Sulfide exposure results in enhanced sqr transcription through upregulating the expression and activation of HSF1 in echiuran worm Urechis unicinctus

Aquatic Toxicology 170 (2016) 229–239

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Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox

Sulfide exposure results in enhanced sqr transcription through upregulating the expression and activation of HSF1 in echiuran worm Urechis unicinctus Xiaolong Liu, Zhifeng Zhang ∗ , Xiaoyu Ma, Xueyu Li, Di Zhou, Beibei Gao, Yajiao Bai Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China

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Article history: Received 23 September 2015 Received in revised form 19 November 2015 Accepted 19 November 2015 Available online 2 December 2015 Keywords: Sulfide exposure Sulfidequinone oxidoreductase (SQR) HSF1 Promoter Transcription regulation Urechis unicinctus

a b s t r a c t Sulfide is a natural, widely distributed, poisonous substance. Sulfide: quinone oxidoreductase (SQR) is responsible for the initial oxidation of sulfide in mitochondria. To study transcriptional regulation of sqr after sulfide exposure, a 2.6-kb sqr upstream sequence from echiuran worm Urechis unicinctus was cloned by genome walking. Bioinformatics analysis showed 3 heat shock elements (HSEs) in proximal promoter region of the sqr upstream sequence. Moreover, an Hsf1 cDNA in U. unicinctus (UuHsf1) was isolated with a full-length sequence of 2334 bp and its polyclonal antibody was prepared using U. unicinctus HSF1 (UuHSF1) expressed prokaryotically with whole sequence of its open reading frame (ORF). In vivo ChIP and in vitro EMSA assays revealed UuHSF1 could interact with the sqr proximal promoter region. Transient transfection and mutation assays indicated that UuHSF1 bound specifically to HSE (−155 bp to −143 bp) and enhanced the transcription of sqr. Furthermore, sulfide treatment experiments demonstrated that sulfide could increase the expression of HSF1 protein, and induce trimerization of the protein which binds to HSEs and then activate sqr transcription. Quantitative real-time PCR analysis revealed sqr mRNA level increased significantly after U. unicinctus was exposed to sulfide for 6 h, which corresponded to content changes of both trimeric HSF1 and HSF1-HSE complex. We concluded that UuHSF1 is a transcription factor of sqr and sulfide could induce sqr transcription by upregulating the expression and activation of HSF1 in U. unicinctus exposed to sulfide. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Exogenous sulfide is a well-known toxin to organisms, which can induce reversible inhibition of cytochrome c oxidase (Nicholls and Kim, 1982; Dorman et al., 2002; Cooper and Brown, 2008; Di

Abbreviations: SQR, sulfidequinone oxidoreductase; HSF1, heat shock factor 1; DBD, DNA binding domain; HR, heptad repeats domain; HSE, heat shock element; PSU, practical salinity units; RACE, rapid amplification of cDNA ends; IPTG, isopropyl ␤-D-1-thiogalactopyranoside; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assays; BSA, bovine serum albumin; HRP, streptavidin–horseradish peroxidase; PAGE, polyacrylamide gel electrophoresis; CMV, cytomegalovirus; ORF, open reading frame; HEK293, human embryonic kidney 293; qRT-PCR, quantitative real-time PCR; DPE, downstream promoter element; UTR, untranslated region; NLS, nuclear localization signal; Inr, initiator; DPE, downstream promoter element; aip-1, ALG-2-interacting protein; Lif, leukaemia inhibitory factor; HYPK, Huntingtin yeast partner K; hsp, heat shock protein; Bis, Bcl-2 interacting cell death suppressor. ∗ Corresponding author. Fax: +86 532 82031647. E-mail address: [email protected] (Z. Zhang). http://dx.doi.org/10.1016/j.aquatox.2015.11.021 0166-445X/© 2015 Elsevier B.V. All rights reserved.

Meo et al., 2011), decrease of hemoglobin oxygen affinity (Carrico et al., 1978), sulfhemoglobin formation (Bagarinao, 1992; Kraus et al., 1996), mitochondrial depolarization (Julian et al., 2005), coelomocyte death, decreased cell proliferation (Hance et al., 2008), inhibition of almost 20 enzymes involved in aerobic metabolism (Bagarinao, 1992), and oxidative damage to RNA and DNA (AtteneRamos et al., 2007; Baskar et al., 2007; Joyner-Matos et al., 2010). Meanwhile, endogenous hydrogen sulfide is known as a signaling molecule to participate in cell physiological processes (Abe and Kimura, 1996; Wang, 2002; Elsey et al., 2010; Li et al., 2011; Kabil et al., 2014; Hine et al., 2015). Therefore, studies on sulfide metabolism and its role in organisms are very significant. Mitochondrial sulfide oxidation is an important mechanism for detoxification of sulfide, and sulfide: quinone reductase (SQR) is a key enzyme which can oxidize sulfide into thiosulfate (Hildebrandt and Grieshaber, 2008; Jackson et al., 2012; Tiranti and Zeviani, 2013). Characteristics of SQR in protein structure, enzyme property, catalytic sites and catalytic capacity has been demonstrated (Wakai et al., 2007; Hildebrandt and Grieshaber, 2008; Theissen

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and Martin, 2008; Chan et al., 2009; Jackson et al., 2012; Tiranti and Zeviani, 2013). Recently, Ma et al. (2012a) reported that level of sqr mRNA expression increases significantly under sulfide exposure in Urechis unicinctus. However, no information is known about the relation between sulfide and sqr transcriptional regulation. Members of heat shock factor (HSF) family are highly conserved with a DNA binding domain (DBD) for target gene recognition and hydrophobic heptad repeats domain (HR-A/B) for HSF trimerization (Damberger et al., 1994; Harrison et al., 1994; Vuister et al., 1994; Wu, 1995). In plants and vertebrates, multiple HSFs are reported, while only HSF1 is identified in invertebrates (Wu, 1995; Nover et al., 1996, 2001; Morano and Thiele, 1998; Nakai, 1999; Åkerfelt et al., 2007). HSF1 can be activated from inactive monomeric state in cytoplasm to trimeric HSF1 complex which enters nucleus and regulates transcription of downstream genes in eukaryotic cells (Pelham, 1982; Orosz et al., 1996; Westwood et al., 1991; Wu, 1995). HSF1 is originally named based on its function of regulating chaperone protein (Hsp) expression under thermo stress (Sorger et al., 1987). Baird et al. (2006) identified that HSF1 is activated and enhances the expression of Hsps in Drosophila melanogaster during hypoxia and reoxygenation. Sakurai et al. (2013) confirmed the maintenance of HYPK expression depends on the increase of Hsf1 mRNA and HSF1 protein as well as its activation under thermal stress conditions. However, recent studies revealed that HSF1’s functions as transcription factor are not only in regulating thermo reaction but also in many biological processes, such as lifespan regulation, organ development, cell growth and differentiation, inflammation, autophagy and tumorigenesis (Morley and Morimoto, 2004; Dai et al., 2007; Christians, 2011; Xi et al., 2012; Desai et al., 2013), as well as in response to a diverse environmental factors including oxidant, hypoxia and heavy metals (Lee et al., 2000; Sakurai et al., 2013; Neef et al., 2014; Yan et al., 2014). Bruce et al. (1993) indicated oxidative injury can rapidly activate HSF1. Deletion experiments of the D. melanogaster Hsf revealed defective oogenesis and larvae development (Jedlicka et al., 1997). A series of research on aging regulated by HSF1 has also been elucidated (Hsu et al., 2003; Morimoto, 2011; Murshid et al., 2013). The echiuran worm U. unicinctus, inhabiting a “U-shaped” burrow in intertidal and subtidal mudflats, distributes widely in Russia, Japan, the Korean Peninsula and China (Li, 1997). Previous studies have revealed that U. unicinctus can survive well in sulfide-rich habitats and tolerate high concentration of sulfide depending on the mitochondrial sulfide oxidation detoxification system (Zhang et al., 2006, 2013; Ma et al., 2010, 2011, 2012a,b; Wang et al., 2010). Specially, Ma et al. (2012a,b) reported that sqr mRNA level in U. unicinctus exposed to sulfide increases significantly in a timeand concentration-dependent manner. In this study, we identified major cis-elements of U. unicinctus sqr promoter and demonstrated HSF1 as a transcription factor could bind to sqr promoter and activate its expression for the first time. Furthermore, we revealed that HSF1 played a key role in up-regulating expression of sqr mRNA in the hindgut of U. unicinctus exposed to sulfide. This work will provide a launch point for future investigations in mechanism of mitochondrial sulfide metabolism in U. unicinctus.

2. Materials and methods

2.2. Animals and sampling U. unicinctus (mean fresh mass of 33.4 ± 10.4 g) were purchased from an aquatic product market, which were collected from a coastal intertidal flat in Yantai, China. The worms were maintained temporarily in aerated seawater (17.6 ± 0.3 ◦ C, pH 8.01 ± 0.02, salinity 32 PSU) for three days. Sixty healthy worms were averagely assigned to six tanks which contained 30 l of seawater for each tank and sealed with cling film. During the experiment, three tanks were used as sulfide treat group in which a sulfide concentration of 150 ␮M was set and maintained, respectively, and other three tanks as control without any added exogenous sulfide. The sulfide concentration was detected using the methylene blue method (Cline, 1969), and maintained by adding the sulfide stock solution (10 mM NaHS, pH 8.0) at a 2-h interval according to the method described by Ma et al. (2012a). One worm per tank was sampled at 0, 0.5, 1, 1.5, 2, 6, 24, 48 and 72 h after sulfide exposure, respectively. Hindguts of the worms were dissected, rinsed with PBS (pH7.4), frozen in liquid nitrogen immediately, and then stored at −80 ◦ C for DNA, RNA and protein extraction. 2.3. Cloning of sqr gene promoter and sequence analysis The genomic DNA of U. unicinctus hindgut was prepared according to the standard protocol from the manufacture’s instruction with the universal genomic DNA extraction kit Ver3.0 (TaKaRa, Japan). The promoter sequence of sqr gene was amplified using a genome walking kit (TaKaRa, Japan) with universal primers (AP1-AP4) and the gene-specific primers SP1-SP3 (Table S1) which were designed according to the U. unicinctus sqr complete cDNA sequence (GenBank accession number: EF487538). In the first round of PCR amplification, the SP1 primer was used together with AP1-AP4 primers, respectively, with 1 ␮l (containing 1 ␮g DNA) of the genomic DNA as template. The reaction condition was as follows: denaturation at 94 ◦ C for 1 min, 98 ◦ C for 1 min, 5 cycles of 30 s at 94 ◦ C, 1 min at 65 ◦ C, and 4 min at 72 ◦ C, then 94 ◦ C for 30 s, 25 ◦ C for 3 min and 4 min at 72 ◦ C and at last, 15 cycles of 94 ◦ C for 30 s, 1 min at 65 ◦ C, 72 ◦ C for 2 min, 94 ◦ C for 30 s, 1 min at 65 ◦ C, 72 ◦ C for 2 min, 94 ◦ C for 30 s, 1 min at 44 ◦ C and 4 min at 72 ◦ C, followed by a 10-min extension at 72 ◦ C. The second and third amplifications were conducted with 1 ␮l of the former round PCR product as template, and primers (SP2 and SP3 to AP1–AP4, respectively). Analysis of the PCR products was carried out by electrophoresis on a 1.2% agarose gel. The purified product of the third round PCR was cloned into a pMD18-T vector (TaKaRa, Japan) and sequenced. The upstream sequence of U. unicinctus sqr gene was assembled with DNAMAN software and predicted using the online tool, Network Promoter Prediction (http://www.fruitfly.org/seq tools/ promoter.html). Putative binding sites of the main transcription factors in the promoter region of the U. unicinctus sqr gene were predicted using TESS and TFSEARCH programs. 2.4. Total RNA isolation and cDNA synthesis Total RNA from hindgut was extracted with TRIzol® Reagent (Invitrogen, USA) following the manufacturer’s instruction and digested by RNase-free DNase I (TaKaRa, Japan) to remove residual genomic DNA. The cDNA template was prepared using a SMARTerTM RACE cDNA Amplification Kit (Clontech, USA) for the rapid amplification of cDNA ends (RACE).

2.1. Ethics statement Each of the procedures that were used to handle and treat the Echiuran worms during this study was approved by the Ocean University of China Institutional Animal Care and Use Committee (OUC-IACUC) prior to the initiation of the study.

2.5. Cloning of U. unicinctus Hsf1 cDNA and polyclonal antibody acquisition A 298-bp fragment was obtained by homologous cloning strategy using the cDNA template and degenerate primers DPHSF1-F

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Fig. 1. Nucleotide sequence of U. unicinctus sqr proximal promoter. The sequence of exon 1 is written in capital letters. The sqr transcription start site is indicated in box. Putative transcription factor binding sites are underlined. Double underline indicates the core sequence of downstream promoter element.

and DPHSF1-R (Table S1) designed based on the conserved domain of HSF1s from Homo sapiens, Mus musculus, Denio rerio, Ciona intestinalis and Crassostrea gigas. The 3 - and 5 -RACE amplifications of the target cDNA were conducted using the SMARTerTM RACE cDNA Amplification Kit (Clontech, USA) following the manufacture’s instruction. The first amplification for 5 - and 3 -RACE was performed respectively with reverse-transcribed cDNA as template and the specific primers HSF1-5 RACE1 and HSF1-3 RACE1 (Table S1) designed according to the cDNA fragment under the following conditions: denaturation at 94 ◦ C for 5 min; 35 cycles of 94 ◦ C for 30 s, 62 ◦ C for 30 s, and 72 ◦ C for 3 min; and a final extension at 72 ◦ C for 10 min before temporarily stored at 4 ◦ C. The second amplification for 5 - and 3 -PCR was carried out using HSF1-5 RACE2 and HSF1-3 RACE2 (Table S1) and the first PCR product as template with the same reaction condition as the first amplification. The PCR products were purified and cloned into the pMD18-T vector and then sequenced. The 5 -RACE fragment and 3 -RACE fragment were finally assembled with DNASTAR software (DNAstar, WI, USA) to get a full length cDNA of U. unicinctus Hsf1 (UuHsf1). Sequence similarity of UuHSF1 with other known HSF1s were analyzed by BLAST program at the National Center for Biotechnology Information (NCBI) server. The deduced amino acid sequence of UuHSF1 was analyzed using the simple modular architecture research tool (http://smart.embl-heidelberg.de). Multiple sequence alignment was performed using the Clustal X 2.0 program.

The ORF of UuHsf1 cDNA was amplified using HSF1ORF-sense and HSF1ORF-antisense primers (Table S1), and then ligated to linearized pET28a vector. The recombinant HSF1ORF-pET28a vector was transformed to BL21 (DE3) competent cells, and UuHSF1 protein was expressed inductively using 1 mM isopropyl ␤-D-1thiogalactopyranoside (IPTG) for 5 h. The expressed HSF1 protein as inclusion body was dissolved with 8 M urea, purified using NiNTA agarose (Novagen, Germany), and then produced the UuHSF1 polyclonal antibody (Sangon Biotech, China). 2.6. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA) The ChIP assays were performed according to the manufacture’s instruction using an agarose ChIP kit (No. 26156, Pierce) with slight modifications. The U. unicinctus hindgut was cut into pieces and digested with 0.25% trypsin solution into single cell suspension, and then treated with 0.4% formaldehyde for 10 min at room temperature to crosslink DNA and protein. Crosslinking was stopped by the addition of 0.125 M glycine solution. Nuclei pellets were collected by centrifugation at 3000 × g for 5 min, and then resuspended in nuclei buffer. Sonication was carried out to shear the chromatin of nuclei pellets, and 10% of the total sample was stored as input control. The rest sample was then incubated with 2 ␮g of UuHSF1 antibody or pre-immune serum for 2 h at 4 ◦ C followed by incubation at 4 ◦ C with protein A/G agarose beads overnight. The protein-DNA complex was incubated and digested with 10 mg/ml

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Fig. 2. Multiple sequence alignment of HSF1 proteins from various species. Multiple sequence alignment is conducted using the Clustal X 2.0 program. The identical amino acid residues are shaded in black, and the similar residues (75% identities) are shaded in gray. The putative conserved domains of DBD, HR-A/B domains and NLS signal sequence are in box.

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Fig. 3. Identification of interaction between HSF1 and sqr proximal promoter in U. unicinctus through (a) ChIP assay and (b) EMSA and supershift assays. (a) ChIP assay showing HSF1 interaction in vivo with −160 bp to −49 bp of sqr proximal promoter. Sheared chromatin was immunoprecipitated using 10 ␮l anti-HSF1 antibody or 10 ␮l pre-immune serum (as a negative control). Input DNA and immunoprecipatated DNA were analyzed by PCR. The three regions, −385 to −203, −160 to −49 and −16 to +160, were amplified by three pairs of ChIP primers, ChIP-F1/R1, ChIP-F2/R2 and ChIP-F3/R3 (Table S1), respectively. (b) In EMSA assays, biotinylated probe was incubated with untreated hindgut nuclear extract as described in Section 2. Lane 1, positive group using biotin-labeled EMSA probe and nuclear extract, * represents the band of protein-probe complex; lane 2, negative group without biotin-labeled probe; lane 3, cold competition group using biotin-labeled probe and 100-fold unlabeled probe; lane 4, super shift assay with addition of anti-HSF1 antibody in positive group; ** represents the super shift band of probe-HSF1-HSF1 antibody complex. Both images are representative of 3 independent experiments.

proteinase K at 65 ◦ C for 6 h. The DNA fragments were then isolated from DNA-protein complex and analyzed by PCR using three pairs of specific primer pairs, ChIP-F1/R1, ChIP-F2/R2 and ChIP-F3/R3 (Table S1) against the proximal region (sites from −391 to +194, Fig. 1) of U. unicinctus sqr promoter. Moreover, to detect the influence of sulfide on the interaction between sqr promoter and HSF1 in U. unicinctus, another set of ChIP assays were performed with the hindguts of U. unicinctus exposed 150 ␮M sulfide for 0, 0.5, 1, 1.5, 2, 6, 24, 48 and 72 h, respectively with the method as described above. The Chemiluminescent EMSA kit (Beyotime, China) was used to perform the EMSA. Nuclear protein extract from U. unicinctus hindgut was prepared using Nuclear and Cytoplasmic Extraction Kit (CwBiotech, China) and stored at −80 ◦ C until use. Protein concentration was measured according to the Bradford method (Bradford, 1976) with bovine serum albumin (BSA) as a standard. Two oligonucleotides, EMSAoligo and anti-EMSAoligo (Table S1) which correspond to HSE element in the promoter of U. unicinctus sqr gene were synthesized, labeled with biotin at the 5 -end using EMSA Probe Biotin Labeling Kit (Beyotime, China), and then annealed into biotin-labeled probe according to the manufacture’s instruction. Unlabeled probe was made by annealing the two oligonucleotides. The reaction system contains EMSA binding buffer (5X), nuclear extract and biotin labeled probe. Cold competition experiment was executed using excessive unlabeled double-stranded probe (100-fold higher than biotin labeled probe), which was added to the reaction mixture prior to the labeled probe. Super shift assays were carried out with the addition of HSF1 antibody. DNA–protein complex was electrophoresed on a 4% polyacrylamide gel in 0.5 × Tris borate/EDTA electrophoresis buffer (pH8.3) at 125 V, and then transferred to a positive nylon membrane, UV-cross-linked with the membrane, probed with streptavidin–horseradish peroxidase (HRP) conjugate, and incubated with the substrate. Photos were captured using ChemiDoc MP imaging system (Biorad, USA).

2.7. Western blotting Total proteins from the hindguts of U. unicinctus exposed to 150 ␮M sulfide for 0, 0.5, 1, 1.5, 2, 6, 24, 48 and 72 h were isolated with tissue protein extraction kit (CWbiotech, China) according to the manufacture’s instruction. The protein extracts were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to PVDF membranes (Roche) for 20 min in a rate of 5 mA/cm2 using Trans-Blot Semi-Dry Electrophoretic Transfer system (Biorad, USA). After blocked with 5% (w/v) BSA dissolved in 0.01 M PBS (pH 7.4) containing 0.05% (v/v) Tween 20, the membranes were incubated with UuHSF1 antibody, and then incubated with second antibody (goat anti-rabbit, HRP conjugated), finally incubated with substrate and pictured on ChemiDoc MP imaging system (Biorad, USA). The gray values of HSF1 bands were processed with ImageJ software. The expression level of ␤-Actin is used as internal control and the expression level of HSF1 at 0 h was regarded as baseline. 2.8. Native polyacrylamide gel electrophoresis (Native PAGE) Native PAGE was performed to detect the oligomerization of HSF1. Hindgut protein extracts from U. unicinctus exposed to 150 ␮M sulfide for 0, 0.5, 1, 1.5, 2, 6, 24, 48 and 72 h were modified to the same loading amount by Western blotting with ␤-Actin, and then separated by native PAGE using the Novex Bis-Tris gel system according to the manufacturer’s specification (Invitrogen). Native gels were then transferred to PVDF membrane (Roche), and monomeric and trimeric HSF1 proteins were visualized by Western blotting. 2.9. Promoter activity assays of U. unicinctus sqr Construction of Pcmv-EGFP plasmid: the original vector pEGFP-N1 (Clontech, USA) was double digested with Ase I and Bgl

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II to remove the original CMV promoter. Cohensive ends of the linearized pEGFP-N1 were filled with Klenow polymerase and ligated using T4 ligase, and the negative control vector, Pcmv-EGFP, was at last generated. Construction of Psqr-EGFP plasmid: the proximal region (from −391 to +194, Fig. 1) of U. unicinctus sqr promoter was amplified using primers, Fwd-391 and Rev + 194 (Table S1) and cloned into Pcmv-EGFP plasmid before the EGFP coding sequence. The constructed vector, Psqr-EGFP plasmid, was used to analyze the activity of sqr promoter. Construction of sqr promoter mutant plasmids: three HSEs (regions from −230 bp to −211 bp, −170 bp to −150 bp and +40 bp to +58 bp, respectively) were predicted in the proximal region of U. unicinctus sqr promoter. Three types of single-HSE-mutated plasmids (Psqr-EGFP-M1, Psqr-EGFP-M2 and Psqr-EGFP-M3, Fig. 4a) were generated based on Psqr-EGFP plasmid using three pairs of primers (Psqr Mut1-F/R, Psqr Mut2-F/R, Psqr Mut3-F/R, Table S1) with the Fast Mutagenesis System (TransGen Biotech, Beijing) according to the manufacture’s instruction. Construction of the HSF1 expression vector Pcmv-HSF1: the pEGFP-N1 vector was digested with enzymes, EcoR I and Not I, to remove the EGFP coding sequence. The ORF of UuHsf1 was excised from the pMD18T-HSF1 plasmid with EcoR I and Not I, ligated to the linearized pEGFP-N1EGFP after it was PCR-amplified with primers HSF1ORF-F and HSF1ORF-R (Table S1), cloned into pMD18T vector (TaKaRa, Japan) and confirmed with sequencing. The constructed Pcmv-HSF1 vector was expected to express UuHSF1 efficiently in HEK293 cells. Transient transfection and promoter activity assays: HEK293 cells were cultured at 37 ◦ C under humidified air containing 5% CO2 in Dulbecco’s modified Eagle’s medium (Gibco, Rockville, MD) with 10% fetal bovine serum (Gibco, Rockville, MD). Each type of sqr promoter plasmids was transiently transfected alone or with Pcmv-HSF1 into HEK293 cells in 24-well plates with Lipofactamine 2000 reagent (Invitrogen) according to the manufacture’s instruction. Each transfection was repeated three times. The pEGFP-N1 and Pcmv-EGFP were transfected as internal controls. 48 h after transfection, the GFP expression levels in HEK293 cells were detected using fluorescence microscope Nikon E80i (Japan). The fluorescence intensities in the different wells were processed with Nikon software and online ImageJ software. In addition, the expression of UuHSF1 in HEK293 cells was also detected by Western blotting to make sure the effectiveness of the co-transfection system. 2.10. Quantitative real-time PCR Total RNAs from U. unicinctus hindgut sampled at different time of sulfide exposure were extracted as described above, and then reverse-transcribed into first-strand cDNA using a PrimeScriptTM First Strand cDNA Synthesis Kit (TaKaRa, Japan). Quantitative realtime PCR (qRT-PCR) was performed to determine expression level of sqr in the hindgut of U. unicinctus exposed to sulfide using Roche 480 detection system (Roche, USA) with SYBR Green Master Mix (TaKaRa, Japan). Two pairs of primers, sqr-sense and sqr-antisense (Table S1) for amplification of 102-bp gene-specific product, and actin-sense and actin-antisense (Table S1) for amplification of 150bp reference gene, were designed and used to conduct qRT-PCR according to the method described previously (Ma et al., 2012a). The 2−Ct method was employed to analyze the relative expression level of U. unicinctus sqr (Livak and Schmittgen, 2001). 2.11. Statistical analysis Data were presented as mean ± SEM (n = 3). Statistical analysis was performed using Student’s t-test. A value of p < 0.05 and p < 0.01

was considered as significant difference and extremely significant difference, respectively. 3. Results 3.1. Cloning and analysis of U. unicinctus sqr upstream sequence A 2.6-kb fragment of the upstream sequence of U. unicinctus sqr from the transcription start site was obtained by genome walking. Bioinformatics analysis on its proximal promoter region (from −391 bp to +196 bp) indicated that it was a typical TATA-less promoter form, and a downstream promoter element (DPE) which was similar to TATA box in function was found precisely at +28 bp to +34 bp downstream of the initiator (Fig. 1). The proximal region also contained several potential transcription factor binding sites (Fig. 1), three HSF1 binding sites (HSEs) which lied at −222 to −209 bp, −155 to −143 bp and +47 to +58 bp, respectively, were concerned specially, suggesting HSF1 protein may play a potential role in the sqr transcriptional regulation. 3.2. Sequence analysis of U. unicinctus Hsf1 The full-length cDNA of Hsf1 (GenBank accession number: KF976398) was 2334 bp including a 5 untranslated region (UTR) of 128 bp, a 3 UTR of 654 bp with a poly(A) tail and a 1554bp ORF encoding a putative protein of 517 amino acids with a calculated molecular mass of 57.14 kDa and Isolectric Point of 4.421. Structural analysis indicated that the deduced amino acid sequence contained two domains, the DNA binding domain (DBD) and trimerization domain HR-A/B, which were highly conserved in HSF family, and a nuclear localization signal (NLS) sequence was also found in UuHSF1 (Fig. 2). Moreover, the target amino acid sequence was highly homologous with other known HSF1s, 65% identity to Capitella teleta, 40% to Halitotis asinina, 38% to Crassostrea gigas, 38% to Octopus vulgaris, 36% to Danio rerio and 40% to Takifugu rubripes, respectively. 3.3. HSF1 binds to HSE element of U. unicinctus sqr promoter Three pairs of primers, ChIP-F1/R1, ChIP-F2/R2 and ChIP-F3/R3 (Table S1) were used to amplify three fragments of U. unicinctus sqr proximal promoter, each of which contained one predicted HSE element. A 111-bp sequence (−160 bp to −49 bp, Fig. 1) containing a predicted HSE element (−155 bp to −143 bp) was amplified using ChIP-F2 and ChIP-R2 primers, and ChIP assay confirmed that the HSF1 protein could bind in vivo to the region of U. unicinctus sqr. However, the other two HSE elements (−222 bp to −209 bp and +47 bp to +58 bp) didn’t interact with UuHSF1 (Fig. 3a). Furthermore, results of EMSA and supershift assays showed that the UuHSF1 was able to bind specifically to the HSE element (−155 bp to−143 bp) using nuclear extract from U. unicinctus hindgut (Fig. 3b, lane1). The specificity of HSF1-HSE binding was confirmed based on the disappearance of the HSE-protein complex when an excess of unlabeled probe competing with the biotin-labeled probe for HSF1 binding (Fig. 3b, lane3) and further identified by supershift assay with the addition of specific anti-HSF1 antibody (Fig. 3b, lane 4). To further identify the HSE sites in the proximal promoter of sqr interacted with HSF1, Psqr-EGFP and three kinds of HSE mutant plasmids (Psqr-EGFP-M1, Psqr-EGFP-M2 and Psqr-EGFP-M3, Fig. 4a) were transfected into the HEK293 cells alone or in combination with HSF1 expression vector, Pcmv-HSF1. Results showed that activities of sqr promoter which were detected by fluorescence intensity in the HEK293 cells had no significant difference when the transfections were conducted with the Psqr-EGFP and three kinds of HSE mutant plasmids, respectively (Fig. S1). However, when PsqrEGFP, Psqr-EGFP-M1 or Psqr-EGFP-M3 was co-transfected with

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Fig. 4. Identification of effective HSE region of sqr promoter interacted with HSF1. (a) Schematic representation of three predicted HSEs in the proximal region of sqr promoter. Three HSE core sequences for the original (boxed) and mutated sequences are shown; mutated nucleotides are underlined. (b) Activity of U. unicinctus sqr promoter in HEK-293 cells was detected according to the EGFP expression using fluorescence microscope and analyzed using ImageJ software after the plasmids are transfected for 48 h; the promoter activities are presented as fluorescence intensity per well. Data are indicated as mean ± SEM from triplicate experiments. * p < 0.01 for Psqr-EGFP, Psqr-EGFP-M1 and Psqr-EGFP-M3 co-transfected with Pcmv-HSF1 vs. single transfection of Psqr-EGFP, Psqr-EGFP-M1 and Psqr-EGFP-M3, and co-transfection of Psqr-EGFP-M2 with Pcmv-HSF1. (c) Expressions of UuHSF1 are detected by Western blotting in HEK 293 cells transfected with the plasmids. Human ␤-Actin is used as internal control.

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Pcmv-HSF1, the activities of sqr promoter in HEK293 cells increased significantly (p < 0.01), with a 5.6 fold, 5.2 fold and 5.5 fold higher than that of the corresponding single plasmid transfection, respectively; meanwhile no significant difference was detected in HEK293 cells co-transfected with mutant Psqr-EGFP-M2 and Pcmv-HSF1 (Fig. 4b). Subsequently, the expression of UuHSF1 in the HEK293 cells transfected with various plasmids was detected by Western blotting (Fig. 4c). Results confirmed that UuHSF1 enhanced the transcription activity of sqr promoter as a transcription factor by binding to HSE (−155 bp to −143 bp) of sqr proximal promoter. 3.4. Sulfide up-regulates HSF1 expression and activates HSF1 in U. unicinctus hindgut When U. unicinctus were challenged in 150 ␮M sulfide, expression levels of the HSF1 protein in the hindgut increased gradually with extension of exposure time, and a significant increase (p < 0.05) firstly occurred at 2 h post exposure, and then reached a maximum at 24 h with 6 folds higher than that of the control at the same time (Fig. 5). After that, the HSF1 content maintained in an extremely significant level until 72 h compared with that of control (Fig. 5). The trimerization of HSF1 is necessary for the HSF1 activation to bind the specific DNA sequence. To examine the activity of HSF1 regulating sqr transcription in the hindgut of U. unicinctus to sulfide, the monomeric HSF1 and trimeric HSF1 were detected by Native PAGE. Results showed that both monomeric and trimeric HSF1 in the hindgut cells existed before sulfide exposure only in a small amount. Trimeric HSF1 increased obviously in the hindgut cells after U. unicinctus exposed to 150 ␮M sulfide for 6 h, while content of the monomeric HSF1 increased slightly (Fig. 6a). Furthermore, content of the UuHSF1 binding to the sqr HSE element (−155 bp to −143 bp) increased obviously in the hindgut of U. unicinctus exposed to sulfide after 6 h (Fig. 6b). Results of qRT-PCR analysis demonstrated that the sqr mRNA expression had no significant difference during early sulfide exposure (before 2 h). A significant increase of sqr expression was firstly detected at 6 h post exposure, with about 6 folds higher than that of the control, and the mRNA level reached the maximum after U. unicinctus was exposed for 48 h (Fig. 7). 4. Discussion SQR is a key enzyme in mitochondrial sulfide oxidation detoxification, and its structural and catalytic characteristics have been revealed (Wakai et al., 2007; Hildebrandt and Grieshaber, 2008; Theissen and Martin, 2008; Ma et al., 2011; Jackson et al., 2012). In this study, we focused on the transcription regulation of sqr and demonstrated HSF1, as a transcription factor up-regulated the expression of sqr in U. unicinctus response to sulfide exposure. 4.1. U. unicinctus sqr promoter lacks the typical TATA element Transcription is centrally involved in many biological processes such as growth, development and response to external stimuli (Burke and Kadonaga, 1997). Many protein-coding genes are transcribed by the RNA polymerase II transcriptional machinery, which comprises RNA polymerase II and other factors that are required for basal and regulated transcription. Transcription initiation requires the precise positioning of RNA polymerase II on core promoter sequence (within about −40 to +40 relative to the transcription start site). At present, three common DNA elements, the initiator (Inr), the TATA box and the downstream promoter element (DPE) have been well-known in the core promoter. In this study, a 2.6kb fragment of the upstream sequence of U. unicinctus sqr from the transcription start site was obtained by genome walking. An

Inr was identified with a sequence of TCACTC, which was similar with the Inr motif of Drosophila genes. The adenine (A) site in this sequence was considered as +1. Bioinformatics analysis on its proximal promoter region (from −391 bp to +196 bp) indicated that it was a typical TATA-less promoter form, and a downstream promoter element (DPE) which was similar to TATA box in function was found precisely at +28 bp to +34 bp downstream of the initiator (Fig. 1). These data suggest that sqr promoter lacks the TATA element, and DPE as a cis-acting element of sqr promoter mediates its basal transcription. Moreover, the proximal region also contained several potential transcription factor binding sites (Fig. 1), three HSF1 binding sites (HSEs) which lied at −222 to −209 bp, −155 to −143 bp and +47 to +58 bp, respectively, were concerned specially, suggesting HSF1 protein may play a potential role in the sqr transcriptional regulation. 4.2. HSF1 is the transcription factor of U. unicinctus sqr gene HSF1 widely exists in plants and animals, and plays a key role as transcription factor in regulating transcription of many genes, such as aip-1 (Hsu et al., 2003), Lif (Takaki et al., 2006), HYPK (Sakurai et al., 2013; Das and Bhattacharyya, 2014), hsp (Sasi et al., 2014; Yan et al., 2014), Bis (Yoo and Wendel, 2014), Cactus (Yan et al., 2014), and Dystrophin 71 (Tan et al., 2015), which involve stress response, cell differentiation and development, cancer, apoptosis, etc. In the present study, we focused on transcription factor HSF1 based on the fact that three HSF1 binding sites were predicted in the proximal region of U. unicinctus sqr promoter (Fig. 1) to reveal its role in transcription regulation of sqr . In vivo ChIP assay determined that the U. unicinctus HSF1 interacted with the proximal region (−160 bp to −49 bp) of the sqr promoter (Fig. 3a), and only the second HSE (sites−155 bp to −143 bp) in the proximal promoter was bound with the UuHSF1 by in vitro EMSA assays (Fig. 3b) and reporter assays. In addition, the promoter activity of sqr proximal region increased after bound by HSF1, with approximate 5.5 folds higher than that without Pcmv-HSF1 transfection (Fig. 4b). Our results suggested that U. unicinctus HSF1 may play a role as a transcription factor for regulating the sqr transcriptional expression. 4.3. HSF1 up-regulates the sqr transcription in U. unicinctus exposed to sulfide Generally, HSF1 in the cytoplasm is an inactive monomer, while it can be transformed into activated trimeric HSF1 which enters nucleus and binds to the HSE element to regulate gene transcription (Adachi et al., 2003). Xue et al. (2012) reported that when mouse colonic cells suffered from heat shock, the content of both trimeric HSF1 and hsp70 mRNA increased significantly at 2 h, indicating heat shock significantly increased the expression of heat shock genes by inducing the monomeric HSF1 into trimeric HSF1. In this study, we determined that content of the trimeric UuHSF1 and the HSF1-HSE complex increased gradually with the sulfide exposure (Fig. 6). The change trend is similar between the trimeric HSF1 and the HSF1-HSE complex, i.e. both contents increased slightly in the hindgut of U. unicinctus at 2 h post sulfide exposure, and then they increased significantly after the worm was exposed to sulfide for 6 h, and maintained at a high level after that, indicating the increased trimeric UnHSF1 binds to HSE element. Furthermore, qRT-PCR results revealed that mRNA level of the U. unicinctus sqr in the hindgut had no significant difference between the sulfide group and the control group within 2 h after sulfide exposure, while it increased significantly after sulfide exposure for 6 h, and reached a peak at 48 h (Fig. 7, Ma et al., 2012a). Comparatively, the content increases of both HSF1-HSE complex and trimeric HSF1 were

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Fig. 5. Expression of HSF1 detected by Western blotting in the hindgut of U. unicinctus exposed to sulfide. (a) Western blotting analysis for the expression of HSF1 in sulfide (150 ␮M) treated and untreated groups. Expression of ␤-Actin was taken as loading control; (b) the relative expression level of HSF1 is normalized to ␤-Actin according to the result of (a). Data are indicated as mean ± SEM (n = 3). Statistical significance is calculated using Student’s t-test. * indicates p < 0.05 and ** indicates p < 0.01 compared with the control at the same time.

Fig. 6. Sulfide increases content of trimeric HSF1 and HSF1-HSE complex. (a) Native PAGE. Activated trimeric HSF1 increases with extension of 150 ␮M sulfide exposure time. (b) ChIP assays. Content of the HSF1-HSE complex examined by amplifying the precipitated fragments with primers ChIP-F2/R2 is obviously increased in the hindgut of U. unicinctus after sulfide exposure for 6 h. Input is used as a standard. Both images are representative of 3 independent experiments.

Fig. 7. Relative sqr mRNA expression in hindgut of U. unicinctus exposed to 150 ␮M sulfide. Values are shown as mean ± SEM (n = 3); the expression level of sqr mRNA at 0 h after sulfide exposure was set as 1.00 to calibrate the relative level at different time of sulfide treatment. * represents statistically significant difference with p < 0.05 and ** as p < 0.01 between sulfide exposure group and control at the same time.

concordant with that of sqr mRNA after sulfide exposure, which increased significantly at 6 h post-exposure and reached the maximum at 48 h. Therefore, we suggest that HSF1 should be a pivotal transcription factor to up-regulate sqr transcription in U. unicinctus response to sulfide stimuli. Traditionally, it is widely believed that expression of HSF1 protein is non-inducible, and the HSF1-mediated up-regulation of the stress-related genes expression occurs primarily via posttranslational mechanism (Wu, 1995). However, new evidence has suggested that HSF1 expression can be induced (Metzler et al., 2003; Sundar et al., 2005; Yang et al., 2008; Zhou et al., 2008; Sonna et al., 2010; Xue et al., 2012). For example, Yang et al. (2008) demonstrated that riluzole, a drug for the treatment of amyotrophic lateral

sclerosis, increases HSF1 content in Hela cells, and this increased HSF1 content leads to a more robust heat shock response during stress. Similar results have also been identified under hyperthermia (Ding et al., 1996; Sonna et al., 2010), laser therapy (Zhou et al., 2008), and hemorrhagic shock (Sundar et al., 2005), suggesting the regulation of HSF1 may occur at pre-translational level. In this study, a significant (p < 0.05) increase of HSF1 protein was detected firstly in the hindgut of U. unicinctus exposed to sulfide for 2 h (Fig. 5), and the increase reached the maximum at 24 h and maintained until 72 h. The initial time of significant increase for HSF1 protein is slightly earlier than that for sqr mRNA, indicating a close relationship between HSF1 protein increase and sqr transcription. Furthermore, most of the expressed HSF1s were activated and

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used for up-regulating sqr transcription (Fig. 6). All the evidences suggested sulfide increases sqr transcription by increasing not only HSF1 activity but also its protein expression level, and the increased monomeric UuHSF1 is transformed immediately into trimeric HSF1 which binds to HSE of U. unicinctus sqr promoter. 5. Conclusion We predicted that U. unicinctus sqr promoter lacks the typical TATA element but owns a DPE element instead. UuHSF1 can bind to an HSE element in U. unicinctus sqr proximal promoter at −155 bp to −143 bp, and up-regulate the transcription expression of sqr as a transcription factor in response to environmental sulfide. Conflict of interest The authors have declared that they have no potential conflicts of interest. Acknowledgement This work was supported by Natural Science Foundation of China (31072191 and 31372506). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aquatox.2015.11. 021. References Abe, K., Kimura, H., 1996. The possible role of hydrogen sulfide as an endogenous neuromodulator. J. Neurosci. Nurs. 16, 1066–1071. Adachi, H., Katsuno, M., Minamiyama, M., Sang, C., Pagoulatos, G., Angelidis, C., Kusakabe, M., Yoshiki, A., Kobayashi, Y., Doyu, M., 2003. Heat shock protein 70 chaperone overexpression ameliorates phenotypes of the spinal and bulbar muscular atrophy transgenic mouse model by reducing nuclear-localized mutant androgen receptor protein. J. Neurosci. Nurs. 23, 2203–2211. Åkerfelt, M., Trouillet, D., Mezger, V., Sistonen, L., 2007. Heat shock factors at a crossroad between stress and development. Ann. N.Y. Acad. Sci. 1113, 15–27. Attene-Ramos, M., Wagner, E., Gaskins, H., Plewa, M., 2007. Hydrogen sulfide induces direct radical-associated DNA damage. Mol. Cancer Res. 5, 455–459. Bagarinao, T., 1992. Sulfide as an environmental factor and toxicant: tolerance and adaptations in aquatic organisms. Aquat. Toxicol. 24, 21–62. Baird, N., Turnbull, D., Johnson, E., 2006. Induction of the heat shock pathway during hypoxia requires regulation of heat shock factor by hypoxia-inducible factor-1. J. Biol. Chem. 281, 38675–38681. Baskar, R., Li, L., Moore, P., 2007. Hydrogen sulfide-induces DNA damage and changes in apoptotic gene expression in human lung fibroblast cells. FASEB J. 21, 247–255. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72 (1), 248–254. Bruce, J., Price, B., Coleman, C., Calderwood, S., 1993. Oxidative injury rapidly activates the heat shock transcription factor but fails to increase levels of heat shock proteins. Cancer Res. 53, 12–15. Burke, T., Kadonaga, J., 1997. The downstream core promoter element, DPE, is conserved from Drosophila to humans and is recognized by TAF(II) 60 of Drosophila. Gene Dev. 11, 3020–3031. Carrico, R.J., Blumberg, W.E., Peisach, J., 1978. The reversible binding of oxygen to sulfhemoglobin. J. Biol. Chem. 253, 7212–7217. Chan, L., Morgan-Kiss, R., Hanson, T., 2009. Functional analysis of three sulfide: quinone oxidoreductase homologs in Chlorobaculum tepidum. J. Bacteriol. 191, 1026–1034. Christians, E., 2011. HSF1 knock-out. J. Biol. Chem. 286, le26. Cline, J., 1969. Spectrophotometric determination of hydrogen sulfide in natural waters. Limnol. Oceanogr. 14, 454–458. Cooper, C., Brown, G., 2008. The inhibition of mitochondrial cytochrome oxidase by the gases carbon monoxide, nitric oxide, hydrogen cyanide and hydrogen sulfide: chemical mechanism and physiological significance. J. Bioenerg. Biomembr. 40, 533–539. Dai, C., Whitesell, L., Rogers, A., Lindquist, S., 2007. Heat shock factor 1 is a powerful multifaceted modifier of carcinogenesis. Cell 130, 1005–1018. Damberger, F., Pelton, J., Harrison, C., Nelson, H., Wemmer, D., 1994. Solution structure of the DNA-binding domain of the heat shock transcription factor

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