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Molecular cloning of metal-responsive transcription factor-1 (MTF-1) and transcriptional responses to metal and heat stresses in Pacific abalone, Haliotis discus hannai

Molecular cloning of metal-responsive transcription factor-1 (MTF-1) and transcriptional... Background: Metal-responsive transcription factor-1 (MTF-1) is a key transcriptional regulator playing crucial roles in metal homeostasis and cellular adaptation to diverse oxidative stresses. In order to understand cellular pathways associated with metal regulation and stress responses in Pacific abalone (Haliotis discus hannai), this study was aimed to isolate the genetic determinant of abalone MTF-1 and to examine its expression characteristics under basal and experimentally stimulated conditions. Results: The abalone MTF-1 shared conserved features in zinc-finger DNA binding domain with its orthologs; however, it represented a non-conservative shape in presumed transactivation domain region with the lack of typical motifs for nuclear export signal (NES) and Cys-cluster. Abalone MTF-1 promoter exhibited various transcription factor binding motifs that would be potentially related with metal regulation, stress responses, and development. The highest messenger RNA (mRNA) expression level of MTF-1 was observed in the testes, and MTF-1 transcripts were detected during the entire period of embryonic and early ontogenic developments. Abalone MTF-1 was found to be Cd inducible and highly modulated by heat shock treatment. Conclusion: Abalone MTF-1 possesses a non-consensus structure of activation domains and represents distinct features for its activation mechanism in response to metal overload and heat stress. The activation mechanism of abalone MTF-1 might include both indirect zinc sensing and direct de novo synthesis of transcripts. Taken together, results from this study could be a useful basis for future researches on stress physiology of this abalone species, particularly with regard to heavy metal detoxification and thermal adaptation. Keywords: Abalone, Haliotis discus hannai, MTF-1, Heavy metal, Heat shock Background transcription factor has also been known to be closely Metal-responsive transcription factor-1 (MTF-1; also involved in cellular adaptation and protection against termed metal-regulatory transcription factor-1 or metal- oxidative stresses through regulating the transcription responsive-element-binding transcription factor-1) is a key of diverse genes related with host defense-related path- transcriptional regulator playing pivotal roles in metal ways (Günther et al. 2012a; Lichtlen and Schaffner homeostasis and detoxification (Laity and Andrews 2007; 2001). They include metal reservation/detoxification Günther et al. 2012a). In addition to its fundamental role (e.g., metallothionein, MT; the main target of MTF-1), for homeostatic metal regulation, this multitasking metal ion transport (e.g., Zn or Cu transporters), iron homeostasis/anti-microbial responses (e.g., hepcidin), cellular redox homeostasis (e.g., selenoproteins and * Correspondence: yoonknam@pknu.ac.kr Department of Marine Bio-Materials & Aquaculture, Pukyong National thioredoxin reductase), and glutathione biosynthesis University, Busan 48513, South Korea © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lee and Nam Fisheries and Aquatic Sciences (2017) 20:9 Page 2 of 13 (e.g., glutamate cysteine ligase) (Günther et al. 2012a; related pollutants contaminated in both water and Lichtlen et al. 2001; Stoytcheva et al. 2010). sediments could be significant factors to provoke cel- In a structural viewpoint, MTF-1 has been considered lular toxicity and oxidative stress in farmed abalones as a conserved transcription factor to possess six C H - (Kim et al. 2007). 2 2 type zinc fingers as the DNA-binding domain to recognize However, despite its importance, adaptive or defensive metal responsive elements (MREs) (Giedroc et al. 2001). functions to such environmental perturbations have As a cellular metal and stress sensor, the activity regula- been limitedly investigated in this abalone species, and tion of MTF-1 is generally characterized by the three suc- almost no information has been available with respect to cessive steps, i.e., nuclear-cytoplasmic shuttling upon the coordinated regulations of genes involved in cellular stress exposure, DNA-binding, and the interaction(s) with pathways associated with metal regulation and oxidative other coactivators to modulate the target gene transcrip- stress responses (Kim et al. 2007; Lee and Nam 2016a). tion (Li et al. 2008). To execute the transcriptional regula- For this reason, understanding of MTF-1 from abalone tion, MTF-1 binds to the specific site, called MRE (core species would be much useful to better comprehend or- sequence = TGCRCNC), in the promoter region of target chestrated and coordinated regulations of host defense gene (Günther et al. 2012a). Accordingly, the transcrip- genes in this abalone species. Based on this need, this tional expression of MTF-1 gene itself has been reported study, as a startpoint research, was aimed to characterize to be constitutive and not to be affected by heavy metal the genetic determinant of MTF-1, the superordinate and other stressor treatments because its regulatory func- regulator for diverse host defense genes, from the Pacific tions should be controlled mainly at post-translational abalone (H. discus hannai). For this, we isolated and char- levels (Auf der Maur et al. 2000; Bi et al. 2006). Structural acterized the full-length complementary DNA (cDNA) scheme and functional context of MTF-1 above-described encoding the abalone MTF-1, bioinformatically dissected are believed to be widespread in the vertebrate lineage, its 5′-upstream regulatory region, and also scrutinized ex- although the majority of empirical information has come pression patterns of MTF-1 under both non-stimulated from mammalian MTF-1s. and stress-challenged (i.e., heavy metal exposure and heat However, in contrast to richness of knowledge on shock) conditions. mammalian orthologs, molluscan MTFs have been nar- rowly explored barring only couples of previous reports Methods (Qiu et al. 2013; Meng et al. 2015). Nevertheless, it is Abalone samples and molecular cloning of MTF-1 noteworthy that currently available molluscan MTF-1 Abalones (H. discus hannai) used in this study were sequences from public databases have suggested that experimental stocks maintained at Experimental Fish mollusc species may show non-canonical features in Culture Station, Pukyong National University (PKNU), their MTF-1 structures, which may differ from verte- Busan, South Korea. Abalones were maintained with brate orthologs. For instances, unlike vertebrate ortho- semi-water recirculation system equipped with 3-ton cap- logs, molluscan MTF-1s often lack several typical motifs acity of rectangular culture tanks, in which the tanks were in presumed transactivation domains. Furthermore, a re- connected with protein skimmers, custom-designed mech- cent study has claimed that the nuclear-cytoplasmic anical filters, and 1-μm-mesh filter. Throughout the experi- shuttling, a key prerequisite step for vertebrate MTF-1s, ment, water temperature and dissolved oxygen were kept to might not always be an absolute precondition in certain be ranged within 20 ± 1 °C and 8 ± 1 ppm, respectively. mollusc species (Meng et al. 2015). Abalones were fed with frozen or dried seaweeds until 2 days Pacific abalone, Haliotis discus hannai, is a highly val- before stress exposure treatments. Daily water exchange rate ued seafood mollusc not only in Korea but also in other was about 20%, and in-tank wastes including feces and deb- East-Asian countries. Intensive aquacultural operation ris on the bottom were removed twice every day. for abalone farming using the marine net-cage system Based on the NGS-transcriptome analysis of the has been established in Korean aquaculture domain. juvenile abalone tissues (unpublished data), partial NGS During the last decade, the remarkable growth of aba- clones representing the significant homology to known lone production in a quantitative term has been achieved animal MTF-1s were selected and assembled into a con- (Park and Kim 2013). However, more recently, the tig. In order to get full-length cDNA version, rapid sustainable progress of abalone culture has been con- amplifications of cDNA ends (RACE) at both 5′-and siderably hurdled by the depressed productivity mainly 3′-directions were carried out using total RNA isolated in relation to frequent outbreaks of high mortality from a whole body sample and SMARTer® RACE 5′/3′ and physiological deformity in many abalone farms Kit (Clontech Laboratories Inc., Mountain View, CA, (Park and Kim 2013; Kang et al. 2015). Considering that USA) according to the manufacturer’s instructions. abalone farming in Korea mainly relies on the net-cage Oligonucleotide primers used in this study are listed in facility installed in coastal areas, the heavy metals or other Additional file 1: Table S1. The amplified fragments were Lee and Nam Fisheries and Aquatic Sciences (2017) 20:9 Page 3 of 13 sequenced and again subjected to contig assembly. Based juveniles (21.5 ± 4.1 g for total weight; n = 12), six kinds on the assembled sequence in a contig, full-length abalone of tissues including the gill, gut, heart, hemolymph, hep- MTF-1 cDNA was re-isolated by RT-PCR amplification atopancreas, and muscle (foot muscle) were surgically using the same total RNA aforementioned. Amplified RT- removed individually. For hemolymph, centrifugation PCR products were directly sequenced at both forward (2500 rpm for 10 min at 4 °C) was carried out in order and reverse directions to obtain a representative cDNA se- to collect hemocyte pellet. Second, from 3-year-old sexu- quence for abalone MTF-1. ally mature adults showing a clear sign of ovarian and From the cDNA sequence, the 5′-upstream region of testicular maturation (96.4 ± 13.1 g for total weight; eight abalone MTF-1 gene was cloned by genome walking each for female and male), same tissue types abovemen- method. Using the genomic DNA prepared from an indi- tioned were obtained and additional ovary and testis vidual muscle, genome walking to 5′-upstream region was were obtained. Upon surgically removed, biological conducted with designated pairs of gene-specific primers samples were immediately frozen on dry ice and stored and Universal Genome Walker® Kit (Clontech Laboratories at −80 °C until used for RNA isolation. Inc., USA) according to the manufacturer’s instructions. In order to obtain developing embryos and early lar- Amplified fragments were TA cloned into pGEM-T® easy vae, artificial insemination of sperm (from three males) vector (Promega, Madison, WI, USA), sequenced and as- to eggs (from eight females) was conducted by using the sembled into a single contig. Afterward, the continuous, conventional induced spawning method including an air genomic fragment containing the 5′-flanking region was exposure and ultraviolet-irradiated seawater treatment. again PCR isolated from the genomic DNA template Insemination was made with wet-method at 20 °C and abovementioned and directly subjected to the sequencing incubated at the same temperature until the end of sam- to confirm the representative sequence of the abalone pling. An aliquot of developmental samples each consist- MTF-1 proximal promoter region. ing of approximately 20,000~30,000 embryos or larvae was sampled at six time points: just before insemination Bioinformatic sequence characterization (unfertilized eggs), early cleavages (i.e., 2~8 cells stage; at With the ORF Finder program (https://www.ncbi.nlm.nih. 2 h post insemination; 2 hpi), morula (5 hpi), gov/orffinder/), the open reading frame (ORF) of abalone trochophore (12 hpi), early veliger (18 hpi), and late veli- MTF-1was predictedand deducedaminoacidsequence ger (42 hpi), based on the microscopic examination. was obtained. Based on the homology search using NCBI Upon sampling, embryos and larvae were also frozen on BLASTx (http://blast.ncbi.nlm.nih.gov/Blast.cgi), sequence dry ice and stored at −80 °C until used. Two replicate homology of abalone MTF-1 with its orthologs was samplings were carried out for each time point. examined. Parameter scores for the primary structure of MTF-1 were estimated using ExPASy ProtParam tool Experimental in vivo stimulatory challenges (http://web.expasy.org/protparam/). Multiple sequence Two independent stress exposure experiments were carried alignment was done using CLUSTALW program (http:// out: one was with heavy metal exposure and the other with www.genome.jp/tools-bin/clustalw). Identification of puta- heat shock treatment. For heavy metal exposure, eight ju- tive zinc finger domains was carried out with Simple venile individuals (24.5 ± 3.6 g; approximately 1 year old) Modular Architecture Research Tool (SMART; http://smar- were assigned into one of five experimental tanks (70-L t.embl.de/). Predictions of potential nuclear localization sig- capacity containing 50 L of 1-μm filtered seawater at 20 °C) nal (NLS) and nuclear export signal (NES) were conducted and acclimated to the tank conditions for 24 h before heavy with cNLS Mapper (http://nls-mapper.iab.keio.ac.jp/cgi- metal treatment. The heavy metals used for exposure were bin/NLS_Mapper_form.cgi) and NetNES 1.1 Server (http:// of analytical grade reagents (Sigma-Aldrich, St. Louis, MO, www.cbs.dtu.dk/services/NetNES/), respectively. The puta- USA). After 24 h, two tanks were treated with 0.02 mg/L tive zinc finger-DNA binding regions from selected MTF-1 (i.e., 20 ppb) and 0.1 mg/L (i.e., 100 ppb) cadmium (Cd), orthologs were subjected to molecular phylogenetic analysis while two tanks with 20 and 100-ppb zinc (Zn) (Lee and using Molecular Evolutionary Genetics Analysis tool (ver. Nam 2016a). Nominal concentration of the metal for each 7.0.21; http://www.megasoftware.net/). Putative transcrip- metal-exposed group was adjusted by using CdC1 or tion factor binding motifs in the abalone MTF-1 promoter ZnCl stock solution. Remaining one tank was treated with were predicted with TRANSFAC® software (http://genex- only 1-mL distilled water that had been used for reconstitu- plain.com/transfac; GeneXplain GmbH, Wolfenbüttel, tion of the metals (i.e., for non-exposed control). For each Germany). group, two replicate tanks were prepared identically. Treatment duration was 24 h. At the end of exposure, gill, Tissue collection from different developmental stages hemocyte, hepatopancreas, and foot muscle were sampled Tissue distribution assay was conducted with two age individually from six randomly chosen individuals as classes of abalones. First, from 1-year-old immature described above. Lee and Nam Fisheries and Aquatic Sciences (2017) 20:9 Page 4 of 13 On the other hand, for heat shock treatment, 22 indi- assays and stress exposure treatments), while RPL7 and viduals (21.1 ± 3.1 g; same-aged as above) were assigned RPL8 (KP698947) were used to normalize MTF-1 expres- into one of four 100-L tanks (two for heat-stressed sion across developmental samples (embryos and larvae). groups and two for non-stressed groups) at 20 °C. Each Additionally, for heavy-metal exposure groups, the mes- tank was equipped with a custom-designed apparatus for senger RNA (mRNA) expression levels of MTF-1 were mechanical filtration. After 24 h of acclimation period, compared with those of MT (the known target gene of water temperature of the two tanks (heat-stressed groups) MTF-1) in order to examine whether or not there might was elevated using the adjustable thermostat-assisted aquar- be any positive or proportional relationship in the metal- ium heaters (400 W) with an increment rate of 1 °C/h. mediated modulation patterns between MTF-1 and MT When the temperature reached 30 °C, the temperature genes. PCR efficiency of primer pair for each gene was vali- was kept to be constant at 30 °C for additional 24 h. dated to be at least higher than 95% based on the standard Samplings were made at 20 °C (just before thermal curve prepared using a fivefold serial dilution of cDNA elevation), 25 °C (5 h after elevation started), 30 °C (10 h), mix. For each cDNA sample, triplicate assays were carried 30 °C+12 h (12 h after reaching 30 °C; 22 h after elevation out in an independent fashion. started), and 30 °C+24 h (24 h after reaching 30 °C; 34 h Quantitative PCR-based MTF-1 mRNA expression after elevation started). Four individuals were randomly levels across tissue types and developmental stages selected from each tank to constitute eight individuals per under non-stressed conditions were presented as ΔCt temperature group at each sampling point. Tissues (Ct of the MTF-1 gene subtracted from the Ct of each sampled were gill, hemocyte, hepatopancreas, and foot internal control gene). On the other hand, differential muscle. Meanwhile, non-stressed control groups were also expression levels of metal-exposed or heat shock-treated identically prepared with heat-shock groups, but the group relative to their corresponding non-stressed con- temperature (20 °C) was kept to be constant until the end trol groups were presented as the fold difference to the −ΔΔCt of experiment. At the same sampling point, the identical non-stressed controls by using the formula 2 number of abalones (n = 4 per tank) was also obtained (Schmittgen and Livak 2008). Expression levels between from non-stressed control groups. Temperature of each or among groups were tested using Student’s t test or tank was confirmed to be ranged within ±0.5 °C. Dissolved one-way ANOVA (followed by Duncan’s multiple ranged oxygen levels were adjusted to be ranged from 7.5 to tests). Difference was considered to be significant when 8.5 ppm for all the experimental tanks. Abalones were not P < 0.05. fed during stimulatory challenge experiments. Results and discussion RT-qPCR assay and statistics Characteristics of abalone MTF-1 cDNA and deduced Total RNA was extracted using TriPure® Reagent (Roche amino acid sequences Applied Science, Mannheim, Germany) and then The full-length cDNA of abalone MTF-1 was comprised purified using RNeasy Mini Plus Kit (Qiagen, Hilden, of 48-bp 5′-untranslated region (UTR), a 1509-bp single Germany) including DNase I treatment step. An aliquot open reading frame (ORF) encoding a polypeptide of (2 μg) of total RNA prepared was reverse transcribed into 503 amino acids, and 582-bp 3′-UTR including a stop cDNA by using the Omniscript® Reverse Transcription Kit codon and 19-bp poly(A+) tail. A putative polyadenyla- (Qiagen, Germany) including oligo-dT primer according to tion signal (AATAAA) was found at 21 bp prior to the the manufacturer’s instruction. Synthesized cDNA was poly(A+) tail (GenBank accession number; KT895224) fourfold diluted with sterile distilled water, and an aliquot (Additional file 1: Figure S1). The MTF-1 protein based of 2 μL was included in a qPCR reaction as a template. on the deduced amino acid sequence was estimated to The qPCR reaction was conducted with a LightCycler® 480 have 54.86 kDa of calculated molecular mass and 5.51 of Real-Time PCR System and LightCycler® 480 SYBR Green theoretical pI value, respectively. Abalone MTF-1 repre- I Master (Roche Applied Science, Germany), according to sented quite a low sequence homology to its vertebrate the manufacturer’s instructions. Thermal cycling condition and invertebrate orthologs where the maximum se- for each gene (i.e., MTF-1 and normalization control quence identity at amino acid level was found to be only genes) can be referred to Additional file 1: Table S1. Based 27% with Biomphalaria glabrata (air-breathing fresh- on our preliminary study to evaluate candidate housekeep- water snail; Gastropoda; Mollusca). Non-conservative ing genes for the normalization of RT-qPCR amplification feature without any appreciable sequence similarity to (unpublished data; see also (Lee and Nam 2016a; Lee and other MTF-1s is also found in the putative activation. Nam 2016b)), abalone ribosomal proteins L5 (RPL5; Abalone MTF-1 was likely to possess only a shortened ABO26701) and L7 (RPL7; KP698945) genes were used as fragment (44-aa; pI = 4.06) presumed for the acidic reference genes to normalize expression levels of MTF-1 domain and to lack almost entire region corresponding transcripts in tissue samples (i.e., for basal tissue expression to the proline-rich domain and serine/threonine-rich Lee and Nam Fisheries and Aquatic Sciences (2017) 20:9 Page 5 of 13 domain of vertebrate MTF-1 orthologs (Additional that MTF-1s have been largely divergent in the molluscan file 1: Figure S2). phylum, which is apparently different form the mono- In contrast, the abalone MTF-1 was proven to share a phyletic clustering of orthologs from Chordata phylum high structural homology with other MTF-1s in the DNA (Additional file 1: Figure S3). Functional partition of the binding domain (Fig. 1). All the MTF-1 proteins including zinc finger domains in abalone MTF-1 has been remained the abalone MTF-1 (but except for the shortest Octopus to be further characterized; however, from the mammalian bimaculoides MTF-1 having only five zinc fingers) were studies, the region from the second to fourth zinc fingers found to show highly conserved six C H -zinc fingers in have been proposed to constitute the core DNA binding 2 2 their DNA binding domains. In their DNA binding do- domain while the first finger has been reported to serve as mains, 12 cysteine residues and histidine residues were a metal-sensing domain (Bittel et al. 2000). Within each clearly conserved, in which the zinc finger domain of aba- zinc finger, the His-X-Arg/Lys-X-His [H-X-(R/K)-X-H lone MTF-1 showed the highest sequence identity (77%) where X is any amino acid] motif has been known as a with that of orthologue isoforms from Crassostrea gigas key site for zinc binding (Günther et al. 2012a), and it is (Bivalve, Mollusca). However, the molecular phylogenetic preserved in the 1st to 5th zinc fingers for all the species analysis using the DNA binding domains has indicated examined in the present study. However, in the last finger Fig. 1 Multiple amino acid sequence alignment of conserved zinc-finger DNA-binding domain from the abalone MTF-1 along with those from representative orthologs. In the alignment, conserved amino acid residues are indicated by red-colored letters, and two cysteines and two histidines typical of the C H structure for each zinc finger are boxed. The putative auxiliary nuclear localization signal (aNLS) conserved in the 2 2 front of the first zinc finger is indicated by upper line. The accession code for each sequence is noted at the end of alignment. The alignment with full-length polypeptide sequences and the full name of each species can be referred to Additional file 1: Figure S2 Lee and Nam Fisheries and Aquatic Sciences (2017) 20:9 Page 6 of 13 (i.e., the 6th finger), the Arg/Lys residue inside the 5- established in mammalian MTF-1), NLS and NES are amino-acid stretch was found to be conserved only in ver- responsible for balanced subcellular distributions of tebrate MTF-1s. Meanwhile, all the molluscan MTF-1s MTF-1 proteins (i.e., import and export, respectively) showed a phenylalanine (Phe) as the first amino acid of under both stressed and non-stressed conditions the first zinc finger, whereas vertebrate and brachiopod (Günther et al. 2012a). The NES motif in vertebrate orthologs possessed a tyrosine (Tyr) (Cheung et al. 2010). MTF-1 is usually embedded in the acidic activation do- Abalone MTF-1 showed both conserved and unique main (Günther et al. 2012a; Cheung et al. 2010). How- features in the peptide linkers connecting zinc fingers. ever, the abalone MTF-1 does not show any typical NES Conserved peptide linkers were found between 1st and motif, although a putative NLS motif is predicted in the 2nd fingers (Arg-Gly-Glu-Tyr-Thr), between 2nd and front of the first zinc-finger domain that are also con- 3rd fingers (Thr-Lys-Glu-Lys-Pro), and between 4th and served all MTF-1s examined (i.e., conserved auxiliary 5th fingers (Thr-Gly-Glu-Lys-Pro). On the other hand, NLS). The absence of acidic domain-embedded NES is unique linkers were found between 3rd and 4th fingers not limited to abalone MTF-1, i.e., all molluscan MTF- [Ser-Gly-Asn-Thr in abalone vs. Thr-Gly-Lys-Thr in ver- 1s are also likely to lack the NES at the corresponding tebrates vs. Thr-Gly-(Glu/Asn/Asp)-Thr in other mol- region. Hence, our finding may suggest that molluscan luscs and a brachiopod species] and between 5th and MTF-1s could be different from mammalian MTF-1s in 6th fingers [Ser-Gly-Glu-Lys-Pro in abalone vs. Thr-Gly- their subcellular localization control under both basal and Glu-(Lys/Arg)-Pro in vertebrates vs. Thr-Gly-(Asp/Gly/ stimulated conditions. A recent study with an oyster spe- Glu)-(Lys/Arg)-Pro in others]. Peptide linkers in multi- cies (C. gigas) has claimed that the MTF-1 would primar- zinc finger domains have been reported to play roles in ily localize in the nucleus even under unstressed not only structural stabilization but also interfinger in- conditions and nuclear translocation might be uncritical teractions for DNA-binding affinity of the zinc finger for the activation of the oyster MTF-1 (Meng et al. 2015). domains (Li et al. 2006). Particularly in the MTF-1, the In addition, the typical motif of cysteine cluster linker between 1st and 2nd fingers has been known to (consensus sequence = Cys-Gln-Cys-Gln-Cys-Ala-Cys) be crucial in the zinc-sensing ability of the MTF-1 with that could be commonly found in the region immedi- regard to the formation of the ternary (MTF-1-zinc- ately following the serine/threonine-rich domain of ver- DNA) complex for activating MT gene transcription tebrate MTF-1s was not detected in the abalone MTF-1 (Li et al. 2006). Taking this into account, the abalone (Additional file 1: Figure S2). The cysteine cluster has MTF-1 is thought to preserve a fundamental property of been reported to be essentially necessary for metal- zinc-sensing function similarly with mammalian orthologs induced transcriptional activity and homodimerization since it conserves a completely identical linker (i.e., the of mammalian MTF-1 (Günther et al. 2012a; Günther et Arg-Gly-Glu-Tyr-Thr linker between 1st and 2nd fingers) al. 2012b). Like with NES abovementioned, none of mol- with mammalian MTF-1s. However, the present abalone luscan MTF-1 represents a canonical C-terminus cyst- MTF-1 showed apparent dissimilarity with vertebrate eine cluster, suggesting that molluscan MTF-1s might orthologs in the linkers between 3rd and 4th fingers and have different mechanism(s) for metal-induced tran- between 5th and 6th fingers. Particularly because the scription (i.e., recruitments of transcriptional cofactor linker between 3rd and 4th fingers has been reported to partners in the promoter/enhancer of target genes). be important for the in vivo and in vitro sensitivity of zinc-dependent activation of MTF-1 (Li et al. 2006), this Prediction of transcription factor binding sites linker could be a good target for future studies to examine From the bioinformatic prediction, the 1691-bp 5′-up- the potential difference in the linker-mediated zinc-finger stream region from the ATG translation start site of the function between abalone MTF-1 and mammalian/verte- abalone MTF-1 gene represented various transcription brate orthologs. factor binding sites (Fig. 2) (Additional file 1: Figure S4). In the abalone MTF-1, a putative auxiliary nuclear The abalone MTF-1 promoter revealed a canonical localization signal (aNLS) was identifiable in the front of TATA box (TATAAA) at −474 bp (from the ATG). Im- the first zinc finger, as similarly with all other MTF-1 portantly, it represented a copy of MRE (TGCRCNC; orthologs. Of seven amino acid residues to comprise the −959 bp), suggesting the possible modulation of MTF-1 auxiliary NLS, the positions of two amino acids ( Glu itself by heavy metal-driven cellular stressors, which is and Arg) are conserved in all the MTF-1s examined clearly inconsistent with the lack of MRE in many mam- (Fig. 1). However, due to the potential deletion in the malian MTF-1 promoters (Auf der Maur et al. 2000; Bi putative acidic domain, no canonical nuclear export et al. 2006). In addition, a xenobiotic response element signal (NES) was observable in the abalone MTF-1 (XRE; TNGCGTG; −736 bp) was predicted in the unlike vertebrate orthologs. Within a concept of MTF-1 abalone MTF-1 promoter. XRE is an aryl hydrocarbon activation (i.e., a nuclear-cytoplasmic shuttling function receptor (AhR)-targeted motif involved in the ligand Lee and Nam Fisheries and Aquatic Sciences (2017) 20:9 Page 7 of 13 Fig. 2 Schematic drawing to show the putative transcription factor-binding motifs predicted in the 5′-upstream region of the abalone MTF-1 gene. Binding motifs are searched by TRANSFAC® search (GeneXplain GmbH, Germany). For detailed representation including the 5′-upstream sequence, reader is referred to Additional file 1: Figure S4 (i.e., 2,3,7,8-tetrachlorodibenzodioxin (TCDD))-activated and fundamental roles in most cell types (Auf der Maur pathway to detoxify the effects of TCDD-related com- et al. 2000; O’Shields et al. 2014). The MTF-1 mRNAs pounds. However, because invertebrate AhR homologues were robustly expressed in hemocytes (P < 0.05), and this have been reported to lack the ability to bind TCDD dir- highest expression level was followed by muscles and ectly (Hahn et al. 2006), the molecular mechanism on the gills, whereas the least mRNA expression was found in potential interconnection between MRE/MTF-1 and heart (P < 0.05). However, this expression pattern was XRE/AhR paths should be further explored. Nevertheless, not fully reproducible when measured with the sexually recent mammalian studies have also highlighted multi- mature adults, although the broad pattern was consist- tasking roles of AhR in various signaling pathways associ- ent with findings from juveniles. Unlike in juveniles, the ated with cell cycle control and antioxidant protection expression level of MTF-1 in the gut was found to be as against oxidative stresses (Jackson et al. 2015). Besides, high as that in the hemocytes in both female and male several transcription factor binding sites such as heat adults. More noticeably, matured adult abalones dis- shock element (HSE; GAANRTTC; −1003 and −32 bp; played a strikingly apparent difference in the MTF-1 targeted by heat shock factor (HSF)), hypoxia response expression in gonads where the extraordinarily high ex- element (HRE; RCGTG; −895 and −285 bp; by hypoxia- pression level was observed in testis while only minute inducible factor-1 alpha (HIF-1α)), and other sites recog- expression in ovary (more than 150-fold difference; nized by cyclic AMP response element binding protein P < 0.05). Testis-predominant expression pattern of (CREBP; TGACGY; −1253 bp) and nuclear factor for acti- abalone MTF-1 in this study is similar with previous vated T-cells (NF-AT1; WGGAAA; −941, −169, and findings made in mouse (Auf der Maur et al. 2000) and −33 bp) were predicted. All of these factors have been hybrid tilapia (Oreochromis aurea × Oreochromis nilotica) known to be related with stress responses of animals (Cheung et al. 2010), collectively suggesting the possible (Saydam et al. 2003; Dubé et al. 2011). Abalone MTF-1 involvement of MTF-1 in the male reproduction. Yet, the promoter also revealed motifs that might be targeted by mechanism underlying the robust expression of MTF-1 in transcription factors generally known to be involved in de- the abalone testes is currently unknown and open to velopment, signal transduction, cell proliferation and/or hypothesize. In mouse, the reason for the high MTF-1 ex- organ development. They included Smad4, hepatocyte nu- pression in testis has been explained by that sexually ma- clear factor (HNF), CCAAT-enhancer binding protein (C/ ture mice need to accumulate a large quantity of MTs in EBP), E-box binding protein, and estrogen receptor (ER). their testes (Auf der Maur et al. 2000; De et al. 1991). However, this hypothesis is unlikely to be adopted to this Tissue distribution and basal expression levels abalone species since the virtual increase of MT expres- Based on the RT-qPCR analysis with immature juvenile sion in sexually mature abalones has been observed in abalones, MTF-1 mRNAs were detected in all the tissue ovary rather than in testis (Lee and Nam 2016a), suggest- types examined; however, basal expression levels were ing the molecular mechanism for the boosted expression quite variable among tissues (Fig. 3). The ubiquitous de- of MTF-1 in abalone testis might be distinct from the ones tection of MTF-1 transcripts across all the tissues is not in mammals. Hence, further study to monitor the MTF-1 surprising when taken into account its housekeeping expression in line with the testis development and Lee and Nam Fisheries and Aquatic Sciences (2017) 20:9 Page 8 of 13 ab Fig. 3 Tissue distribution patterns and basal expression levels of abalone MTF-1 mRNAs in a immature juvenile and b sexually matured adult tissues, based on RT-qPCR assays using the normalization against RPL5 and PPL7 references. Tissue abbreviations are gill (GI), gut (GU), heart (HE), hemocyte (HC), hepatopancreas (HP), muscle (MU), and gonad (GON; i.e., ovary or testis). In a, histograms with different letters indicate the significantly different means based on ANOVA followed by Duncan’s multiple ranged tests at P <0.05. In b, the significantly different means within a sex are indicated by a–e (for females) and v–z (for males) base on ANOVA followed by Duncan’s multiple ranged tests at P < 0.05. The significantly different means between sexes observed in heat, hepatopancreas, and gonad tissues are noted by asterisks based on Student’s t test analysis at P <0.05 maturation cycle would be valuable to get a deeper insight developments, as inferred from mammalian and teleos- into this phenomenon. Besides the gonadal expression, tean cases (Günes et al. 1998; Chen et al. 2002; Chen matured abalones showed a sex-specific difference in the et al. 2007). The expression pattern of MTF-1 during the basal expression of MTF-1 in heart and hepatopancreas. development was also in agreement with the modulation Male abalones displayed higher expression in heart than pattern of its primary target (e.g., MT) in the same aba- females did whereas female abalones showed higher ex- lone species (Lee and Nam 2016a). Previously, the func- pression in hepatopancreas than males did. This finding is tional involvements of MTF-1 in the development and similar with a previous observation with zebrafish where organogenesis have been highlighted by the lethality of males should have a higher MTF-1 mRNA expression in “MTF-1-knockout” mice (Günes et al. 1998; Wang et al. the heart than females (O’Shields et al. 2014). Although it 2004) and by induced inhibition of MTF-1 signaling has been still inconclusive for such a sex-related dimorph- followed by transcriptomic profiling in zebrafish em- ism, the response of zebrafish MTF-1 to Cd exposure has bryos (O’Shields et al. 2014). been reported to be gender dependent in some tissues (O’Shields et al. 2014). Expression during Zn and Cd exposure There was an apparent difference between the two metal Developmental expression ions in the modulation of MTF-1 gene expression. In The MTF-1 mRNAs were found to be already present in overall, Cd induced potently the mRNA expression of unfertilized eggs based on RT-qPCR assay, which could be considered as a typical indicative sign of the maternal contribution of MTF-1 to offspring. Fine regulation of metal homeostasis should be one of the prerequisite re- quirements for developmental success of marine mollusc embryos that undergo external development in metal- residing sea water (Roesijadi et al. 1996; Jenny et al. 2006; Mao et al. 2012). The initial expression level was decreased down in early cleavage stages and rebounded to the initial level at morula stage. Although there was a trend toward increase of MTF-1 mRNAs with the devel- opmental progress up to early veliger stage, the degree of upregulation was only modest. When the develop- Fig. 4 Expression of abalone MTF-1 in developing embryos and early ment progressed to late veliger stage, the mRNA expres- larvae assessed by RT-qPCR analysis using RPL7 and PPL8 reference sion level of MTF-1 was significantly elevated (P < 0.05) genes as normalization controls. Abbreviations for developmental (Fig. 4). Developmental expression of MTF-1 in marine samples are unfertilized eggs (UF), eggs at early cleavages (EC; 2~8 molluscan animals has not been yet characterized previ- cells stage), eggs at morula (MO), trochophore (TR), early veliger (EV), and late veliger (LV). Histograms with different letters indicate the ously. However, the expression pattern observed in this significantly different means based on ANOVA followed by Duncan’s study was generally in congruent with the anticipated multiple ranged tests at P < 0.05 roles of MTF-1 in embryonic and early ontogenic Lee and Nam Fisheries and Aquatic Sciences (2017) 20:9 Page 9 of 13 MTF-1 while on the contrary, Zn repressed the MTF-1 showing the downregulation upon Zn exposure in most expression (Fig. 5). Differential expression patterns of instances, MT was found to be consistently upregulated MTF-1 in response to Zn and Cd (20 and 100 ppb for by Zn in both 20 and 100-ppb exposure treatments both metals) were also dependent upon tissue types (P < 0.05). Although the induced folds were variable examined (gill, hepatopancreas, muscle, and hemocyte). among tissues, MT gene expression was unfailingly in- In the gill, the exposure with 20-ppb Cd strongly in- duced in all the four tissues by exposure treatments with duced the MTF-1 (P < 0.05) but higher exposure dose 100-ppb Cd, 20-ppb Zn, and 100-ppb Zn, but not by 20- (100 ppb) did not give rise to the significant modulation ppb Cd. Collectively, the modulation patterns upon metal of MTF-1 (P > 0.05). However, exposure with both doses exposure were apparently different between MTF-1 and of Zn significantly downregulated the MTF-1 in the gill MT genes, and the degree of Cd-mediated upregulation in (P < 0.05). On the other hand, in the hepatopancreas, each tissue was much higher for MT than MTF-1 (Fig. 5). both doses of Cd elevated the MTF-1 mRNA levels in a Previous studies have indicated that mammalian MTF- dose-dependent fashion (P < 0.05). Although the 20-ppb 1 should be a constitutively expressed gene with a Zn exposure exhibited the small increase of MTF-1 TATA-less promoter and that mammalian MTF-1s mRNA levels in the hepatopancreas, 100-ppb Zn did not would not show any appreciable response to experimen- show any significant difference as compared to the level tally designed heavy metal and other stress factors. The observed in non-exposed control (P > 0.05). Unlike in plausible reason for the absence of metal (or stress) re- other three tissues, Cd exposure was unable to induce sponsiveness has been explained by the lack of MRE motif the MTF-1 expression in muscle tissue. In the muscle, in the mammalian (e.g., mouse and human) MTF-1 gene Zn exposure depressed the MTF-1 mRNA expression promoters (Auf der Maur et al. 2000; Bi et al. 2006). (more significant downregulation of MTF-1 in the Hence, the MTF-1 activity in mammals is widely agreed 100-ppb exposed group than in the 20-ppb exposed to be largely induced at a post-translation level (Günther group). For hemocytes, the group exposed with 100-ppb et al. 2012a; Smirnova et al. 2000; Saydam et al. 2001). Cd displayed a small, but statistically significant, increase However, in the present study, this abalone MTF-1 gene of MTF-1 expression, while again, Zn-exposure resulted was proven to possess a putative MRE copy as well as a in the rapid downregulation of MTF-1 irrespective of ex- canonical TATA box in its promoter region. Accordingly, posure doses (P <0.05). the present abalone MTF-1 gene displayed the Cd- In contrast to the variable or opposite regulation of mediated induction in multiple tissues (in gill, hepatopan- MTF-1 by Cd and Zn, the transcriptional response of MT creas and hemocytes, but not in muscles). Similarly, the (the main target of MTF-1) to the metal exposure treat- Cd-induced expression of MTF-1 gene has also been re- ments was relatively uniform. Further, unlike MTF-1 ported in other cold-blooded animals such as zebrafish Fig. 5 Transcriptional responses of abalone MTF-1 (a) and MT (b) to experimental heavy metal exposures. Metal exposures were done with cadmium or zinc with the dose strengths of 20 and 100 ppb for 24 h. RT-qPCR data were presented as fold changes of MTF-1 mRNAs in expressed groups relative to that of non-exposed control (Con) based on the normalization against two internal control genes (RPL5 and PPL7). Histograms with different letters indicate the significantly different means based on ANOVA followed by Duncan’s multiple ranged tests at P <0.05 Lee and Nam Fisheries and Aquatic Sciences (2017) 20:9 Page 10 of 13 (Dubé et al. 2011; Cheuk et al. 2008) and carp (Ferencz when the temperature reached 30 °C (i.e., designated and Hermesz 2009), which is obviously different from the 30 °C+0 h) (P < 0.05). Afterward, the MTF-1 expression principle for the MTF-1 regulation in mammals (Bi et al. levels began to be decreased down with the continued 2006; Saydam et al. 2001). incubation at 30 °C (i.e., 30 °C+12 h and 30 °C+24 h); However, the transcription of abalone MTF-1 was not however, the expression level at the end of thermal treat- induced by zinc except only a minute increase in the ment was still significantly higher than that of 20 °C group hepatopancreas; furthermore, Zn exposure even down- (P < 0.05). On the other hand, in the hepatopancreas, the regulated the mRNA expression of abalone MTF-1 in beginning of MTF-1 upregulation was more or less lagged other tissues. This finding is similar with no induction of as compared to that in the gill. Significant induction of MTF-1 mRNA observed for zebrafish cells exposed to MTF-1 in the hepatopancreas became evident from the Zn (Cheuk et al. 2008), although Zn exposure in zebra- 30 °C+12 h group, and the elevated expression level fish was reported to be able to activate the nuclear trans- remained constant at 30 °C+24 h (P < 0.05). In muscles, location of MTF-1 from cytoplasm (Chen et al. 2007). the significant induction of MTF-1 was found at On the other hand, a recent study with oyster C. gigas 30 °C+0 h and 30 °C+12 h but soon returned to the initial has shown that mRNA expression level of MTF-1 could level observed at 20 °C group. Meanwhile, hemocyte dis- be increased by Zn exposure, but the induced amount played the rapid induction of MTF-1 mRNA at 25 °C; was only modest even treated with high dose of Zn however, the expression dropped sharply at 30 °C+0 h in (Meng et al. 2015). In the same report, authors have in- which the decreased expression level was even lower than dicated that RNA interference of MTF-1 would result in that of 20 °C group (P < 0.05). Afterward, the MTF-1 ex- the depression of Zn-mediated induction of MT (the pression rebounded at 30 °C+0 h group. Then, the re- known target of MTF-1), consequently confirming that bound was followed by a further increase at 30 °C+12 h MTF-1 is a superordinate regulator of the MT (Meng group (P <0.05) (Fig. 6). et al. 2015). However, in this study, the inducible pattern As aforementioned, the zinc pool sensing mechanism of MTF-1 was not in agreement with that of MT by MTF-1 upon exposure to stress factors might be expression. Both Cd (inducer of abalone MTF-1) and Zn adopted for the involvement of MTF-1 in host defense (non-inducer of abalone MTF-1) were found to be able pathways against oxidative stress, since the abrupt and to induce MT expression in all the tissues examined. substantial changes of water temperature might be a Further, the quantitative relationship between MTF-1 causative factor to generate oxidative stress in poikilo- and MT expression was not proportional. Currently, it thermal invertebrates (Kim et al. 2007; Attig et al. 2014; has been widely agreed that Zn-mediated induction of Banni et al. 2014). Accordant with this explanation, the MT could be achieved by direct binding of Zn to the potential involvement of MT protein in the heat shock MTF-1 fingers, and other metals such as Cd and Cu response has already been reported in this abalone spe- may not replace Zn in zinc finger binding (Schmittgen cies and also in other aquatic animals (Lee and Nam and Livak 2008; Chen et al. 1999; Zhang et al. 2001). 2016a; Negri et al. 2013; Jarque et al. 2014). Within this Based on this, the Cd-mediated induction of MT gene context, the cold shock-induced MTF-1 has been reported might be considered as an indirect consequence. Pos- in the common carp (Cyprinus carpio)brain, with an ex- sibly, Cd load might give rise to the release of Zn from planation that sudden temperature drop gave rise to the MTs (also from other metalloproteins), and the increase alteration of physiologically accessible Zn concentrations of cellular Zn levels may activate MTF-1 through the in that tissue (Ferencz and Hermesz 2008). Zn-binding. This zinc pool hypothesis can also be Meanwhile, a series of mammalian studies has pro- applied to the MT induction upon exposed to diverse posed that MT and heat shock protein (HSP) genes oxidative stresses (Günther et al. 2012a). Hence, the con- might work in a non-cooperative way in their transcrip- troversy between MTF-1 and MT responses to zinc tional responses to stress treatments. Experimental evi- should be challenged in future study by examining cellu- dences for this proposal may include that (1) HSF lar Zn concentrations (or tissue burden) under various should boost the activity of HSP gene promoter but Zn-exposure conditions. hardly affect an MRE-containing promoter of MT gene upon heat shock and metal exposure, (2) heat shock- Response to heat shock treatment induced nuclear translocation of MTF-1 has been shown Abalone MTF-1 was proven an early phase, heat-shock to be insufficient to activate a MT gene promoter, (3) di- responsive protein as evidenced by the rapid modulation verse target gene searches for MTF-1 have been indica- upon thermal increases. The MTF-1 mRNA levels in the tive of HSP genes as non-affected genes, and (4) MTF-1 gill were rapidly increased (fourfold relative to 20 °C; has been likely to repress HSF-regulated genes through P < 0.05) as early as when water temperature reached a direct protein-protein interaction (Lichtlen et al. 2001; 25 °C (Fig. 6). The expression level was further elevated Saydam et al. 2003; Uenishi et al. 2006). However, on the Lee and Nam Fisheries and Aquatic Sciences (2017) 20:9 Page 11 of 13 Fig. 6 Differential mRNA expression patterns of abalone MTF-1 in response to heat shock treatments. During heat elevation with an increase rate of 1 °C/h, abalones were sampled at 20 °C (just before the elevation), 25 °C, and 30 °C. After reaching 30 °C, abalones were exposed at the constant 30 °C and sampled after 12 h (30 °C+12 h) and 24 h (30 °C+24 h), respectively. RT-qPCR data were presented as fold changes of MTF-1 mRNAs in expressed groups relative to that of non-heat shocked group (i.e., 20 °C control) based on the normalization with RPL5 and PPL7 reference genes. Histograms with different letters indicate the significantly different means based on ANOVA followed by Duncan’s multiple ranged tests at P < 0.05 contrary, the present study has shown that the transcrip- mature males. Abalone MTF-1 was expressed during the tion of abalone MTF-1 could be directly activated by entire period of embryonic and early ontogenic develop- heat shock. Hence, our finding suggests that the thermal ment. From the heavy metal exposure, abalone MTF-1 stress-mediated activation of MTF-1 in abalone may be was found to be Cd inducible (but not by Zn); however, achieved not only at a post-translation level (i.e., indir- the induced amounts were only modest as compared to ectly based on cellular zinc sensing abovementioned) that of MT. Abalone MTF-1 was highly modulated in re- but also at a transcriptional level (i.e., de novo synthesis). sponsive to heat shock potentially via both indirect zinc The presence of potential HRE motifs in the abalone pool sensing and direct de novo transcription. Data from MTF-1 gene regulatory region is also in congruent with this study could be a useful basis to approach various re- the present hypothesis. Possibly, the activation of MTF-1 searches regarding the stress responses in this abalone from the dual routes is likely to be advantageous for species particularly including detoxification of heavy permitting the poikilothermal animals to prepare more metals and adaptation to thermal stresses. efficiently antioxidant components (i.e., antioxidant enzyme genes containing MREs in their promoters as Additional file well as MT) against the oxidative stress caused by Additional file 1: Table S1. Summarized information on oligonucleotide thermal fluctuations (Kim et al. 2007; Cho et al. 2009). primersusedin thisstudy. Figure S1. Full-length cDNA and deduced amino However, further efforts are needed to get direct evi- acid sequences of abalone Haliotis discus hannai metal responsive transcription dence on the cooperativity between HSF and MTF-1 in factor-1 (MTF-1). In the nucleotide sequence, stop codon is indicated by an asterisk and putative polyadenylation signal is underlined. On the other this abalone species. hand, in the amino acid sequence (in a singlet code), presumed nuclear localization signal (NLS) is underlined (in the front of the first zinc finger), Conclusions while six putative C H -zinc fingers in the DNA-binding domain are boxed. 2 2 Figure S2. Multiple sequence alignments of abalone MTF-1 along with its Novel MTF-1 was isolated and characterized from a representative orthologs. Putative NLS, six C H -zinc fingers, NES embedded 2 2 commercially important marine mollusc species, Pacific in transactivation domain, and C-terminal Cys-cluster are indicated. Figure abalone (H. discus hannai). The abalone MTF-1 was S3. Neighbor-joining phylogenetic tree based on amino acid sequences of zin-finger DNA-binding domain and NLS region in MTF-1 orthologs. Tree found to share a conserved feature in zinc finger, DNA- was computed by poisson correction method using MEGA 7.0 program. binding domain with its orthologs; however, it repre- Confidence level of each clade was evaluated with bootstrap testing (1000 sented non-conservative features in remaining other replicates). Figure S4. Putative transcription factor-binding motifs predicted in the 5′-flanking upstream region of the abalone MTF-1 gene. Transcription factor parts. 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Molecular cloning of metal-responsive transcription factor-1 (MTF-1) and transcriptional responses to metal and heat stresses in Pacific abalone, Haliotis discus hannai

Fisheries and Aquatic Sciences , Volume 20 (1) – Jul 3, 2017

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Life Sciences; Fish & Wildlife Biology & Management; Marine & Freshwater Sciences; Zoology; Animal Ecology
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Abstract

Background: Metal-responsive transcription factor-1 (MTF-1) is a key transcriptional regulator playing crucial roles in metal homeostasis and cellular adaptation to diverse oxidative stresses. In order to understand cellular pathways associated with metal regulation and stress responses in Pacific abalone (Haliotis discus hannai), this study was aimed to isolate the genetic determinant of abalone MTF-1 and to examine its expression characteristics under basal and experimentally stimulated conditions. Results: The abalone MTF-1 shared conserved features in zinc-finger DNA binding domain with its orthologs; however, it represented a non-conservative shape in presumed transactivation domain region with the lack of typical motifs for nuclear export signal (NES) and Cys-cluster. Abalone MTF-1 promoter exhibited various transcription factor binding motifs that would be potentially related with metal regulation, stress responses, and development. The highest messenger RNA (mRNA) expression level of MTF-1 was observed in the testes, and MTF-1 transcripts were detected during the entire period of embryonic and early ontogenic developments. Abalone MTF-1 was found to be Cd inducible and highly modulated by heat shock treatment. Conclusion: Abalone MTF-1 possesses a non-consensus structure of activation domains and represents distinct features for its activation mechanism in response to metal overload and heat stress. The activation mechanism of abalone MTF-1 might include both indirect zinc sensing and direct de novo synthesis of transcripts. Taken together, results from this study could be a useful basis for future researches on stress physiology of this abalone species, particularly with regard to heavy metal detoxification and thermal adaptation. Keywords: Abalone, Haliotis discus hannai, MTF-1, Heavy metal, Heat shock Background transcription factor has also been known to be closely Metal-responsive transcription factor-1 (MTF-1; also involved in cellular adaptation and protection against termed metal-regulatory transcription factor-1 or metal- oxidative stresses through regulating the transcription responsive-element-binding transcription factor-1) is a key of diverse genes related with host defense-related path- transcriptional regulator playing pivotal roles in metal ways (Günther et al. 2012a; Lichtlen and Schaffner homeostasis and detoxification (Laity and Andrews 2007; 2001). They include metal reservation/detoxification Günther et al. 2012a). In addition to its fundamental role (e.g., metallothionein, MT; the main target of MTF-1), for homeostatic metal regulation, this multitasking metal ion transport (e.g., Zn or Cu transporters), iron homeostasis/anti-microbial responses (e.g., hepcidin), cellular redox homeostasis (e.g., selenoproteins and * Correspondence: yoonknam@pknu.ac.kr Department of Marine Bio-Materials & Aquaculture, Pukyong National thioredoxin reductase), and glutathione biosynthesis University, Busan 48513, South Korea © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lee and Nam Fisheries and Aquatic Sciences (2017) 20:9 Page 2 of 13 (e.g., glutamate cysteine ligase) (Günther et al. 2012a; related pollutants contaminated in both water and Lichtlen et al. 2001; Stoytcheva et al. 2010). sediments could be significant factors to provoke cel- In a structural viewpoint, MTF-1 has been considered lular toxicity and oxidative stress in farmed abalones as a conserved transcription factor to possess six C H - (Kim et al. 2007). 2 2 type zinc fingers as the DNA-binding domain to recognize However, despite its importance, adaptive or defensive metal responsive elements (MREs) (Giedroc et al. 2001). functions to such environmental perturbations have As a cellular metal and stress sensor, the activity regula- been limitedly investigated in this abalone species, and tion of MTF-1 is generally characterized by the three suc- almost no information has been available with respect to cessive steps, i.e., nuclear-cytoplasmic shuttling upon the coordinated regulations of genes involved in cellular stress exposure, DNA-binding, and the interaction(s) with pathways associated with metal regulation and oxidative other coactivators to modulate the target gene transcrip- stress responses (Kim et al. 2007; Lee and Nam 2016a). tion (Li et al. 2008). To execute the transcriptional regula- For this reason, understanding of MTF-1 from abalone tion, MTF-1 binds to the specific site, called MRE (core species would be much useful to better comprehend or- sequence = TGCRCNC), in the promoter region of target chestrated and coordinated regulations of host defense gene (Günther et al. 2012a). Accordingly, the transcrip- genes in this abalone species. Based on this need, this tional expression of MTF-1 gene itself has been reported study, as a startpoint research, was aimed to characterize to be constitutive and not to be affected by heavy metal the genetic determinant of MTF-1, the superordinate and other stressor treatments because its regulatory func- regulator for diverse host defense genes, from the Pacific tions should be controlled mainly at post-translational abalone (H. discus hannai). For this, we isolated and char- levels (Auf der Maur et al. 2000; Bi et al. 2006). Structural acterized the full-length complementary DNA (cDNA) scheme and functional context of MTF-1 above-described encoding the abalone MTF-1, bioinformatically dissected are believed to be widespread in the vertebrate lineage, its 5′-upstream regulatory region, and also scrutinized ex- although the majority of empirical information has come pression patterns of MTF-1 under both non-stimulated from mammalian MTF-1s. and stress-challenged (i.e., heavy metal exposure and heat However, in contrast to richness of knowledge on shock) conditions. mammalian orthologs, molluscan MTFs have been nar- rowly explored barring only couples of previous reports Methods (Qiu et al. 2013; Meng et al. 2015). Nevertheless, it is Abalone samples and molecular cloning of MTF-1 noteworthy that currently available molluscan MTF-1 Abalones (H. discus hannai) used in this study were sequences from public databases have suggested that experimental stocks maintained at Experimental Fish mollusc species may show non-canonical features in Culture Station, Pukyong National University (PKNU), their MTF-1 structures, which may differ from verte- Busan, South Korea. Abalones were maintained with brate orthologs. For instances, unlike vertebrate ortho- semi-water recirculation system equipped with 3-ton cap- logs, molluscan MTF-1s often lack several typical motifs acity of rectangular culture tanks, in which the tanks were in presumed transactivation domains. Furthermore, a re- connected with protein skimmers, custom-designed mech- cent study has claimed that the nuclear-cytoplasmic anical filters, and 1-μm-mesh filter. Throughout the experi- shuttling, a key prerequisite step for vertebrate MTF-1s, ment, water temperature and dissolved oxygen were kept to might not always be an absolute precondition in certain be ranged within 20 ± 1 °C and 8 ± 1 ppm, respectively. mollusc species (Meng et al. 2015). Abalones were fed with frozen or dried seaweeds until 2 days Pacific abalone, Haliotis discus hannai, is a highly val- before stress exposure treatments. Daily water exchange rate ued seafood mollusc not only in Korea but also in other was about 20%, and in-tank wastes including feces and deb- East-Asian countries. Intensive aquacultural operation ris on the bottom were removed twice every day. for abalone farming using the marine net-cage system Based on the NGS-transcriptome analysis of the has been established in Korean aquaculture domain. juvenile abalone tissues (unpublished data), partial NGS During the last decade, the remarkable growth of aba- clones representing the significant homology to known lone production in a quantitative term has been achieved animal MTF-1s were selected and assembled into a con- (Park and Kim 2013). However, more recently, the tig. In order to get full-length cDNA version, rapid sustainable progress of abalone culture has been con- amplifications of cDNA ends (RACE) at both 5′-and siderably hurdled by the depressed productivity mainly 3′-directions were carried out using total RNA isolated in relation to frequent outbreaks of high mortality from a whole body sample and SMARTer® RACE 5′/3′ and physiological deformity in many abalone farms Kit (Clontech Laboratories Inc., Mountain View, CA, (Park and Kim 2013; Kang et al. 2015). Considering that USA) according to the manufacturer’s instructions. abalone farming in Korea mainly relies on the net-cage Oligonucleotide primers used in this study are listed in facility installed in coastal areas, the heavy metals or other Additional file 1: Table S1. The amplified fragments were Lee and Nam Fisheries and Aquatic Sciences (2017) 20:9 Page 3 of 13 sequenced and again subjected to contig assembly. Based juveniles (21.5 ± 4.1 g for total weight; n = 12), six kinds on the assembled sequence in a contig, full-length abalone of tissues including the gill, gut, heart, hemolymph, hep- MTF-1 cDNA was re-isolated by RT-PCR amplification atopancreas, and muscle (foot muscle) were surgically using the same total RNA aforementioned. Amplified RT- removed individually. For hemolymph, centrifugation PCR products were directly sequenced at both forward (2500 rpm for 10 min at 4 °C) was carried out in order and reverse directions to obtain a representative cDNA se- to collect hemocyte pellet. Second, from 3-year-old sexu- quence for abalone MTF-1. ally mature adults showing a clear sign of ovarian and From the cDNA sequence, the 5′-upstream region of testicular maturation (96.4 ± 13.1 g for total weight; eight abalone MTF-1 gene was cloned by genome walking each for female and male), same tissue types abovemen- method. Using the genomic DNA prepared from an indi- tioned were obtained and additional ovary and testis vidual muscle, genome walking to 5′-upstream region was were obtained. Upon surgically removed, biological conducted with designated pairs of gene-specific primers samples were immediately frozen on dry ice and stored and Universal Genome Walker® Kit (Clontech Laboratories at −80 °C until used for RNA isolation. Inc., USA) according to the manufacturer’s instructions. In order to obtain developing embryos and early lar- Amplified fragments were TA cloned into pGEM-T® easy vae, artificial insemination of sperm (from three males) vector (Promega, Madison, WI, USA), sequenced and as- to eggs (from eight females) was conducted by using the sembled into a single contig. Afterward, the continuous, conventional induced spawning method including an air genomic fragment containing the 5′-flanking region was exposure and ultraviolet-irradiated seawater treatment. again PCR isolated from the genomic DNA template Insemination was made with wet-method at 20 °C and abovementioned and directly subjected to the sequencing incubated at the same temperature until the end of sam- to confirm the representative sequence of the abalone pling. An aliquot of developmental samples each consist- MTF-1 proximal promoter region. ing of approximately 20,000~30,000 embryos or larvae was sampled at six time points: just before insemination Bioinformatic sequence characterization (unfertilized eggs), early cleavages (i.e., 2~8 cells stage; at With the ORF Finder program (https://www.ncbi.nlm.nih. 2 h post insemination; 2 hpi), morula (5 hpi), gov/orffinder/), the open reading frame (ORF) of abalone trochophore (12 hpi), early veliger (18 hpi), and late veli- MTF-1was predictedand deducedaminoacidsequence ger (42 hpi), based on the microscopic examination. was obtained. Based on the homology search using NCBI Upon sampling, embryos and larvae were also frozen on BLASTx (http://blast.ncbi.nlm.nih.gov/Blast.cgi), sequence dry ice and stored at −80 °C until used. Two replicate homology of abalone MTF-1 with its orthologs was samplings were carried out for each time point. examined. Parameter scores for the primary structure of MTF-1 were estimated using ExPASy ProtParam tool Experimental in vivo stimulatory challenges (http://web.expasy.org/protparam/). Multiple sequence Two independent stress exposure experiments were carried alignment was done using CLUSTALW program (http:// out: one was with heavy metal exposure and the other with www.genome.jp/tools-bin/clustalw). Identification of puta- heat shock treatment. For heavy metal exposure, eight ju- tive zinc finger domains was carried out with Simple venile individuals (24.5 ± 3.6 g; approximately 1 year old) Modular Architecture Research Tool (SMART; http://smar- were assigned into one of five experimental tanks (70-L t.embl.de/). Predictions of potential nuclear localization sig- capacity containing 50 L of 1-μm filtered seawater at 20 °C) nal (NLS) and nuclear export signal (NES) were conducted and acclimated to the tank conditions for 24 h before heavy with cNLS Mapper (http://nls-mapper.iab.keio.ac.jp/cgi- metal treatment. The heavy metals used for exposure were bin/NLS_Mapper_form.cgi) and NetNES 1.1 Server (http:// of analytical grade reagents (Sigma-Aldrich, St. Louis, MO, www.cbs.dtu.dk/services/NetNES/), respectively. The puta- USA). After 24 h, two tanks were treated with 0.02 mg/L tive zinc finger-DNA binding regions from selected MTF-1 (i.e., 20 ppb) and 0.1 mg/L (i.e., 100 ppb) cadmium (Cd), orthologs were subjected to molecular phylogenetic analysis while two tanks with 20 and 100-ppb zinc (Zn) (Lee and using Molecular Evolutionary Genetics Analysis tool (ver. Nam 2016a). Nominal concentration of the metal for each 7.0.21; http://www.megasoftware.net/). Putative transcrip- metal-exposed group was adjusted by using CdC1 or tion factor binding motifs in the abalone MTF-1 promoter ZnCl stock solution. Remaining one tank was treated with were predicted with TRANSFAC® software (http://genex- only 1-mL distilled water that had been used for reconstitu- plain.com/transfac; GeneXplain GmbH, Wolfenbüttel, tion of the metals (i.e., for non-exposed control). For each Germany). group, two replicate tanks were prepared identically. Treatment duration was 24 h. At the end of exposure, gill, Tissue collection from different developmental stages hemocyte, hepatopancreas, and foot muscle were sampled Tissue distribution assay was conducted with two age individually from six randomly chosen individuals as classes of abalones. First, from 1-year-old immature described above. Lee and Nam Fisheries and Aquatic Sciences (2017) 20:9 Page 4 of 13 On the other hand, for heat shock treatment, 22 indi- assays and stress exposure treatments), while RPL7 and viduals (21.1 ± 3.1 g; same-aged as above) were assigned RPL8 (KP698947) were used to normalize MTF-1 expres- into one of four 100-L tanks (two for heat-stressed sion across developmental samples (embryos and larvae). groups and two for non-stressed groups) at 20 °C. Each Additionally, for heavy-metal exposure groups, the mes- tank was equipped with a custom-designed apparatus for senger RNA (mRNA) expression levels of MTF-1 were mechanical filtration. After 24 h of acclimation period, compared with those of MT (the known target gene of water temperature of the two tanks (heat-stressed groups) MTF-1) in order to examine whether or not there might was elevated using the adjustable thermostat-assisted aquar- be any positive or proportional relationship in the metal- ium heaters (400 W) with an increment rate of 1 °C/h. mediated modulation patterns between MTF-1 and MT When the temperature reached 30 °C, the temperature genes. PCR efficiency of primer pair for each gene was vali- was kept to be constant at 30 °C for additional 24 h. dated to be at least higher than 95% based on the standard Samplings were made at 20 °C (just before thermal curve prepared using a fivefold serial dilution of cDNA elevation), 25 °C (5 h after elevation started), 30 °C (10 h), mix. For each cDNA sample, triplicate assays were carried 30 °C+12 h (12 h after reaching 30 °C; 22 h after elevation out in an independent fashion. started), and 30 °C+24 h (24 h after reaching 30 °C; 34 h Quantitative PCR-based MTF-1 mRNA expression after elevation started). Four individuals were randomly levels across tissue types and developmental stages selected from each tank to constitute eight individuals per under non-stressed conditions were presented as ΔCt temperature group at each sampling point. Tissues (Ct of the MTF-1 gene subtracted from the Ct of each sampled were gill, hemocyte, hepatopancreas, and foot internal control gene). On the other hand, differential muscle. Meanwhile, non-stressed control groups were also expression levels of metal-exposed or heat shock-treated identically prepared with heat-shock groups, but the group relative to their corresponding non-stressed con- temperature (20 °C) was kept to be constant until the end trol groups were presented as the fold difference to the −ΔΔCt of experiment. At the same sampling point, the identical non-stressed controls by using the formula 2 number of abalones (n = 4 per tank) was also obtained (Schmittgen and Livak 2008). Expression levels between from non-stressed control groups. Temperature of each or among groups were tested using Student’s t test or tank was confirmed to be ranged within ±0.5 °C. Dissolved one-way ANOVA (followed by Duncan’s multiple ranged oxygen levels were adjusted to be ranged from 7.5 to tests). Difference was considered to be significant when 8.5 ppm for all the experimental tanks. Abalones were not P < 0.05. fed during stimulatory challenge experiments. Results and discussion RT-qPCR assay and statistics Characteristics of abalone MTF-1 cDNA and deduced Total RNA was extracted using TriPure® Reagent (Roche amino acid sequences Applied Science, Mannheim, Germany) and then The full-length cDNA of abalone MTF-1 was comprised purified using RNeasy Mini Plus Kit (Qiagen, Hilden, of 48-bp 5′-untranslated region (UTR), a 1509-bp single Germany) including DNase I treatment step. An aliquot open reading frame (ORF) encoding a polypeptide of (2 μg) of total RNA prepared was reverse transcribed into 503 amino acids, and 582-bp 3′-UTR including a stop cDNA by using the Omniscript® Reverse Transcription Kit codon and 19-bp poly(A+) tail. A putative polyadenyla- (Qiagen, Germany) including oligo-dT primer according to tion signal (AATAAA) was found at 21 bp prior to the the manufacturer’s instruction. Synthesized cDNA was poly(A+) tail (GenBank accession number; KT895224) fourfold diluted with sterile distilled water, and an aliquot (Additional file 1: Figure S1). The MTF-1 protein based of 2 μL was included in a qPCR reaction as a template. on the deduced amino acid sequence was estimated to The qPCR reaction was conducted with a LightCycler® 480 have 54.86 kDa of calculated molecular mass and 5.51 of Real-Time PCR System and LightCycler® 480 SYBR Green theoretical pI value, respectively. Abalone MTF-1 repre- I Master (Roche Applied Science, Germany), according to sented quite a low sequence homology to its vertebrate the manufacturer’s instructions. Thermal cycling condition and invertebrate orthologs where the maximum se- for each gene (i.e., MTF-1 and normalization control quence identity at amino acid level was found to be only genes) can be referred to Additional file 1: Table S1. Based 27% with Biomphalaria glabrata (air-breathing fresh- on our preliminary study to evaluate candidate housekeep- water snail; Gastropoda; Mollusca). Non-conservative ing genes for the normalization of RT-qPCR amplification feature without any appreciable sequence similarity to (unpublished data; see also (Lee and Nam 2016a; Lee and other MTF-1s is also found in the putative activation. Nam 2016b)), abalone ribosomal proteins L5 (RPL5; Abalone MTF-1 was likely to possess only a shortened ABO26701) and L7 (RPL7; KP698945) genes were used as fragment (44-aa; pI = 4.06) presumed for the acidic reference genes to normalize expression levels of MTF-1 domain and to lack almost entire region corresponding transcripts in tissue samples (i.e., for basal tissue expression to the proline-rich domain and serine/threonine-rich Lee and Nam Fisheries and Aquatic Sciences (2017) 20:9 Page 5 of 13 domain of vertebrate MTF-1 orthologs (Additional that MTF-1s have been largely divergent in the molluscan file 1: Figure S2). phylum, which is apparently different form the mono- In contrast, the abalone MTF-1 was proven to share a phyletic clustering of orthologs from Chordata phylum high structural homology with other MTF-1s in the DNA (Additional file 1: Figure S3). Functional partition of the binding domain (Fig. 1). All the MTF-1 proteins including zinc finger domains in abalone MTF-1 has been remained the abalone MTF-1 (but except for the shortest Octopus to be further characterized; however, from the mammalian bimaculoides MTF-1 having only five zinc fingers) were studies, the region from the second to fourth zinc fingers found to show highly conserved six C H -zinc fingers in have been proposed to constitute the core DNA binding 2 2 their DNA binding domains. In their DNA binding do- domain while the first finger has been reported to serve as mains, 12 cysteine residues and histidine residues were a metal-sensing domain (Bittel et al. 2000). Within each clearly conserved, in which the zinc finger domain of aba- zinc finger, the His-X-Arg/Lys-X-His [H-X-(R/K)-X-H lone MTF-1 showed the highest sequence identity (77%) where X is any amino acid] motif has been known as a with that of orthologue isoforms from Crassostrea gigas key site for zinc binding (Günther et al. 2012a), and it is (Bivalve, Mollusca). However, the molecular phylogenetic preserved in the 1st to 5th zinc fingers for all the species analysis using the DNA binding domains has indicated examined in the present study. However, in the last finger Fig. 1 Multiple amino acid sequence alignment of conserved zinc-finger DNA-binding domain from the abalone MTF-1 along with those from representative orthologs. In the alignment, conserved amino acid residues are indicated by red-colored letters, and two cysteines and two histidines typical of the C H structure for each zinc finger are boxed. The putative auxiliary nuclear localization signal (aNLS) conserved in the 2 2 front of the first zinc finger is indicated by upper line. The accession code for each sequence is noted at the end of alignment. The alignment with full-length polypeptide sequences and the full name of each species can be referred to Additional file 1: Figure S2 Lee and Nam Fisheries and Aquatic Sciences (2017) 20:9 Page 6 of 13 (i.e., the 6th finger), the Arg/Lys residue inside the 5- established in mammalian MTF-1), NLS and NES are amino-acid stretch was found to be conserved only in ver- responsible for balanced subcellular distributions of tebrate MTF-1s. Meanwhile, all the molluscan MTF-1s MTF-1 proteins (i.e., import and export, respectively) showed a phenylalanine (Phe) as the first amino acid of under both stressed and non-stressed conditions the first zinc finger, whereas vertebrate and brachiopod (Günther et al. 2012a). The NES motif in vertebrate orthologs possessed a tyrosine (Tyr) (Cheung et al. 2010). MTF-1 is usually embedded in the acidic activation do- Abalone MTF-1 showed both conserved and unique main (Günther et al. 2012a; Cheung et al. 2010). How- features in the peptide linkers connecting zinc fingers. ever, the abalone MTF-1 does not show any typical NES Conserved peptide linkers were found between 1st and motif, although a putative NLS motif is predicted in the 2nd fingers (Arg-Gly-Glu-Tyr-Thr), between 2nd and front of the first zinc-finger domain that are also con- 3rd fingers (Thr-Lys-Glu-Lys-Pro), and between 4th and served all MTF-1s examined (i.e., conserved auxiliary 5th fingers (Thr-Gly-Glu-Lys-Pro). On the other hand, NLS). The absence of acidic domain-embedded NES is unique linkers were found between 3rd and 4th fingers not limited to abalone MTF-1, i.e., all molluscan MTF- [Ser-Gly-Asn-Thr in abalone vs. Thr-Gly-Lys-Thr in ver- 1s are also likely to lack the NES at the corresponding tebrates vs. Thr-Gly-(Glu/Asn/Asp)-Thr in other mol- region. Hence, our finding may suggest that molluscan luscs and a brachiopod species] and between 5th and MTF-1s could be different from mammalian MTF-1s in 6th fingers [Ser-Gly-Glu-Lys-Pro in abalone vs. Thr-Gly- their subcellular localization control under both basal and Glu-(Lys/Arg)-Pro in vertebrates vs. Thr-Gly-(Asp/Gly/ stimulated conditions. A recent study with an oyster spe- Glu)-(Lys/Arg)-Pro in others]. Peptide linkers in multi- cies (C. gigas) has claimed that the MTF-1 would primar- zinc finger domains have been reported to play roles in ily localize in the nucleus even under unstressed not only structural stabilization but also interfinger in- conditions and nuclear translocation might be uncritical teractions for DNA-binding affinity of the zinc finger for the activation of the oyster MTF-1 (Meng et al. 2015). domains (Li et al. 2006). Particularly in the MTF-1, the In addition, the typical motif of cysteine cluster linker between 1st and 2nd fingers has been known to (consensus sequence = Cys-Gln-Cys-Gln-Cys-Ala-Cys) be crucial in the zinc-sensing ability of the MTF-1 with that could be commonly found in the region immedi- regard to the formation of the ternary (MTF-1-zinc- ately following the serine/threonine-rich domain of ver- DNA) complex for activating MT gene transcription tebrate MTF-1s was not detected in the abalone MTF-1 (Li et al. 2006). Taking this into account, the abalone (Additional file 1: Figure S2). The cysteine cluster has MTF-1 is thought to preserve a fundamental property of been reported to be essentially necessary for metal- zinc-sensing function similarly with mammalian orthologs induced transcriptional activity and homodimerization since it conserves a completely identical linker (i.e., the of mammalian MTF-1 (Günther et al. 2012a; Günther et Arg-Gly-Glu-Tyr-Thr linker between 1st and 2nd fingers) al. 2012b). Like with NES abovementioned, none of mol- with mammalian MTF-1s. However, the present abalone luscan MTF-1 represents a canonical C-terminus cyst- MTF-1 showed apparent dissimilarity with vertebrate eine cluster, suggesting that molluscan MTF-1s might orthologs in the linkers between 3rd and 4th fingers and have different mechanism(s) for metal-induced tran- between 5th and 6th fingers. Particularly because the scription (i.e., recruitments of transcriptional cofactor linker between 3rd and 4th fingers has been reported to partners in the promoter/enhancer of target genes). be important for the in vivo and in vitro sensitivity of zinc-dependent activation of MTF-1 (Li et al. 2006), this Prediction of transcription factor binding sites linker could be a good target for future studies to examine From the bioinformatic prediction, the 1691-bp 5′-up- the potential difference in the linker-mediated zinc-finger stream region from the ATG translation start site of the function between abalone MTF-1 and mammalian/verte- abalone MTF-1 gene represented various transcription brate orthologs. factor binding sites (Fig. 2) (Additional file 1: Figure S4). In the abalone MTF-1, a putative auxiliary nuclear The abalone MTF-1 promoter revealed a canonical localization signal (aNLS) was identifiable in the front of TATA box (TATAAA) at −474 bp (from the ATG). Im- the first zinc finger, as similarly with all other MTF-1 portantly, it represented a copy of MRE (TGCRCNC; orthologs. Of seven amino acid residues to comprise the −959 bp), suggesting the possible modulation of MTF-1 auxiliary NLS, the positions of two amino acids ( Glu itself by heavy metal-driven cellular stressors, which is and Arg) are conserved in all the MTF-1s examined clearly inconsistent with the lack of MRE in many mam- (Fig. 1). However, due to the potential deletion in the malian MTF-1 promoters (Auf der Maur et al. 2000; Bi putative acidic domain, no canonical nuclear export et al. 2006). In addition, a xenobiotic response element signal (NES) was observable in the abalone MTF-1 (XRE; TNGCGTG; −736 bp) was predicted in the unlike vertebrate orthologs. Within a concept of MTF-1 abalone MTF-1 promoter. XRE is an aryl hydrocarbon activation (i.e., a nuclear-cytoplasmic shuttling function receptor (AhR)-targeted motif involved in the ligand Lee and Nam Fisheries and Aquatic Sciences (2017) 20:9 Page 7 of 13 Fig. 2 Schematic drawing to show the putative transcription factor-binding motifs predicted in the 5′-upstream region of the abalone MTF-1 gene. Binding motifs are searched by TRANSFAC® search (GeneXplain GmbH, Germany). For detailed representation including the 5′-upstream sequence, reader is referred to Additional file 1: Figure S4 (i.e., 2,3,7,8-tetrachlorodibenzodioxin (TCDD))-activated and fundamental roles in most cell types (Auf der Maur pathway to detoxify the effects of TCDD-related com- et al. 2000; O’Shields et al. 2014). The MTF-1 mRNAs pounds. However, because invertebrate AhR homologues were robustly expressed in hemocytes (P < 0.05), and this have been reported to lack the ability to bind TCDD dir- highest expression level was followed by muscles and ectly (Hahn et al. 2006), the molecular mechanism on the gills, whereas the least mRNA expression was found in potential interconnection between MRE/MTF-1 and heart (P < 0.05). However, this expression pattern was XRE/AhR paths should be further explored. Nevertheless, not fully reproducible when measured with the sexually recent mammalian studies have also highlighted multi- mature adults, although the broad pattern was consist- tasking roles of AhR in various signaling pathways associ- ent with findings from juveniles. Unlike in juveniles, the ated with cell cycle control and antioxidant protection expression level of MTF-1 in the gut was found to be as against oxidative stresses (Jackson et al. 2015). Besides, high as that in the hemocytes in both female and male several transcription factor binding sites such as heat adults. More noticeably, matured adult abalones dis- shock element (HSE; GAANRTTC; −1003 and −32 bp; played a strikingly apparent difference in the MTF-1 targeted by heat shock factor (HSF)), hypoxia response expression in gonads where the extraordinarily high ex- element (HRE; RCGTG; −895 and −285 bp; by hypoxia- pression level was observed in testis while only minute inducible factor-1 alpha (HIF-1α)), and other sites recog- expression in ovary (more than 150-fold difference; nized by cyclic AMP response element binding protein P < 0.05). Testis-predominant expression pattern of (CREBP; TGACGY; −1253 bp) and nuclear factor for acti- abalone MTF-1 in this study is similar with previous vated T-cells (NF-AT1; WGGAAA; −941, −169, and findings made in mouse (Auf der Maur et al. 2000) and −33 bp) were predicted. All of these factors have been hybrid tilapia (Oreochromis aurea × Oreochromis nilotica) known to be related with stress responses of animals (Cheung et al. 2010), collectively suggesting the possible (Saydam et al. 2003; Dubé et al. 2011). Abalone MTF-1 involvement of MTF-1 in the male reproduction. Yet, the promoter also revealed motifs that might be targeted by mechanism underlying the robust expression of MTF-1 in transcription factors generally known to be involved in de- the abalone testes is currently unknown and open to velopment, signal transduction, cell proliferation and/or hypothesize. In mouse, the reason for the high MTF-1 ex- organ development. They included Smad4, hepatocyte nu- pression in testis has been explained by that sexually ma- clear factor (HNF), CCAAT-enhancer binding protein (C/ ture mice need to accumulate a large quantity of MTs in EBP), E-box binding protein, and estrogen receptor (ER). their testes (Auf der Maur et al. 2000; De et al. 1991). However, this hypothesis is unlikely to be adopted to this Tissue distribution and basal expression levels abalone species since the virtual increase of MT expres- Based on the RT-qPCR analysis with immature juvenile sion in sexually mature abalones has been observed in abalones, MTF-1 mRNAs were detected in all the tissue ovary rather than in testis (Lee and Nam 2016a), suggest- types examined; however, basal expression levels were ing the molecular mechanism for the boosted expression quite variable among tissues (Fig. 3). The ubiquitous de- of MTF-1 in abalone testis might be distinct from the ones tection of MTF-1 transcripts across all the tissues is not in mammals. Hence, further study to monitor the MTF-1 surprising when taken into account its housekeeping expression in line with the testis development and Lee and Nam Fisheries and Aquatic Sciences (2017) 20:9 Page 8 of 13 ab Fig. 3 Tissue distribution patterns and basal expression levels of abalone MTF-1 mRNAs in a immature juvenile and b sexually matured adult tissues, based on RT-qPCR assays using the normalization against RPL5 and PPL7 references. Tissue abbreviations are gill (GI), gut (GU), heart (HE), hemocyte (HC), hepatopancreas (HP), muscle (MU), and gonad (GON; i.e., ovary or testis). In a, histograms with different letters indicate the significantly different means based on ANOVA followed by Duncan’s multiple ranged tests at P <0.05. In b, the significantly different means within a sex are indicated by a–e (for females) and v–z (for males) base on ANOVA followed by Duncan’s multiple ranged tests at P < 0.05. The significantly different means between sexes observed in heat, hepatopancreas, and gonad tissues are noted by asterisks based on Student’s t test analysis at P <0.05 maturation cycle would be valuable to get a deeper insight developments, as inferred from mammalian and teleos- into this phenomenon. Besides the gonadal expression, tean cases (Günes et al. 1998; Chen et al. 2002; Chen matured abalones showed a sex-specific difference in the et al. 2007). The expression pattern of MTF-1 during the basal expression of MTF-1 in heart and hepatopancreas. development was also in agreement with the modulation Male abalones displayed higher expression in heart than pattern of its primary target (e.g., MT) in the same aba- females did whereas female abalones showed higher ex- lone species (Lee and Nam 2016a). Previously, the func- pression in hepatopancreas than males did. This finding is tional involvements of MTF-1 in the development and similar with a previous observation with zebrafish where organogenesis have been highlighted by the lethality of males should have a higher MTF-1 mRNA expression in “MTF-1-knockout” mice (Günes et al. 1998; Wang et al. the heart than females (O’Shields et al. 2014). Although it 2004) and by induced inhibition of MTF-1 signaling has been still inconclusive for such a sex-related dimorph- followed by transcriptomic profiling in zebrafish em- ism, the response of zebrafish MTF-1 to Cd exposure has bryos (O’Shields et al. 2014). been reported to be gender dependent in some tissues (O’Shields et al. 2014). Expression during Zn and Cd exposure There was an apparent difference between the two metal Developmental expression ions in the modulation of MTF-1 gene expression. In The MTF-1 mRNAs were found to be already present in overall, Cd induced potently the mRNA expression of unfertilized eggs based on RT-qPCR assay, which could be considered as a typical indicative sign of the maternal contribution of MTF-1 to offspring. Fine regulation of metal homeostasis should be one of the prerequisite re- quirements for developmental success of marine mollusc embryos that undergo external development in metal- residing sea water (Roesijadi et al. 1996; Jenny et al. 2006; Mao et al. 2012). The initial expression level was decreased down in early cleavage stages and rebounded to the initial level at morula stage. Although there was a trend toward increase of MTF-1 mRNAs with the devel- opmental progress up to early veliger stage, the degree of upregulation was only modest. When the develop- Fig. 4 Expression of abalone MTF-1 in developing embryos and early ment progressed to late veliger stage, the mRNA expres- larvae assessed by RT-qPCR analysis using RPL7 and PPL8 reference sion level of MTF-1 was significantly elevated (P < 0.05) genes as normalization controls. Abbreviations for developmental (Fig. 4). Developmental expression of MTF-1 in marine samples are unfertilized eggs (UF), eggs at early cleavages (EC; 2~8 molluscan animals has not been yet characterized previ- cells stage), eggs at morula (MO), trochophore (TR), early veliger (EV), and late veliger (LV). Histograms with different letters indicate the ously. However, the expression pattern observed in this significantly different means based on ANOVA followed by Duncan’s study was generally in congruent with the anticipated multiple ranged tests at P < 0.05 roles of MTF-1 in embryonic and early ontogenic Lee and Nam Fisheries and Aquatic Sciences (2017) 20:9 Page 9 of 13 MTF-1 while on the contrary, Zn repressed the MTF-1 showing the downregulation upon Zn exposure in most expression (Fig. 5). Differential expression patterns of instances, MT was found to be consistently upregulated MTF-1 in response to Zn and Cd (20 and 100 ppb for by Zn in both 20 and 100-ppb exposure treatments both metals) were also dependent upon tissue types (P < 0.05). Although the induced folds were variable examined (gill, hepatopancreas, muscle, and hemocyte). among tissues, MT gene expression was unfailingly in- In the gill, the exposure with 20-ppb Cd strongly in- duced in all the four tissues by exposure treatments with duced the MTF-1 (P < 0.05) but higher exposure dose 100-ppb Cd, 20-ppb Zn, and 100-ppb Zn, but not by 20- (100 ppb) did not give rise to the significant modulation ppb Cd. Collectively, the modulation patterns upon metal of MTF-1 (P > 0.05). However, exposure with both doses exposure were apparently different between MTF-1 and of Zn significantly downregulated the MTF-1 in the gill MT genes, and the degree of Cd-mediated upregulation in (P < 0.05). On the other hand, in the hepatopancreas, each tissue was much higher for MT than MTF-1 (Fig. 5). both doses of Cd elevated the MTF-1 mRNA levels in a Previous studies have indicated that mammalian MTF- dose-dependent fashion (P < 0.05). Although the 20-ppb 1 should be a constitutively expressed gene with a Zn exposure exhibited the small increase of MTF-1 TATA-less promoter and that mammalian MTF-1s mRNA levels in the hepatopancreas, 100-ppb Zn did not would not show any appreciable response to experimen- show any significant difference as compared to the level tally designed heavy metal and other stress factors. The observed in non-exposed control (P > 0.05). Unlike in plausible reason for the absence of metal (or stress) re- other three tissues, Cd exposure was unable to induce sponsiveness has been explained by the lack of MRE motif the MTF-1 expression in muscle tissue. In the muscle, in the mammalian (e.g., mouse and human) MTF-1 gene Zn exposure depressed the MTF-1 mRNA expression promoters (Auf der Maur et al. 2000; Bi et al. 2006). (more significant downregulation of MTF-1 in the Hence, the MTF-1 activity in mammals is widely agreed 100-ppb exposed group than in the 20-ppb exposed to be largely induced at a post-translation level (Günther group). For hemocytes, the group exposed with 100-ppb et al. 2012a; Smirnova et al. 2000; Saydam et al. 2001). Cd displayed a small, but statistically significant, increase However, in the present study, this abalone MTF-1 gene of MTF-1 expression, while again, Zn-exposure resulted was proven to possess a putative MRE copy as well as a in the rapid downregulation of MTF-1 irrespective of ex- canonical TATA box in its promoter region. Accordingly, posure doses (P <0.05). the present abalone MTF-1 gene displayed the Cd- In contrast to the variable or opposite regulation of mediated induction in multiple tissues (in gill, hepatopan- MTF-1 by Cd and Zn, the transcriptional response of MT creas and hemocytes, but not in muscles). Similarly, the (the main target of MTF-1) to the metal exposure treat- Cd-induced expression of MTF-1 gene has also been re- ments was relatively uniform. Further, unlike MTF-1 ported in other cold-blooded animals such as zebrafish Fig. 5 Transcriptional responses of abalone MTF-1 (a) and MT (b) to experimental heavy metal exposures. Metal exposures were done with cadmium or zinc with the dose strengths of 20 and 100 ppb for 24 h. RT-qPCR data were presented as fold changes of MTF-1 mRNAs in expressed groups relative to that of non-exposed control (Con) based on the normalization against two internal control genes (RPL5 and PPL7). Histograms with different letters indicate the significantly different means based on ANOVA followed by Duncan’s multiple ranged tests at P <0.05 Lee and Nam Fisheries and Aquatic Sciences (2017) 20:9 Page 10 of 13 (Dubé et al. 2011; Cheuk et al. 2008) and carp (Ferencz when the temperature reached 30 °C (i.e., designated and Hermesz 2009), which is obviously different from the 30 °C+0 h) (P < 0.05). Afterward, the MTF-1 expression principle for the MTF-1 regulation in mammals (Bi et al. levels began to be decreased down with the continued 2006; Saydam et al. 2001). incubation at 30 °C (i.e., 30 °C+12 h and 30 °C+24 h); However, the transcription of abalone MTF-1 was not however, the expression level at the end of thermal treat- induced by zinc except only a minute increase in the ment was still significantly higher than that of 20 °C group hepatopancreas; furthermore, Zn exposure even down- (P < 0.05). On the other hand, in the hepatopancreas, the regulated the mRNA expression of abalone MTF-1 in beginning of MTF-1 upregulation was more or less lagged other tissues. This finding is similar with no induction of as compared to that in the gill. Significant induction of MTF-1 mRNA observed for zebrafish cells exposed to MTF-1 in the hepatopancreas became evident from the Zn (Cheuk et al. 2008), although Zn exposure in zebra- 30 °C+12 h group, and the elevated expression level fish was reported to be able to activate the nuclear trans- remained constant at 30 °C+24 h (P < 0.05). In muscles, location of MTF-1 from cytoplasm (Chen et al. 2007). the significant induction of MTF-1 was found at On the other hand, a recent study with oyster C. gigas 30 °C+0 h and 30 °C+12 h but soon returned to the initial has shown that mRNA expression level of MTF-1 could level observed at 20 °C group. Meanwhile, hemocyte dis- be increased by Zn exposure, but the induced amount played the rapid induction of MTF-1 mRNA at 25 °C; was only modest even treated with high dose of Zn however, the expression dropped sharply at 30 °C+0 h in (Meng et al. 2015). In the same report, authors have in- which the decreased expression level was even lower than dicated that RNA interference of MTF-1 would result in that of 20 °C group (P < 0.05). Afterward, the MTF-1 ex- the depression of Zn-mediated induction of MT (the pression rebounded at 30 °C+0 h group. Then, the re- known target of MTF-1), consequently confirming that bound was followed by a further increase at 30 °C+12 h MTF-1 is a superordinate regulator of the MT (Meng group (P <0.05) (Fig. 6). et al. 2015). However, in this study, the inducible pattern As aforementioned, the zinc pool sensing mechanism of MTF-1 was not in agreement with that of MT by MTF-1 upon exposure to stress factors might be expression. Both Cd (inducer of abalone MTF-1) and Zn adopted for the involvement of MTF-1 in host defense (non-inducer of abalone MTF-1) were found to be able pathways against oxidative stress, since the abrupt and to induce MT expression in all the tissues examined. substantial changes of water temperature might be a Further, the quantitative relationship between MTF-1 causative factor to generate oxidative stress in poikilo- and MT expression was not proportional. Currently, it thermal invertebrates (Kim et al. 2007; Attig et al. 2014; has been widely agreed that Zn-mediated induction of Banni et al. 2014). Accordant with this explanation, the MT could be achieved by direct binding of Zn to the potential involvement of MT protein in the heat shock MTF-1 fingers, and other metals such as Cd and Cu response has already been reported in this abalone spe- may not replace Zn in zinc finger binding (Schmittgen cies and also in other aquatic animals (Lee and Nam and Livak 2008; Chen et al. 1999; Zhang et al. 2001). 2016a; Negri et al. 2013; Jarque et al. 2014). Within this Based on this, the Cd-mediated induction of MT gene context, the cold shock-induced MTF-1 has been reported might be considered as an indirect consequence. Pos- in the common carp (Cyprinus carpio)brain, with an ex- sibly, Cd load might give rise to the release of Zn from planation that sudden temperature drop gave rise to the MTs (also from other metalloproteins), and the increase alteration of physiologically accessible Zn concentrations of cellular Zn levels may activate MTF-1 through the in that tissue (Ferencz and Hermesz 2008). Zn-binding. This zinc pool hypothesis can also be Meanwhile, a series of mammalian studies has pro- applied to the MT induction upon exposed to diverse posed that MT and heat shock protein (HSP) genes oxidative stresses (Günther et al. 2012a). Hence, the con- might work in a non-cooperative way in their transcrip- troversy between MTF-1 and MT responses to zinc tional responses to stress treatments. Experimental evi- should be challenged in future study by examining cellu- dences for this proposal may include that (1) HSF lar Zn concentrations (or tissue burden) under various should boost the activity of HSP gene promoter but Zn-exposure conditions. hardly affect an MRE-containing promoter of MT gene upon heat shock and metal exposure, (2) heat shock- Response to heat shock treatment induced nuclear translocation of MTF-1 has been shown Abalone MTF-1 was proven an early phase, heat-shock to be insufficient to activate a MT gene promoter, (3) di- responsive protein as evidenced by the rapid modulation verse target gene searches for MTF-1 have been indica- upon thermal increases. The MTF-1 mRNA levels in the tive of HSP genes as non-affected genes, and (4) MTF-1 gill were rapidly increased (fourfold relative to 20 °C; has been likely to repress HSF-regulated genes through P < 0.05) as early as when water temperature reached a direct protein-protein interaction (Lichtlen et al. 2001; 25 °C (Fig. 6). The expression level was further elevated Saydam et al. 2003; Uenishi et al. 2006). However, on the Lee and Nam Fisheries and Aquatic Sciences (2017) 20:9 Page 11 of 13 Fig. 6 Differential mRNA expression patterns of abalone MTF-1 in response to heat shock treatments. During heat elevation with an increase rate of 1 °C/h, abalones were sampled at 20 °C (just before the elevation), 25 °C, and 30 °C. After reaching 30 °C, abalones were exposed at the constant 30 °C and sampled after 12 h (30 °C+12 h) and 24 h (30 °C+24 h), respectively. RT-qPCR data were presented as fold changes of MTF-1 mRNAs in expressed groups relative to that of non-heat shocked group (i.e., 20 °C control) based on the normalization with RPL5 and PPL7 reference genes. Histograms with different letters indicate the significantly different means based on ANOVA followed by Duncan’s multiple ranged tests at P < 0.05 contrary, the present study has shown that the transcrip- mature males. Abalone MTF-1 was expressed during the tion of abalone MTF-1 could be directly activated by entire period of embryonic and early ontogenic develop- heat shock. Hence, our finding suggests that the thermal ment. From the heavy metal exposure, abalone MTF-1 stress-mediated activation of MTF-1 in abalone may be was found to be Cd inducible (but not by Zn); however, achieved not only at a post-translation level (i.e., indir- the induced amounts were only modest as compared to ectly based on cellular zinc sensing abovementioned) that of MT. Abalone MTF-1 was highly modulated in re- but also at a transcriptional level (i.e., de novo synthesis). sponsive to heat shock potentially via both indirect zinc The presence of potential HRE motifs in the abalone pool sensing and direct de novo transcription. Data from MTF-1 gene regulatory region is also in congruent with this study could be a useful basis to approach various re- the present hypothesis. Possibly, the activation of MTF-1 searches regarding the stress responses in this abalone from the dual routes is likely to be advantageous for species particularly including detoxification of heavy permitting the poikilothermal animals to prepare more metals and adaptation to thermal stresses. efficiently antioxidant components (i.e., antioxidant enzyme genes containing MREs in their promoters as Additional file well as MT) against the oxidative stress caused by Additional file 1: Table S1. Summarized information on oligonucleotide thermal fluctuations (Kim et al. 2007; Cho et al. 2009). primersusedin thisstudy. Figure S1. Full-length cDNA and deduced amino However, further efforts are needed to get direct evi- acid sequences of abalone Haliotis discus hannai metal responsive transcription dence on the cooperativity between HSF and MTF-1 in factor-1 (MTF-1). In the nucleotide sequence, stop codon is indicated by an asterisk and putative polyadenylation signal is underlined. On the other this abalone species. hand, in the amino acid sequence (in a singlet code), presumed nuclear localization signal (NLS) is underlined (in the front of the first zinc finger), Conclusions while six putative C H -zinc fingers in the DNA-binding domain are boxed. 2 2 Figure S2. Multiple sequence alignments of abalone MTF-1 along with its Novel MTF-1 was isolated and characterized from a representative orthologs. Putative NLS, six C H -zinc fingers, NES embedded 2 2 commercially important marine mollusc species, Pacific in transactivation domain, and C-terminal Cys-cluster are indicated. Figure abalone (H. discus hannai). The abalone MTF-1 was S3. Neighbor-joining phylogenetic tree based on amino acid sequences of zin-finger DNA-binding domain and NLS region in MTF-1 orthologs. Tree found to share a conserved feature in zinc finger, DNA- was computed by poisson correction method using MEGA 7.0 program. binding domain with its orthologs; however, it repre- Confidence level of each clade was evaluated with bootstrap testing (1000 sented non-conservative features in remaining other replicates). Figure S4. Putative transcription factor-binding motifs predicted in the 5′-flanking upstream region of the abalone MTF-1 gene. Transcription factor parts. Bioinformatic analysis of the 5′-upstream region sites were predicted with perfect and imperfect matches to consensus core has predicted diverse transcription factor binding motifs sequences. TRANSFAC search (GeneXplain GmbH, Germany) was carried out that are potentially related with metal regulation, stress with the cut-offs scores >0.99 (core score) and >0.95 (matrix score). responses, and development. Abalone MTF-1 was ubiquitously detected in various tissues, in which the Acknowledgements highest expression level was observed in the testes of Not applicable. Lee and Nam Fisheries and Aquatic Sciences (2017) 20:9 Page 12 of 13 Funding Ferencz Á, Hermesz E. Identification of a splice variant of the metal-responsive This study was supported by the grant from the Golden Seed Project (GSP), transcription factor MTF-1 in common carp. Comp Biochem Physiol C Toxicol Ministry of Oceans and Fisheries, Republic of Korea. Pharmacol. 2009;150:113–7. Giedroc DP, Chen X, Apuy JL. 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