BOR Papers in Press. Published on April 30, 2014 as DOI:10.1095/biolreprod.113.117200 1

Title: The Shrimp Heat Shock Cognate 70 Functions as a Negative Regulator in Vitellogenin Gene Expression1 Running title: Heat Shock Cognate, shrimp vitellogenesis Summary sentence: The study has provided evidence for the role of Heat shock cognate 70 and heat shock factor in the negative regulation of shrimp vitellogenesis Keywords: Vitellogenin, Heat shock Cognate 70, Molecular chaperons Siuming Francis Chan,2,4 Jian-Guo He,5 Ka Hou Chu,6 and Cheng Bo Sun3,4 4 Fisheries College, Guangdong Ocean University, Zhanjiang, PR China 5 School of Life Sciences, Zhongshan University, Guangzhou, PR China 6 Simon F.S. Li Marine Science Laboratory, School of Life Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, PR China 1

S.F.C is supported by a start-up fund from Guangdong Ocean University, Zhanjiang, PR China. Correspondence: Siuming Francis Chan, Fisheries College, Guangdong Ocean University, 40 E, Jie Fang Road, Zhanjiang, 524025, PR China. E-mail: [email protected] 3 Correspondence: Cheng Bo Sun, Fisheries College, Guangdong Ocean University, 40 E, Jie Fang Road, Zhanjiang, 524025, PR China. E-mail: [email protected] 2

ABSTRACT Within the 2.6 kb 5’ flanking region of the shrimp (Metapenaeus ensis) vitellogenin gene (MeVg2), several clusters of putative heat shock factor (HSF) response elements were identified. Deletion of these response elements has caused significant increases in MeVg2 promoter activity suggesting the HSF and Hsc70 complex may regulate vitellogenin gene expression in a negative manner. To confirm the role of Hsc70 in the regulation of vitellogenin gene expression, the ovary cDNA for Hsc70 was cloned and characterized. Hsc70 transcript level was high in the ovary and hepatopancreas of female at early vitellogenic stage but dropped during ovarian maturation. In addition, Western blot analysis revealed the presence of Hsc70 in the nuclear but not in cytoplasmic fraction during early stage of ovary maturation. Electrophoretic mobility shift assay (EMSA) result showed that ovary nuclear extract contained a factor that bind to the HSF response element. Since addition of ATP caused a decrease in binding of Hsc70, Hsc70 may form a repressor complex with HSF to inhibit MeVg2 expression. An in vitro RNA interference technique was used to study the gene function of hsc70. Hsc70 gene knock-down resulted in increased MeVg2 mRNA level in the ovary (54%) and hepatopancreas (62%). In summary, this report describes the first study of vitellogenin gene regulation at transcription level in crustaceans and provides strong evidence that Hsc70 acts as a molecular chaperon to negatively regulate MeVg2 gene expression in shrimp. INTRODUCTION Shrimp aquaculture has become an important contributor in the supply of high quality protein for human consumption. One of the constraints in shrimp aquaculture is insufficient gravid females to produce large quantity of shrimp fry. Consequently, it is important to acquire more knowledge on the regulation of shrimp reproductive maturation before more promising

Copyright 2014 by The Society for the Study of Reproduction.

2

aquaculture techniques can be developed [1]. Like other oviparous animals, gonad maturation in shrimp is characterized by rapid production of egg yolk protein in the process called vitellogenesis [2]. In the study of vitellogenesis in the shrimp Metapenaeus ensis, we have cloned and characterized two cDNA encoding for two vitellogenin, MeVg1, MeVg2 [3]. We have also cloned the 2.6 kb 5’ flanking region of the MeVg2 gene. Inspection of this region revealed the presence of several potential heat shock transcription factor binding elements (HSE). The findings suggest that the heat shock transcription factor (HSF), together with the heat shock cognate 70 (Hsc70), might regulate cellular processes during reproductive event, such as the ovarian maturation in shrimp. Because of the importance as a molecular chaperone in the cell, members of the heat shock protein 70 (Hsp70) family, including the constitutively expressed heat shock cognate 70 (Hsc70) and the inducible heat shock protein 70 (Hsp70) are extensively studied in many organisms [4–6]. In decapod crustaceans, several studies of Hsc70/Hsp70 genes have been initiated [7–10]. Hsc70 cDNA had been cloned and sequenced in many shrimp including the tiger shrimp Penaeus monodon, and evidence had been accumulated that the Hsc70 might function as a chaperone [11]. In the giant freshwater prawn Macrobrachium rosenbergii, differential expression of Hsc70 and Hsp70 were reported when they were in heat-stress conditions [12]. Additionally, Hsc70 and Hsp70 were molt cycle-dependent molecular chaperones in the lobster Homarus americanus [12]. The interaction of HSF and Hsc70/Hsp70 in gene regulation had been demonstrated in many organisms. This complex appeared to be a potential transcriptional regulation complex bound on the promoter elements and regulated the transcription of several genes [13, 14]. In an earlier study, we have cloned the partial cDNA and reported the potential role of hsp70 as vitellogenin repressor before the onset of vitellogenesis in M. ensis [15]. Further study reveal that translated protein of the cDNA consist of feature more closer belongs to the Heat shock cognate (see result and discussion below) It was reported that HSF monomer would compete with nascent peptide for the interaction with molecular chaperone Hsc70/Hsp70 [13, 14]. To elucidate the function of Hsc70, the molecular characterization of the M. ensis Hsc70 gene was first performed. In this paper, focus would be put on the molecular cloning of M. ensis Hsc70 gene. Hsc70 genomic sequence and cDNA ORF would be cloned, sequenced and compared with Hsc70 of other species. Deduced amino acid sequence and the peptide domains would also be compared with other species to confirm the identity of Hsc70. The expression of Hsc70 in different tissues and reproductive stages of shrimp would be performed by Northern blot analysis. Western blot analysis was also used to identify Hsc70 protein in cytoplasmic protein or nuclear extract from different tissues. To determine if HSF-Hsc70 complex are involved in MeVg2 gene regulation, one of the approaches is to show the involvement of Hsc70 in a DNA-protein complex using oligonucleotides containing the potential HSF binding sites (HSE) designed according to the MeVg2 gene promoter. This is achieved by electrophoretic mobility shift assay (EMSA). To further demonstrate the negative regulation of Hsc70 on MeVg2 gene expression, an RNA interference (RNAi) technique was developed to knock down Hsc70 gene expression and to study its effect on MeVg2 expression. MATERIALS AND METHODS Identification of the putative heat shock factor binding sites on MeVg2 gene The 5’ promoter upstream region of the MeVg2 gene was cloned from a genomic DNA

3

library screening with MeVg2 gene specific promoter as a promoter [3]. For the production of promoter deletion clones, the 5’ flanking region of MeVg2 gene was excised by restriction digestion with KpnI and BamHI and subcloned into the pGL3-Basic vector (Invitrogen, USA). After ligation and subcloning, potential positive clones were selected for DNA sequence determination to confirm the DNA insert is ligated correctly in frame and in correct orientation. DNA plasmid preparation was performed to obtain enough starting materials for the generation of promoter deletion clones by a deletion kit (Pharmacia, USA). Appropriate deletion clones were selected for use in the transfection assay (see Supplemental Fig S1; Supplemental Data are available online at www.biolreprod.org). Before transfection, cells were pelleted by centrifugation at 300 g for 5 min. and resuspended in ExCell420 serum free medium at 2 x 106 cell/ml. For transfection, the IPLB-LdFB cell derived from Lymantria dispar 5th larval instar fat body cell was used. Transfection was performed with Tfx-20 reagent (Promega, USA). Deleted plasmid constructs (4 g) was mixed with internal control plasmid pRL-SV40 (50:1) in 0.5 ml of Tfx-20 reagent and with the DNA mixture at room temperature for 15 min (i.e. final ratio of Tfx-20 to DNA is 4:1). The above mixture was added to 0.5 ml of cell in each well of the 6-well plate (Costar, USA) at 37ºC for 48 hours. The luciferase activities were analyzed with the Dual-luciferase Reporter Assay kit (Promega, USA) and measured by a Luminometer (EG&G Berthold Lumat LB9507). Cells were rinsed twice with PBS and lysed for 15 min with 100 l of Passive lysis buffer. The lysate was centrifuged and 20 l of supernatant was transferred into a 1.5 ml Eppendorf tube for detection of luciferase activity. Equal volume (100 l) firefly luciferase assay reagent and stop & Glo substrate solvent were added into the Eppendorf tube and the activity was normalized from the ratio of firefly luciferase activity to Renilla luciferase activity related to the negative control (pGL-basic) Cloning of MeHsc70 cDNA Total RNA (3 μg) was used in the reverse transcription (RT) reaction to generate first strand complementary DNA (cDNA) according to a modified MMLV Reverse Transcriptase Tested User Friendly First Strand cDNA Synthesis Protocol. The RT reaction was performed in a final volume of 50 μl containing 1 First strand buffer, 0.2 mM dNTP mix (Amersham), 1.25 μg oligo (dT)12 , 0.5 μl RNase-inhibitor (31750 U/ml, Amersham) and 0.5 μl MMLV (200 U/μl, USB). The RT was performed at 37oC for 3 hr and finally 70oC for 15 min. Gene-specific primers (based on other shrimp Hsp70 including Penaeus monodon and Litopenaeus vannamei) HSP-F1 (10 μM) – 5’ CGA ATT CAT GAG TAA GGC ATC AGC AG 3’; HSP-R1 (10 μM) – 5’ GAA GCT TTA GTC GAC CTC CTC AAT GG 3’) were designed for PCR amplification using RT reaction product as template. A PCR reaction was performed in a final volume of 20 μl containing 1/10 RT reaction product, 1 reaction buffer, 1.5 mM MgCl2, 0.2 mM dNTP mix (Amersham), 0.5 μM each of a pair of forward and reverse primers, and 0.1 μl Taq DNA polymerase (5 U/μl). 10 μl mineral oil was added on top of each PCR reaction mixture. PCR amplification was performed according to the thermal cycle: 95oC for 3 min. 35 cycles of [95oC for 1 min; 58oC for 1 min; 72oC for 2 min]. 72oC for 10 min. PCR products were analyzed by performing 1% agarose gel electrophoresis. Experimental animals and RNA preparation and Northern blot analysis Shrimp purchased from local seafood markets were acclimated in the laboratory at 26oC. Total RNA was prepared by a modified guanidine isothiocyanate extraction method [16]. Reverse

4

transcription polymerase chain reaction (RT-PCR), was performed in a final volume of 50 μl containing 1 First strand buffer, 0.2 mM dNTP mix (Amersham, USA), 1.25 μg oligo (dT)12 , 0.5 μl RNase-inhibitor (31750 U/ml, Amersham), 0.5 μl MMLV (200 U/μl, USB), total RNA templates (3 g) at 37oC for 3 hr and then at 70oC for 15 min. For RT-PCR, the mix consists of 1 reaction buffer, 1.5 mM MgCl2, 0.2 mM dNTP mix (Amersham), 0.5 μM each of forward and reverse primers, and 0.1 μl Taq DNA polymerase (5 U/μl) PCR reaction mixture, 1 l of the RT mix was added as template. For Northern blot, RNA were analyzed on a 1% formaldehyde agarose gel and transferred onto the Nylon membrane by capillary action. The membrane was hybridized in a pre-warmed High-SDS hybridization buffer with the Hsc70 probe at 50oC for 16-20 hr. All the washing and detection steps were performed at room temperature except the 0.1 SSC with 0.1% SDS stringency washes. The membrane was transferred to 2 SSC with 0.1% SDS and washed twice for 15 min. 0.1 SSC with 0.1% SDS was used to wash the membrane twice for 15 min at 58oC. The membrane was then incubated in 1:20,000 Anti-DIG-AP conjugate in 1 blocking buffer. For signal detection, 200 μl CDP-Star (Roche) was evenly added on the membrane which was then sealed by plastic wrap. The luminescent signal was exposed to an X-ray film for 5 min to 1 hr depending on signal intensities, and the film was developed by a Kodak film processor. Tissue protein and nuclear extract preparation Tissues were dissected and homogenized in an extraction buffer, centrifuged at 3,000 g for 5 min at 4oC. The supernatant was then transferred to a fresh tube and centrifuged again at 17,000 g for 40 min at 4oC to remove subcellular debrides. For nuclear protein extraction, 0.1 g tissue was added to 1 ml 1 hypotonic buffer with 1 mM dithiothreitol (DTT) and 1.5 μl Detergent (Active Motif) was used to grind the tissue in a Dounce homogenizer (Wheaton) with Pestle B. The homogenate was incubated on ice for 15 min and then centrifuged at 850 g for 10 min at 4oC. The pellet was then resuspended in 1 ml 1 hypotonic buffer and incubated on ice for 15 min. About 50 μl Detergent (Active Motif) was added to the resuspension and vortex. After centrifugation at 14,000 g for 30 sec at 4oC, the pellet was resuspended in 100 μl lysis buffer (Active Motif) and vortex was again 10 sec. The resuspension was incubated on ice for 30 min with gentle shaking at 150 rpm. Vigorous vortex was then applied to the resuspension for 30 sec and then centrifuged at 14,000 g for 10 min at 4oC. The supernatant, which was the nuclear protein extract, was collected and divided into aliquot of 10 μl for storage in -80oC. For MeHsc70 production, enzyme-restricted PCR fragment containing the open reading frame (ORF) of the Hsc70 was inserted into the expression vector pRSET-B (Invitrogen) previously digested with the same Eco RI and Hind III cut vector in a ligation reaction overnight at 16oC. The ligation product was then transformed into E. coli XL1-Blue cells by electroporation. The potential positive subclones were purified for DNA sequencing determination to confirm correct orientation and in frame insertion of the construct. The overnight culture was then diluted (1:100) in LB broth and inoculated at 37oC for about 3 hr at 250 rpm until the OD600 0.4-0.6. Then a final concentration of 1 mM of IPTG (isopropylthio-β-D- galactoside) was added to the culture for a further inoculation for 3 hr at 37oC or 8 hr at 30oC with vigorous shaking (250 rpm). At one-hour interval, 1 ml samples of culture were collected, they were centrifuged at 5,000 rpm for 5 min at 4oC and 100 μl 1 PBS was used to resuspend the bacterial pellet. The recombinant rHsc70 that appeared in the inclusion body was purified with Ni-nitrilotriacetic acid (NTA) agarose (Qiagen, Germany) under denaturing conditions (8 M urea). For refolding, purified rHsc70 protein was

5

serially diluted in cold dialysis buffer (10 mM Tris–HCl, 140 mM NaCl, 20 mM CaCl2, 0.05% Tween-20, pH = 7.9) and incubated for 2 h at 4 °C for each dilution step. The rHsc70 was dialyzed against a dialysis buffer with gentle stirring at 4 °C to remove all urea. Precipitated protein was removed by centrifugation at 14,000 rpm for 10 min at 4 °C. The suspension was then frozen in liquid nitrogen and thawed at 42oC. After 2 cycles of freeze-thaw, the lysate was centrifuged at 17,000 g for 10 min at 4oC. 2 SDS sample buffer was added to the supernatant (soluble fraction) while 1 SDS sample buffer was used to dissolve the pellet (insoluble fraction). After denaturation at 95oC for 5 min, denatured proteins were analyzed by SDS-PAGE and Western blot using a monoclonal anti-hsp/hsc antibody (1: 2000). The second antibody used was an alkaline phosphtase conjugated rabbit against the mouse Igg fraction (dilution 1:3000).. Detection of the Hsc70 specific protein was by the nitroblue (NBT) and 5-bromo-4-chloro-3-indoyl phosphate (BCIP) in an alkaline phosphatase (AP) detection buffer. Electrophoretic mobility shift assay (EMSA) The HSF specific oligonucleotide (5’-TCATTTCTGGGAATAAGTT TTTATGGAGAGAAAGGG-3’) was 5’ end labeled with T4 polynucleotide kinase (PNK). In a reaction mixture of 10 μl, 2 μl of oligonucleotides, 1 μl T4 PNK 10 buffer (Promega), 5 μl MilliQ water, 1μl T4 PNK (5–10 U/μl) (Promega) and 1 μl [γ-32P] ATP (3,000 Ci/mmol at 10 mCi/ml) (Amersham) were mixed and incubated at 37oC for 45 min. To remove the unlabeled oligonucleotides, QIAquick Nucleotide Removal Kit (QIAGEN) was used. The activity of the labeled probe was determined by scintillation counting and. 5,000–10,000 cpm per 10 fmole were used in the EMSA. For DNA-protein binding reactions (15 l), the reaction mix consisted of 3–5 μg nuclear extracts, 20 mM HEPES (pH 7.8), 50 mM NaCl, 0.5 mM EDTA, 1 mM MgCl2, 10% (v/v) glycerol, 0.5 mM DTT, 1 μg poly (dI.dC) and 0.1% NP-40. The samples were incubated at room temperature for 10 minutes and 0.2 pmole of 32P-labeled oligonucleotide probe was added for further incubation (20 min) at room temperature. For non-specific competition, the oligo (i.e. M1-SNOG – 5’ CCTCCACCCCTCAACCATGCCAGA 3’) that did not contain the HSE was added in 10 and 50 molar excess of the labeled HSE-oligonucleotide probe. For specific competition, unlabeled HSE-oligonucleotides were added in 50 and 100 molar excess of the labeled probe to the reaction mixture. To demonstrate the possible involvement of hsc70 in the DNA-protein complex, 10 mM and 20 mM ATP were added to the reaction mixture. At the end of incubation, the samples were analyzed on a 6% non-denaturing polyacrylamide gel and electrophoresis at 150V. After electrophoresis, the gel was dried in vacuum and the signal was exposed to a film with an intensifying screen at -80oC and then developed after 2–3 days. Double strand RNA (dsRNA) interference For the synthesis of dsRNA, the Megascript dsRNA synthesis kit (Ambion, USA) was used. All procedures follow the instructions of the manual and using buffer supplied in the kit. The in vitro transcription reaction at 37ºC overnight, 8 μl (0.5–2 pmoles) T7-linked hsc70 DNA template obtained from the above mentioned PCR amplification, 2 μl each of 10 T7 Reaction Buffer, ATP, CTP, GTP, UTP solution and T7 Enzyme Mix. For DNA removal, 21 μl nuclease-free water, 5 μl 10 digestion buffer, 2 μl DNase I and 2 μl RNase were added to the reaction mix and the digestion reaction was performed at 37oC for 1.5 hr. After nuclease digestion, 50 μl 10 binding buffer, 150 μl nuclease-free water and 250 μl 100% ethanol were added to the mix. The 500 μl reaction mixture was then added to a filter cartridge and then centrifuged at 12,000 g for 2 min to discard the flow through. Wash Solution (500 μl) was added onto the filter and then centrifuged at

6

12,000 g for 2 min. For in vitro dsRNA interference, hepatopancreas and ovary explants were incubated in 1.5 ml M199 culture medium in a 24-well plate with gentle shaking for 2, 4, 6, 8, 10 and 12 hr at room temperature. Serial dilutions of Hsc70 specific dsRNA (in 10 l) were added to the wells of the tissues explant in the culture medium and the incubation was continued for an additional 5 hours. For the controls, 0.3 μg/ml CHH dsRNA (non specific dsRNA) was also added to another explant in the well. RNA was extracted for Northern blot analysis to monitor the change of MeVg2 expression level and also the knockdown of Hsc70 gene expression level. RESULTS To study the promoter region and the factor that may regulate the MeVg2 gene, a genomic DNA clone was isolated from the library screening using a MeVg2 probe derived from the N-terminal end of the cDNA. This clone was eventually confirmed to carry the 5’-end promoter sequence of the MeVg2 gene (Fig.1). When the 2.6 kb 5’ flanking region of MeVg2 gene was analyzed using the TEES (University of Pennsylvania), various putative protein-DNA binding motifs can be identified. Some general transcription factor binding site such as TATA, GC-rich site can be found within the first 200 nt of the proximal region (Figs. 1 and 2a). Several heat shock factor (HSF) binding sites located at -758 to -665, -1349 to 1143 and 2106-1881 were identified (Figs. 1 and 2a). Since there are reports for the formation of a complex by the heat shock factor and heat shock protein that together form the regulatory complex of many genes, we use the deletion promoter assay to further demonstrate its importance in Vg gene regulation (Fig. 2b). When the distal 995 bp fragment which contained the four putative HSP binding sites were deleted, the promoter activity increased 21-folds as compared to the Vg2661-LUC. A further 302 nt deletion caused a decrease of 38% in reporter activity, (Fig. 2b). For the Vg716-Luc deletion clone, two HSF binding sites were found in the deleted region. Next, the deletion of two HSF binding sites produced an even greater increase in promoter activity in the Vg205-Luc deletion clone, being 36% of the positive control. To summarize, the result of the deletion promoter assay demonstrated that the heat shock factor (HSF) are involved in the regulation of the vitellogenin gene. Since the HSF form complex with the heat shock cognate/or heat shock protein and regulate the vitellogenin gene of the mosquito [13], we performed the following experiments to determine if similar regulatory mechanism also occur in the shrimp. Cloning, characterization and expression study of MeHsc70 DNA sequence was obtained and submitted for amino acid homology search in Genbank database at NCBI using BLASTX program (Genbank Accession no. KJ511266). The sequence exhibited a very high homology to hsc70 of other decapod crustaceans. It shared 98% identity to the heat shock protein 70 (Hsp70) of the pacific white shrimp, L. vannamei, 91% to the heat shock cognate 70 (Hsc70) of the black tiger shrimp, P. monodon and 87% to the Hsp70 of the giant freshwater prawn, M. rosenbergii. Based on the homology of MeHsc70 sequences to that of other species, similar approach was used to clone the ORF of the Hsc70 from the hepatopancreas cDNA. The deduced amino acid sequence encoded a 648-amino acid protein with an estimated 71 kDa and pI=5.34 (Fig. 3a). By the Prosite analysis program, three Hsp70 family motif signatures on the deduced amino acid sequence were identified. These include (i) IDLGTTYS (aa 9–16), IFDLGGGTFDVSIL (aa 197–210); (ii) IVLVGGSTRIPKIQK (aa 334–348); (iii) a putative ATP-GTP binding motif with AEAYLGAT (aa 131–138). Two bipartite nuclear localization sequences (KRKYKKDPSENKRSLRR located at aa 246–262) and KRSLRRLRTACERAKRT located at aa 257–273) were located on the deduced amino acid

7

sequence: Two other non-organellar eukaryotic consensus motif (i.e. RARFEEL at aa 299–305) and the cytoplasmic Hsp70 carboxyl terminal region: GPTIEEVD at aa 641–648 (Fig. 3a). When MeHsc70 was aligned with Hsc70 from P. monodon, L. vannamei and M. rosenbergii, a high degree of homology among the sequences was observed. Phylogenetic tree has placed the four shrimp Hsc70/Hsp70 sequences into one branch, well separated from a cluster populated by the fly and hornworm hsc70s and the barnacle hsp70. The zebrafish, chicken and human Hsc70/Hsp70 sequences were clustered into a diverse group (Fig. 3b). In a genomic DNA PCR analysis, a 2.6 kb PCR product was amplified and subcloned into pGEM-T vector and transformed into XLI-Blue cells. DNA sequencing determination confirmed that the Hsc70 ORF and genomic DNA are identical. The result suggested that MeHsc70 gene was intronless (Supplemental Figure S2). A transcript of 2.4 kb representing the MeHsc70 can be detected in many tissues (Fig. 4a). In order of decreasing transcript level, these tissues include the ovary, epidermis, hepatopancreas, nerve cord and eyestalk. Although Mehsc70 mRNA was expressed constitutively in most tissues, a differential expression pattern was also observed. MeHsc70 transcript level was much higher in the ovary as compared to (Fig. 4) the epidermis, hepatopancreas, eyestalk, ventral nerve cord and the muscle. MeHsc70 transcript was highly enriched in stage I ovary and hepatopancreas and then dropped significantly in more the mature stages (stage II–V) (Fig. 4b). The protein lysate from soluble fraction of the Hsc70 expression protein were separated by SDS-PAGE and stained with Coomassie blue to confirm the presence of the recombinant protein of expected size (75.4 kDa) (Fig. 5a). Monoclonal anti-Hsp70 antibody was used in a Western blot analysis to confirm the identity of the protein (Fig. 5b). Using the expression system described in this report, the maximum expression level of the recombinant protein occurred at 8 hr after ITPG stimulation (Fig. 5a). As revealed by Coomassie blue staining (Fig. 5b), nuclear extract could be prepared from most tissues. However nuclear proteins from hepatopancreas appeared as a smear, indicating that the hepatopancreas nuclear protein extract was degraded. Monoclonal anti-heat shock protein 70 antibody was used in a Western blot analysis to detect for the native Hsc70 in the protein samples. In addition to the rHsc70/BL21 positive control, positive signals were detected in the ovary nuclear extract sample (Fig. 5b). Weaker signals were also observed in the epidermis and ventral nerve cord. Although native hsc70 was detected in ovary nuclear extract, no signal was observed in the ovary cytoplasmic protein. Due to protein degradation, no positive signal was obtained in the hepatopancreas sample. For EMSA, oligonucleotides (-760 to -726 on the MeVg2 promoter) harboring these HSEs were designed and used as a probe in the EMSAs. Addition of both 3 μg and 5 μg ovary nuclear extract into the in vitro DNA-protein binding reaction can cause the retardation of the complex on the mobility gel shift assay. This indicated that the ovary nuclear extract contained a factor that bind to the radiolabeled probe. A non-specific band was observed migrating approximately halfway through the gel (Fig. 6a). Unincorporated oligonucleotide was appeared as a dark smear (Fig.6a). When the recombinant Hsc70 was added in the mix, a slightly different size retardation complex was obtained (broken arrows). We reasoned that the size variation in the retardation complex could be due to the difference in sizes of the endogenous Hsc70 and the rHsc70 (Fig. 6a). In the next experiment, addition of 10 and 100 excess of non-specific competitor (i.e. M1-SNOG) could not compete away the retardation complex. However, when the heat shock response element (HSE) was added to 100 folds, no retardation signal was recorded (Fig. 6b). To extend further, when adenosine triphosphate (ATP) was added (1 M and 2 M) in the binding

8

reaction, the retarded band was diminished (Fig. 6b). The result suggested that Hsc70 might be present as one of the partner in the HSE binding complex because dissociation from its peptide substrates by ATP could lead to a decrease in HSE binding affinity of the protein factor(s) in the ovary nuclear extract and thus causing a diminish in the retardation complex (Fig. 6c ). RNA interference of Hsc70 In this study, ovary and hepatopancreas from shrimp at early maturation cycle were used. An initial time course experiment was performed to determine the optimal culture time for the organ explants. The result indicated that a 3-4 hour culture time is best for both tissues and therefore all the organ explants were cultured for 4 hours (Supplemental Fig. S3). Northern blot results indicate that the dose of 0.3 g/ml dsRNA was more effective than 3 g/ml dsRNA (data not shown). Therefore the 0.3 g/ml dsRNA was used as working dose for the subsequent experiments. The Hsc70 gene knock-down effect in the ovary accounts for 52% as compared to the TE treated control (Fig. 7a). Addition of 0.3 g hsc70 dsRNA in the ovary explant led to the largest increase in MeVg2 gene expression. The specificity of the Hsc70 dsRNA was confirmed as the dsRNA for the crustacean hyperglycemic hormone has little or no effect on Hsc70 and MeVg2 gene expression (Fig. 7a). To ascertain the consistent results, the experiments have been repeated three times in which the 0.3 g/ml dsRNA was used in the RNAi experiment. The increase in MeVg2 expression level amounted to 54% as compared to the control. Similarly, in the hepatopancreas, the most effective dsRNA dose was 0.3 g/ml as it was demonstrated in the ovary culture experiment. The knock-down effect of dsRNA amounted to 60% for the Hsc70 and the increase of MeVg2 was about 62% (Fig. 7b). DISCUSSION Vitellogenesis is an importance process for the successful production of eggs and healthy larval development. Currently, only a few recently studies describe the regulation of this gene shrimp. Most of the studies report the endocrine regulation of vitellogenesis by eyestalk vitellogenesis (or gonad) inhibiting hormone (VIH) [17, 18, 19, 20, 21, 22]. For example, in M. ensis, using recombinant protein and RNA interference approach, an eyestalk neuropeptide previously named as MIH-B had shown to have gonad stimulating activity [17]; in Marsupenaeus japonicus, only bilaterial eyestalk ablation is effective to cause upregulation of vitellogenin; in Litopenaeus vannamei [19], several sinus gland neuropeptides of the CHH family were purified and most of these neuropeptides have shown to have vitellogenin inhibiting effect [20, 21]. In P. monodon, RNA interference of the GIH has resulted in vitellogenin gene regulation [22]. In other oviparous animals, information on the structural feature of Vg gene regulation is only limited to several insect such as the Apis mellifera [23], mosquitoes Acedes aegypti [24], and the fly Drosophila [25], the worm Caenorhabditis elegan [26] and the higher vertebrate such as fish Salmo gairdneri [27] and Xenopus [28]. This report describes the first study of Vg gene regulation at transcription level in crustaceans. Because the lack of a suitable crustacean cell line, the use of insect cell described in this study can only provide indirect evidence for the involvement of the factor in vitellogenin regulation. In Xenopus, Wollfe et al. [29] demonstrate that an elevated concentration of heat shock protein caused by thermal stress abolished the estrogen activated transcription and accumulation of vitellogenin mRNA accumulated by prior treatment with estrogen. Molecular cloning of MeHsc70 Although MeHsc70 exhibited a high sequence homology with the Hsp70 of other decapod

9

crustaceans, it was difficult to determine if it was Hsc70 or hsp70. Heat shock protein 70 (Hsp70) family was widely studied in many organisms. In addition to crustaceans, MeHsc70 also showed higher sequence homology with the hsc70 of the honey bee Apis mellifera (83%), the rainbow trout Oncorhynchus mykiss (83%), the silver crucian carp Carassius auratus gibelio (83%) and the fruit fly Drosophila melanogaster (79%). Therefore, it was proposed that the sequence obtained in M. ensis was Hsc70. The ovary is a fast developing organ in which the oocytes undergo rapid proliferation and DNA replication during reproductive maturation. Hsc70 expression level was particularly high in growing cells than in resting cells. Therefore, Hsc70 may be required in cellular processes such as transcription control or protein translocation in shrimp. The interaction between Hsc70 and the heat shock transcription (HSF) had been demonstrated in vitro in human, Drosophila and Arabidopsis [30, 31]. Therefore, the absence of intron for the MeHsc70 gene may facilitate rapid synthesis and accumulation of the protein to exert its function as a molecular chaperone in a series of cellular process in the ovary. In fact, some segments of the eukaryotic Hsc70 sequence, such as ATP/GTP-binding site, cytoplasmic HSP70 C-terminal sequence, and GGMP/GAP repeats, are also found in the putative shrimp Hsc70. The presence of an ATP-GTP binding motif (AEAYLGAT) on the shrimp Hsc70 sequence further confirms that it is a Hsc70 because hsc70 was known to have ATPase activity [32, 33] . The two bipartite nuclear localization signals (KRKYKKDPSENKRSLRR and KRSLRRLRTACERAKRT) consist of an abundance of the basic amino acids lysine (K) and arginine (R) for the selective translocation of Hsc70 into the nucleus [34]. In M. ensis, considerable variations in Hsc70 transcript level were observed in the ovary. Similar enriched pattern of Hsc70 was also found in D. melanogaster [35] and zebrafish D. rerio [36] during embryogenesis. The enrichment of Hsc70 in the ovary might also suggest a critical function played by the protein during the shrimp’s early phase of vitellogenesis. Although Hsc70 mRNA level is the highest in the ovary, the mRNA level in the hepatopancreas was higher than that in the eyestalk, ventral nerve cord and muscle. In M. sexta, Hsc70 was enriched in the fat body [37] suggesting that it might be correlated with vitellogenesis since the fat body is the synthesis site of vitellogenin [38, 39]. Therefore, hsc70 might also play a functional role in the hepatopancreas during the shrimp reproductive cycle. By Western blot analysis, MeHsc70 protein was identified in the nuclear extract prepared from the ovary, epidermis and ventral nerve cord (Fig. 5b). Among the tissues examined, a differential expression pattern was observed in which the Hsc70 protein was highly enriched in the ovary than in other tissues. Thus the result from Western blot is consistent with those from the Northern blot. Taken together, the finding that Hsc70 was localized in the nuclear fraction of the stage I ovary was demonstrated. Hsc70 expression might be negatively correlated with the shrimp reproductive maturation as Hsc70 and vitellogenin (the egg yolk precursor) have a reverse expression pattern. In fact, the result from the present study on the vitellogenin gene promoter provides evidence on the involvement of Hsc70 in the regulation of vitellogenin gene expression. Many reports have demonstrated the modulation of transcriptional activity of HSF by interacting with the Hsc70/Hsp70 protein family member [14]. The study on D. melanogaster provides insight on the role of Hsp70/HSF interaction as a transcriptional regulation complex during oogenesis and larval development. HSF appears to regulate genes involved in growth or developmental processes under normal conditions and the DNA binding affinity of HSF monomer was low unless a HSF homotrimer was formed [15, 40]. However, HSF monomer

10

might bind on certain gene promoters as a heteromeric complex with other protein [14]. Depending on the particular promoter, the HSF could then either stimulate or suppress gene expression. Therefore, Hsc70/Hsp70 may interact with HSF and form a transcriptional complex. Although the antibody was able to recognize the recombinant Hsc70 protein and the MeHsc70 in the ovary nuclear extract in Western blot analysis, it did not cause a supershift of the retardation complex (Fig. 6). This might be attributed to the masking of antibody binding site on the Hsc70 upon interacting with the HSE-binding complex. However, it was overcome by the addition of ATP as ATP disrupted the DNA-protein binding complex by dissociating the Hsp70 from the HSF [41, 42]. The activity of HSF might be enhanced by interacting with the molecular chaperone Hsc/Hsp70, as compared to the HSF monomer itself [43, 44]. Therefore, addition of ATP would dissociate Hsc70/Hsp70 from the HSF, resulting in a decreased DNA binding affinity of monomer HSF, and thus cause the disruption of the DNA-protein binding complex in EMSA. As it is shown in M. ensis that addition of ATP (1 and 2 μM) disrupted the DNA-protein binding complex (Fig. 6c). Although Hsc70 was neither characterized as a DNA binding protein in previous studies nor identified to have any DNA binding domain in the deduced amino acid sequence by conserved domain search, it is logical to say that hsc70 is involved in the HSE-binding complex based on the disruption of DNA-protein binding complex in the ATP-competition EMSA in the shrimp. Regulatory role of Hsc70 on MeVg2 expression in the ovary and hepatopancreas In the regulation of vitellogenesis, we speculate that MeHsc70 interacts with HSF to form a heteromeric complex that represses transcription of the MeVg2 gene in the ovary. As described in many species, there is interaction between Hsc70/Hsp70 with the HSF, and also the conserved transcriptional control of Hsc/Hsp70 gene by the HSF [41, 45]. HSF was an ubiquitous transcription activator of heat shock protein genes in eukaryotes, Hahn et al. [46] used a whole-genome analysis approach to identify the role of the HSF in yeast cells as a transcriptional regulator of genes that encode proteins for signal transduction, carbohydrate metabolism and energy production. Besides, HSF-Hsc70/Hsp70 interaction has been studied extensively by many researchers. Marchler and Wu [30] had reported a modulation of the Drosophila HSF activity by the molecular chaperone Hsc70. The human Hsp70 could interact with the C-terminal transactivation domain of HSF trimers by hydrophobic interaction during attenuation of the heat shock transcriptional response [47]. During heat stresses, heavy metal exposure or starvation, HSF production would be induced. The three monomers of the HSF would form a homotrimer to act as transcriptional activator of gene such as the Hsc70/Hsp70, which was an important protein for cell survival during stress [40, 45]. In non-reproductive seasons, MeHsc70 transcript level in the ovary and hepatopancreas is high. Besides, native Hsc70 was only identified in the ovary nuclear fraction. In mouse NIH-3T3 cells, nuclear localization of Hsc70 could lead to Hsc70/HSF-1 interaction that regulate HSF activity [47]. In fact, HSF was involved in regulating oogenesis and larval development in the Drosophila under normal cell growth conditions [13]. Therefore, elevated levels of Hsc/Hsp70 immature shrimp might be able to recognize and bind to the HSF activation domain and this complex would bind on the HSE-containing MeVg2 gene promoter and subsequently repress the gene transcription. This postulation was further supported by the results from the promoter deletion assay as the lost of HSE led to an increase of the MeVg2 gene promoter activity. Consequently, it is possible that the Hsc70 interacts with HSF to act as a transcriptional repressor to suppress MeVg2 gene expression in the ovary of immature shrimp.

11

To elucidate the role of Hsc70 as a negative regulator of MeVg2 gene expression in both ovary and hepatopancreas, a double strand RNA (dsRNA) mediated RNA interference (RNAi) experiment was carried out to study the effect of Hsc70 gene knockdown on MeVg2 production and this would be discussed in the following sections. The results from ovary and hepatopancreas explant study show that 0.3 μg/ml Hsc70 dsRNA can knockdown the expression of Hsc70 and cause a rise in the MeVg2 expression level by 52% in the ovary and 60% in the hepatopancreas. Although there was a significant reduction in Hsc70 mRNA level, the effect was moderate. This might be attributed to the incomplete penetration by the knockdown or incomplete activation of the RNAi system and thus some cells could escape the effects of RNAi [48]. In addition, the knockdown efficiency, i.e. the percentage of endogenous gene being knocked down after applying RNAi, was higher in the hepatopancreas and that might be attributed to the difference in the Hsc70 expression level in these reproductive organs. Besides, the knockdown efficiency seems to be dependent on the expression level of the target gene as well as the approach of experiment. The decrease in knockdown efficiency of Hsc70 might be due to the constitutive nature and abundant expression level of Hsc70 in the shrimp. As constitutive Hsc70 mRNA expression was demonstrated in various organs of reproductive immature female shrimp, in vivo injection of the dsRNA might lead to systemic silencing. As the physiological role of Hsc70 is highly conserved, its knockdown might cause unpredictable harmful effect on the shrimp. Thus, the in vitro explant RNAi assay system is more appropriate for a short term study. By the knock-down of Hsc70 gene expression to 52% in the ovary, a 54% increase in the MeVg2 gene expression was observed. For the hepatopancreas culture, there was a 62% increase in MeVg2 transcript level for a 60% reduction in Hsc70 expression. These results strongly suggest that there is a suppressing effect of Hsc70 on the MeVg2 expression in the shrimp. During the in vitro organ explant culture, Hsc70 dsRNA in the medium was taken up by the ovary or hepatopancreas explants as the 800 bp can be observed in the Northern blot in the sample treated with 3 μg/ml dsRNA. The smear of signal indicated the cleavage of dsRNA into smaller units by the Dicer enzyme in the organ explant. However, this smear was not observed for the sample treated with 0.3 μg/ml presumably due to complete cleavage of dsRNA. Subsequently, translational repression on Hsc70 mRNA occurs followed by reduction of hsc70 protein synthesis. The reduced level of Hsc70 would cause dissociation of HSF from the transcriptional repressor complex and the HSF would restore to its monomeric form. Given that the DNA binding affinity of the HSF monomer was low and devoid of transcriptional activity [49, 50], the suppressing effect of hsc70-HSF complex on the MeVg2 gene was removed and therefore the MeVg2 expression level increased. Although the mechanism for RNAi in shrimp has yet to be uncovered, recent cloning of gene encoding the Argonaute protein in the tiger shrimp Penaeus monodon might reflect the presence of native RNAi mechanism in the shrimp [51]. Members of this protein family were shown to be involved in siRNA recognition and binding for initiating dsRNA induced gene silencing effect [52, 53]. RNAi was also demonstrated by the researchers that it was a native mechanism to prevent viral replication in the shrimp [54]. Tirasophon et al. [55] had recently shown the silencing of yellow head virus replication in Penaeus monodon cells by dsRNA and this suggested that RNAi-mediated gene silencing was operative in the penaeid shrimp. By RNAi approach, a reduction by 52% and 60% in hsc70 transcript level was observed in ovary and hepatopancreas respectively. Given an approximately 2.7 folds more endogenous Hsc70 transcript in the ovary than in hepatopancreas, the hsc70 transcript level in the ovary after

12

a knockdown by 52% would still be significantly higher than that in the hepatopancreas. However, the increase in MeVg2 transcript level in the ovary could reach 54% which is close to the 62% increase in the hepatopancreas. Therefore, although Hsc70 could serve as a negative regulator of MeVg2 expression in both ovary and hepatopancreas, it might play a more critical role in regulating the MeVg2 expression in the ovary than in hepatopancreas. In short, in vitro knockdown of Hsc70 by dsRNA has caused consistent increase in MeVg2 expression level in both the ovary and hepatopancreas explant experiment. Although previous studies had shown the involvement of Hsc70 in Drosophila oogenesis [13] and salmon vitellogenesis [27] the regulatory mechanism or the target gene under regulation were not reported. However, the present RNAi experiment in the shrimp has demonstrated a novel finding that Hsc70 could act as a negative regulator of the MeVg2 gene transcription in addition to other biological activities. Since two vitellogenin genes had been cloned in M. ensis, it would be interesting to know if MeVg1 gene also consists of similar 5’ upstream promoter region with similar cluster of HSE. In conclusion, the result from this study is important in opening a new research area for transcription control of vitellogenesis and it may provide new insights in developing new techniques to improve gonad maturation in shrimp aquaculture. ACKNOWLEDGEMENTS We thank Mr. Manson Chung for the technical works. REFERENCES 1. FAO. Culture Aquatic species information programme, Penaeus vannamei (Boone, 1931), United Nation pp1-14 2. Kung SY, Chan SM, Hui JH, Tsang WS, Mak A, He JG. Vitellogenesis in the sand shrimp, Metapenaeus ensis: the contribution from the hepatopancreas-specific vitellogenin gene (MeVg2).Biol Reprod. 2004; 71:863-70. 3. Tsang WS, Quackenbush LS, Chow BK, Tiu SH, He JG., Chan, SM. Organization of the shrimp vitellogenin gene: evidence of multiple genes and tissue specific expression by the ovary and hepatopancreas. Gene 2003; 16:99–109. 4. Mosser DD, Duchaine J, Massie B. The DNA-binding activity of the human heat shock transcription factor is regulated in vivo by hsp70. Mol Cell Biol 1993; 13:5427–5438. 5. Demand J, Luders J, Hohfeld J. The carboxy-terminal domain of Hsc70 provides binding sites for a distinct set of chaperone cofactors. Mol Cell Biol 1998; 18:2023–2028. 6. Čvoro A, Korač A, Matić G: Intracellular localization of constitutive and inducible heat shock protein 70 in rat liver after in vivo heat stress. Mol Cell Biochem 2004; 265:27–35. 7. Qian Z, Liu X, Wang L, Wang X, Li Y, Xiang J, Wang P. Gene expression profiles of four heat shock proteins in response to different acute stresses in shrimp, Litopenaeus vannamei. Comp Biochem Physiol C-Toxicol Pharmacol 2012; 156:211–20. 8. Yan F, Xia D, Hu J, Yuan H, Zou T, Zhou Q, Liang L, Qi Y, Xu H. Heat shock cognate protein 70 gene is required for prevention of apoptosis induced by WSSV infection. Arch Virol 2010; 155:1077–1083. 9. Zhou J, Wang WN, He WY, Zheng Y, Wang L, Xin Y, Liu Y, Wang AL. Expression of HSP60 and HSP70 in white shrimp, Litopenaeus vannamei in response to bacterial challenge. J Invertebr Pathol 2010; 103:170–178. 10. Luan W, Li F, Zhang J, Wen R, Li Y, Xiang J.Identification of a novel inducible cytosolic Hsp70 gene in Chinese shrimp Fenneropenaeus chinensis and comparison of its expression

13

11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

with the cognate Hsc70 under different stresses. Cell Stress Chaperones 2010; 15:83–93. Lo WY, Liu KF, Liao IC, Song YL. Cloning and molecular characterization of heat shock cognate 70 from tiger shrimp (Penaeus monodon). Cell Stress Chaperones 2004; 9:332–343. Spees JL, Chang SA, Mykles DL, Snyder MJ, Chang ES. Molt cycle-dependent molecular chaperone and polyubiquitin gene expression in lobster. Cell Stress Chaperones 2003; 8:258–264. Jedlicka P, Mortin MA, Wu C. Multiple functions of Drosophila heat shock transcription factor in vivo. EMBO J 1997; 16:2452–2462. Baler R, Zou J, Voellmy R. Evidence for a role of Hsp70 in the regulation of the heat shock response in mammalian cells. Cell Stress Chaperones 1996; 1:33–39. Lo TS, Cui ZX, Mong LY, Wong WL, Chan SM, Kwan HS, Chu KH. Molecular coordinated regulation of gene expression during ovarian development in the penaeid shrimp. Mar Biotech 2007; 9:459-468 Chomczynski P, Sacchi N. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162:156-159. Tiu SH, Chan SM. The use of recombinant protein and RNA interference approaches to study the reproductive functions of a gonad-stimulating hormone from the shrimp Metapenaeus ensis. FEBS J. 2007;274:4385-4395. Tiu SH, Hui JH, He JG, Tobe SS, Chan SM. Characterization of vitellogenin in the shrimp Metapenaeus ensis: expression studies and hormonal regulation of MeVg1 transcription in vitro. Mol Reprod Dev. 2006, 73:424-36. Okumura T. Effects of bilateral and unilateral eyestalk ablation on vitellogenin synthesis in immature female kuruma prawns, Marsupenaeus japonicus. Zoolog Sci. 2007, 24:233-240. Tsutsui N, Ohira T, Kawazoe I, Takahashi A, Wilder MN. Purification of sinus gland peptides having vitellogenesis-inhibiting activity from the whiteleg shrimp Litopenaeus vannamei. Mar Biotechnol (NY). 2007, 9:360-369. Kang BJ, Okutsu T, Tsutsui N, Shinji J, Bae SH, Wilder MN.Dynamics of Vitellogenin and Vitellogenesis-Inhibiting Hormone Levels in Adult and Subadult Whiteleg Shrimp, Litopenaeus vannamei: Relation to Molting and Eyestalk Ablation. Biol Reprod. 2014 23;90. Treerattrakool S1, Panyim S, Chan SM, Withyachumnarnkul B, Udomkit A.Molecular characterization of gonad-inhibiting hormone of Penaeus monodon and elucidation of its inhibitory role in vitellogenin expression by RNA interference. FEBS J. 2008,275:970-980. Piulachs MD, Guidugli KR, Barchuk AR, Cruz J, Simões ZL, Bellés X. The vitellogenin of the honey bee, Apis mellifera: structural analysis of the cDNA and expression studies. Insect Biochem Mol Biol 2003; 33:459–465. Zhu J, Chen L, Raikhel AS. Distinct roles of Broad isoforms in regulation of the 20-hydroxyecdysone effector gene, Vitellogenin, in the mosquito Aedes aegypti. Mol Cell Endocrinol 2007; 267:97–105. Postlethwait JH, Kunert CJ. Endocrine and genetic regulation of vitellogenesis in Drosophila. Prog Clin Biol Res 1986; 205:33–52. MacMorris M, Broverman S, Greenspoon S, Lea K, Madej C, Blumenthal T, Spieth J. Regulation of vitellogenin gene expression in transgenic Caenorhabditis elegans: short sequences required for activation of the vit-2 promoter. Mol Cell Biol 1992; 12:1652–1662. Le Guellec K, Lawless K, Valotaire Y, Kress M, Tenniswood M. Vitellogenin gene expression in male rainbow trout (Salmo gairdneri). Gen Comp Endocrinol 1988; 71:359–371. Corthésy B, Corthésy-Theulaz I, Cardinaux JR, Wahli W. A liver protein fraction regulating

14

29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47.

hormone-dependent in vitro transcription from the vitellogenin genes induces their expression in Xenopus oocytes. Mol Endocrinol 1991; 5:159–169. Wolffe AP, Perlman AJ, Tata JR. Transient paralysis by heat shock of hormonal regulation of gene expression. EMBO J. 1984; 3:2763–2770. Marchler G, Wu C. Modulation of Drosophila heat shock transcription factor activity by the molecular chaperone DROJ1. EMBO J 2001; 20:499–509. Kim BH, Schöffl F. Interaction between Arabidopsis heat shock transcription factor 1 and 70 kDa heat shock proteins. J Exp Bot. 2002 ;53:371-375. Wilbanks SM, DeLuca-Flaherty C, McKay DB. Structural basis of the 70-kilodalton heat shock cognate protein ATP hydrolytic activity. I. Kinetic analyses of active site mutants. J Biol Chem. 1994; 269:12893-12898. Buxbaum E, Woodman PG Binding of ATP and ATP analogues to the uncoating ATPase Hsc70 (70 kDa heat-shock cognate protein). Biochem J. 1996.;318:923-929. Knowlton AA, Salfity M. Nuclear localization and the heat shock proteins. J Biosci 1996; 21:123–133. Perkins LA, Doctor JS, Zhang K, Stinson L, Perrimon N, Craig EA. Molecular and developmental characterization of the heat shock cognate 4 gene of Drosophila melanogaster. Mol Cell Biol 1990; 10:3232–3238. Santacruz H, Vriz S, Angelier N. Molecular characterization of a heat shock cognate cDNA of zebrafish, hsc70, and developmental expression of the corresponding transcripts. Dev Genet 1997; 21:223–233. Rybczynski R, Gilbert LI. Prothoracicotropic hormone-regulated expression of a hsp 70 cognate protein in the insect prothoracic gland. Mol Cell Endocrinol 1995; 115:73–85. Kawooya JK, Osir EO, Law JH. Physical and chemical properties of microvitellogenin. A protein from the egg of the tobacco hornworm moth, Manduca sexta. J Biol Chem 1986; 15: 261:10844–10849. Wang XY, Cole KD, Law JH. The nucleotide sequence of a microvitellogenin encoding gene from tobacco hornworm, Manduca sexta. Gene 1989; 80:259–268. Morimoto RI. Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev 1998; 12:3788–3796. Abravaya K, Myers MP, Murphy SP, Morimoto RI. The human heat shock protein hsp70 interacts with HSF, the transcription factor that regulates heat shock gene expression. Genes Dev 1992; 6:1153–1164. Bonner JJ, Carlson T, Fackenthal DL, Paddock D, Storey K, Lea K. Complex regulation of the yeast heat shock transcription factor. Mol Cell Biol 2000; 11:1739–1751.43 Thakur P, Nehru B 2014 Long-term heat shock proteins (HSPs) induction by carbenoxolone improves hallmark features of Parkinson's disease in a rotenone-based model. Neuropharmacology. 79:190-200. Shi Y, Mosser DD, Morimoto RI. Molecular chaperones as HSF1-specific transcriptional repressors. Genes Dev 1998; 12:654–666. Schöffl F, Prändl R, Reindl A. Regulation of the heat-shock response. Plant Physiol 1998; 117:1135–1141. Hahn JS, Hu Z, Thiele DJ, Iyer VR. Genome-wide analysis of the biology of stress responses through heat shock transcription factor. Mol Cell Biol 2004; 24:5249–5256. Nunes SL, Calderwood SK. Heat shock factor-1 and the heat shock cognate 70 protein

15

48. 49. 50. 51. 52. 53. 54. 55. 56.

57.

associate in high molecular weight complexes in the cytoplasm of NIH-3T3 cells. Biochem Biophys Res Commun 1995; 213:1–6. Attardo GM, Higgs S, Klingler KA, Vanlandingham DL, Raikhel AS. RNA interference-mediated knockdown of a GATA factor reveals a link to anautogeny in the mosquito Aedes aegypti. Proc Natl Acad Sci USA 2003; 100:13374–13379. Westwood JT, Clos J, Wu C. Stress-induced oligomerization and chromosomal relocalization of heat-shock factor. Nature. 1991; 353:822–827. Kim SJ, Tsukiyama T, Lewis MS, Wu C. Interaction of the DNA-binding domain of Drosophila heat shock factor with its cognate DNA site: a thermodynamic analysis using analytical ultracentrifugation. Protein Sci 1994; 3:1040–1051. Dechklar M, Udomkit A, Panyim S. Characterization of Argonaute cDNA from Penaeus monodon and implication of its role in RNA interference. Biochem Biophys Res Commun 2008; 367:768–774. Lingel A, Sattler M. Novel modes of protein-RNA recognition in the RNAi pathway. Curr Opin Struct Biol 2005; 15:107–115. Matzke MA, Birchler JA. RNAi-mediated pathways in the nucleus. Nat Rev Genet 2005; 6:24–35. Robalino J, Browdy CL, Prior S, Metz A, Parnell P, Gross P, Warr G. Induction of antiviral immunity by double-stranded RNA in a marine invertebrate. J Virol 2004; 78:10442–10448. Tirasophon W, Roshorm Y, Panyim S. Silencing of yellow head virus replication in penaeid shrimp cells by dsRNA. Biochem Biophys Res Commun 2005; 334:102–107. Artimo P, Jonnalagedda M, Arnold K, Baratin D, Csardi G, de Castro E, Duvaud S, Flegel V, Fortier A, Gasteiger E, Grosdidier A, Hernandez C, Ioannidis V, Kuznetsov D, Liechti R, Moretti S, Mostaguir K, Redaschi N, Rossier G, Xenarios I, and Stockinger H. ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res 2012; 40(W1):W597-W603.” Yushmanov SV and Chumakov KM. Algorithms of the maximum topological similarity phylogenetic trees construction [in Russian]. Mol Genet Microbiol Virusol 1988; 3:9-15.

Figure legends Figure 1. Nucleotide sequence of the shrimp (M. ensis) vitellogenin gene promoter. The 2.6 kb 5’-upstream sequence of the MeVg2 gene consists of putative response elements for the binding siteof SP1, TATA, etc. It also contains potential binding site for HNF-3 beta, CEBP alpha, estrogen response element (ERE), GATA transcription factor (GATA) and several clusters of heat shock factor binding site (HSE). Figure 2. a) Diagramatic representation of the vitellogenin (MeVg2) gene promoter. b) Deletion analysis of the MeVg2 gene promoter. Figure 3. a) Amino acid sequence of the heat shock cognate 70 protein. Motif analysis of the ovary derived Hsc70. Domain search program was performed using Expasy server (URL: www.expasy.org) [56] to identify conserved motifs in the shrimp Hsc70 sequence. The amino acids in green indicates the Hsc 70/hsp70 family signature, the amino acid in red indicates the ATP binding site motif, the amino acid in grey and black indicate the bipartite nuclear localization signal, with abundant basic lysine and arginine, for selective localization of hsc70 into the nucleus. The amino acids in blue shows the association motif for the co-factors. b) Phylogenetic tree showing the relationship of MeHsc70 with other Hsp70 family members. The

16

sequences include Hsc70s from Penaeus monodon (AF474375), Litopenaeus vannamei (AY645906), Macrobrachium rosenbergii (AY466445), the barnacle Balanus amphitrite (AY150182), the insect Drosophila melanogaster (NM168568) and Manduca sexta (AF194819), the fish Danio rerio (NM131397), Gallus (NM205003) and Homo sapiens (M11717). The phylogram was generated by the Genebee software and viewed by Treetop (URL: http://www.genebee.msu.su/services/hlp/phtree-hlp.hmtl) [57]. Figure 4. Northern blot analysis of MeHsc70 gene expression in various tissues (a) including hepatopancreas, ovary, eyestalk, epidermis, nerve cord and muscle of the early reproductive female stage and in the hepatopancreas (Hp) and ovary (Ov) of the female (b) at different maturation stage (stage I-V) with stage I being the early vitellogenesis and stage V at the late vitellogenesis. The rRNAs of the corresponding RNA are included to show the loading amount and RNA integrity. Figure 5. Analysis of recombinant protein for rMeHsc70. a) Time course experiment showing the expression of rMeHsc70 in E. coli. 10% SDS-PAGE and Western blot analysis of recombinant hsc70 in pRSET-B/BL21 (DE3) cells. Top: CB staining of proteins from cell lysates of different induction time (1-8 hours) after ITPG induction. Bottom: Western blot analysis using monoclonal antibody of anti-Hsp/Hsc70 antibodies (1:2,000) dilution. All protein samples were from soluble protein fraction of bacterial cell culture. Lysate of pRSET-B vector was used as control (-). b) Detection of hsc70 in nuclear extracts from different tissues. The + lane is the rHsc70 pRSET-B-Hsc70/BL21; Cy, ovary cytoplasmic protein; M, pre-stained protein marker; Hp, hepatopancreas; Ov, ovary; Es, eyestalk; Epi, Epidermis; NC, nerve cord, Mu, muscles. Figure 6. Electrophoretic mobility shift assay (EMSA) to demonstrate the presence of HSF in ovary nuclear///using ovary nuclear extract from shrimp at reproductive stage I. a) A factor in the ovary extract was shown to bind the heat shock transcription factor response elements (HSE) in vitro. b) Competition assays with nuclear extract (NE) and recombinant protein (rHsc70) was performed to confirm the binding specificity. The retardation complex (arrowheads) was significantly competed away by 10- and 100-fold molar excess of un-labeled HSE which served as specific competitor. There was no effect on the retardation complex when 10- and 100-fold cold non-specific competitor (NC). c) Binding reaction with the addition of the ATP in the EMSA. Figure 7. Double strand RNA interference of the Hsc70 in the hepatopancreas (a) and ovary (b) of an early vitellogenic shrimp. Expression of Hsc70 and MeVg2 after administration of Hsc70 dsRNA (0.3 g/ml), dsCHH-B (0.3 g/ml) and TE. The 0-hr control are also included in the test. The top panel shows the Northern blot results from 3 shrimp and the lower panel shows the bar diagram (with standard error bar) of the scanned Northern blot of of the triplicated experiments (N=3 shrimp and each treatment group was duplicated 3 times) The expression values were normalized to the rRNA. Gene expression levels in control were set to 1.0. Each bar represents the mean ± standard deviation (SD) from three samples and each sample was pooled from 3 shrimps. Statistically significant was indicated by different letter indicated on the bar (p < 0.05).

Figure 1

TGTGTAAACAAGAG

CF2-II

963 bp

C/EBPalpha

AGGTGCACTCCCC

-140bp

-162bp

CF2-II

+1

439 bp

158 bp

TTTGATCTGC TTACCAAAT

-601bp

-909bp

-1067bp

Clusters of HSF

-1143bp

-2106bp

-2232bp

HNF-3beta

-2589bp

a)

GATA 3

AGATAAGGGTC GATA

CGTTAGTCATG AP1

C/EBPalpha

b) Deletion clones

pGL3-Basic pGL3-Control Vg2661-Luc Vg1666-Luc Vg1364-Luc Vg1178-Luc Vg883-Luc Vg796-Luc Vg716-Luc Vg205-Luc

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Relative activity

Figure 2

TATATAA TATA

a) MSKASAVGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDTERLIGDAAKNQVA MNPNNTVFDAKRLIGRKFDDATVQSDMKHWPFTIVNESTKPKIQVEYKGDKKTFYPEEIS SMVLIKMKETAEAYLGATVKDAVVTVPAYFNDSQRQATKDAGTISGLNVLRIINEPTAAA IAYGLDKKVGGERNVLIFDLGGGTFDVSILTIEDGIFEVKSTAGDTHLGGEDFDNRMVNH FIQEFKRKYKKDPSENKRSLRRLRTACERAKRTLSSSTQASVEIDSLFEGIDFYTSITRA RFEELCADLFRGTLEPVEKSLRDAKMDKAKIHDIVLVGGSTRIPKIQKLLQDFFNGKELN KSINPDEAVAYGAAVQAAILCGDKSEAVQDLLLLDVTPLSLGIETAGGVMTALIKRNTTI PTKQTQTFTTYSDNQPGVLIQVYEGERAMTKDNNLLGKFELSGIPPAPRGVPQIEVTFDI DANGILNVSAVDKSTGKENKITITNDKGRLSKEEIERMVQDAEKYKADDEKQRDRISAKN SLESYCFNMKSTVEDEKFKDKISEEDRTKILEMCNEAIKWLDGNQLGEKEEYEHKQKEIE QVCNPIITKMYGAAGGPPPGGMPGGMGGAAPGGAGTGGSSGPTIEEVD*

b)

Figure 3

60 120 180 240 300 360 420 480 540 600 648

Muscle

Nerve cord

Epidermis

Eyestalk

Ovary

Hepatopancreas

a) b)

I

Hp

Ov

Figure 4

II III IV V Hsc70

rRNA

Hsc70

rRNA

a)

b) Time of induction (hr)

M 0 1 2

4

6 8 (-)

147 98 64

+ cy M Hp Ov Es Ep Nc Mu 147 98 64

Recombinant hsc70

Figure 5

Nuclear protein extract

b) +5 g NE + 10X HSE +100X HSE + 10 XNC + 100 X NC

No NE

0.2 g rHsc70 0.4 g rHsc 70 Free probe

No NE 3 g NE 5 g NE

a)

Figure 6

No NE + 1 M ATP +2 M ATP +5 g NE

c)

Free probe

level

Relative expression

Hsc70 MeVg2

Figure 7 Hsc70

Test 1

Test 2

Test 3

MeVg2

+dsHsc70

+dsCHH

+TE

0 hr

+dsHsc70

+dsCHH

+TE

0 hr

+dsHsc70

+dsCHH

+TE

0 hr

+dsHsc70

+dsCHH

+TE

0 hr

a) b)

The shrimp heat shock cognate 70 functions as a negative regulator in vitellogenin gene expression.

Within the 2.6-kb 5' flanking region of the shrimp (Metapenaeus ensis) vitellogenin gene (MeVg2), several clusters of putative heat shock factor (HSF)...
2MB Sizes 0 Downloads 4 Views