khosravi-Far, H.Y. Chang, D. Baltimore, Daxx, a novel Fas-binding protein that activates JNK and apoptosis, Cell 89 (1997) 1067e1076.

Please cite this article in press as: L. Qi, Z. Xiang, Molecular cloning and expression analysis of an apoptosis-associated gene Daxx from zebrafish, Danio rerio, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.03.040

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Please cite this article in press as: L. Qi, Z. Xiang, Molecular cloning and expression analysis of an apoptosis-associated gene Daxx from zebrafish, Danio rerio, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.03.040

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Molecular cloning and expression analysis of an apoptosis-associated gene Daxx from zebrafish, Danio rerio.

The death domain-associated protein Daxx exerts many functions including the induction and inhibition of apoptosis, regulation of chromatin remodeling...
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