Turning up the heat: The effects of thermal acclimation on the kinetics of hsp70 gene expression in the eurythermal goby, Gillichthys mirabilis

Comparative Biochemistry and Physiology, Part A 143 (2006) 435 – 446 www.elsevier.com/locate/cbpa

Turning up the heat: The effects of thermal acclimation on the kinetics of hsp70 gene expression in the eurythermal goby, Gillichthys mirabilis Susan G. Lund a,⁎, Marlena R. Ruberté a , Gretchen E. Hofmann a,b a

Department of Ecology, Evolution, and Marine Biology, University of California, Santa Barbara, Santa Barbara, CA 93106-9610, USA b Marine Science Institute, University of California, Santa Barbara, Santa Barbara, CA 93106-9610, USA Received 23 July 2005; received in revised form 16 December 2005; accepted 18 December 2005 Available online 8 February 2006

Abstract Most organisms respond to temperature fluctuations by altering the expression of an evolutionarily conserved family of proteins known as heat shock proteins (Hsps). Studies have shown Hsp expression and the activation of HSF1, one of the primary regulators of Hsp transcription, are highly malleable, varying with the recent thermal history of the organism; however, the mechanisms that confer plasticity to the regulation of this ubiquitous response are not well-understood. This study furthers our knowledge in this area by characterizing the activation kinetics of HSF1 and the corresponding transcription of hsp70 in the liver of the eurythermal goby, Gillichthys mirabilis, following a month-long acclimation at 13, 21 or 28 °C. Our data revealed HSF1 DNA-binding kinetics varied as a function of acclimation temperature and magnitude/duration of exposure, with gobies acclimated at 21 °C exhibiting the most robust response. Hsp70 mRNA followed a similar pattern with induction first occurring in the 13 and 21 °C fish, and then most robustly in the 28 °C group at 36 °C. The hsp70 mRNA induction pattern was corroborated by levels of HSF1 DNA-binding activity in each group and may have been lowest in the 28 °C group due to the 2-fold greater levels of hsp70 protein prior to thermal exposure. This study illustrates the integral role of HSF1 as a key regulator of Hsp induction and helps explain the plasticity of this response in ectothermic organisms. © 2006 Elsevier Inc. All rights reserved. Keywords: Fish; Gene expression; HSF; Heat shock proteins; Hsp70; Stress response; Thermal acclimation; Transcriptional-level regulation

1. Introduction The plasticity of the response to seasonal cyclical changes in the abiotic environment, such as temperature change, is a hallmark of many poikilothermic animals, especially fish. While stenothermal species may never experience environmental temperature fluctuations, eurythermal species have evolved a number of behavioural, biochemical and molecular mechanisms to assist in their continuous adjustment to naturally occurring temperature variation (Hochachka and Somero, 2002). For example, a fish that routinely experiences rapid temperature fluctuations has been shown to alter gene expression patterns in a short period of time (Podrabsky and Somero, 2004). Complex molecular level adaptive mechanisms are initiated when the physical parameters of heat stress are transduced ⁎ Corresponding author. Department of Biology, University of Regina, Regina, Saskatchewan, Canada, S4S 0A2. Fax: +1 306 337 2410. E-mail address: [email protected] (S.G. Lund). 1095-6433/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2005.12.026

and sensed on a cellular level. One of the most universal molecular responses to temperature stress is the induction of a suite of proteins called heat shock proteins (Hsps; Lindquist, 1986). The majority of the work that has been done to delineate the mechanisms of Hsp gene regulation has been accomplished under highly controlled conditions using a limited number of mammalian and invertebrate model systems. In contrast, relatively little is known about the mechanisms that confer plasticity to the regulation of Hsp gene expression in natural populations of poikilothermic organisms in highly variable thermal environments. Until recently, studies examining the heat shock response (HSR) in poikilotherms have been primarily descriptive in nature, focusing on the size class of Hsp induced with different stressors, the change in Hsp expression with increasing temperature and the correlation between Hsp expression and thermotolerance (reviewed in Feder and Hofmann, 1999; Iwama et al., 1998). The purpose of the present study was to build on this descriptive knowledge, and attempt to isolate and examine the

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effects of thermal stress on the temperature thresholds and timing of the regulatory steps involved in transducing heat stress from the environment to the level of cellular activation and synthesis of Hsps in a highly eurythermal fish. The longjaw mudsucker, Gillichthys mirabilis, is a eurthythermal marine gobiid fish that inhabits coastal sloughs, lagoons and estuaries from as far north as Tomales Bay, California, to as far south as Bahía Magdalena, Mexico (Thomson et al., 2000). In its natural environment it routinely experiences a wide span of ecological temperatures ranging from as low as 5 °C in the winter to greater than 38 °C in the summer (Place and Hofmann, 2001). G. mirabilis was chosen as the experimental model for the present study because of its wide thermal tolerance range, and due to the fact that a significant amount of the more descriptive groundwork on Hsp expression has already been done (Dietz and Somero, 1992; Dietz, 1994). These early studies were instrumental in illustrating that the threshold induction temperature for Hsp synthesis is not genetically fixed, but varies depending on the acclimatization or acclimation history of the fish. The higher the acclimation or acclimatization temperature is, the higher is the temperature required to elicit a heat shock response (Dietz and Somero, 1992; Dietz, 1994). More recent studies examining the correlation between HSF1 activation and Hsp induction temperatures in G. mirabilis have shown that the temperature of in vitro heat shock transcription factor 1 (HSF1) activation is positively correlated with the prior acclimation temperature of the fish, although this relationship was not maintained when examined on a seasonal basis (Buckley and Hofmann, 2004). The absence of an increase in HSF1 activity with increasing exposure temperature in the liver of gobies collected during different seasons may be due to the presence of stressors, in addition to heat, that could be present in a goby's natural environment. Buckley and Hofmann (2004) also examined the kinetics of HSF1 activation and hsp70 mRNA transcription in liver tissue extracted from gobies collected from the wild during the month of June and showed that the rapidity of HSF1 activation and of hsp70 mRNA synthesis increased with laboratory exposure temperature (Buckley and Hofmann, 2004). Inducible Hsp expression in all organisms is regulated by the heat shock transcription factors (HSFs), several members of which have been identified to date in animals and plants. Although only one HSF species has been identified in lower organisms such as yeast and Drosophila, higher vertebrates are capable of expressing a total of 4 distinct but related HSFs. HSF 1, 2 and 4 have been found in mammalian cells, whereas HSF 3 appears to be an avian-specific factor. Recent HSF cloning and characterization studies in fish have identified three distinct isoforms of HSF1 in the zebrafish (Råbergh et al., 2000; Wang et al., 2001) and two in rainbow trout (Ojima and Yamashita, 2004). In all vertebrates, HSF1 has been shown to be the primary transcription factor involved in mediating stress-induced heat shock gene expression. Stress does not alter the quantity of HSF produced (Rabindran et al., 1994), but alters the regulation of its activity on a post-transcriptional level through oligomerization, translocation and hyperphosphorylation (Baler et al., 1993; Cotto et al., 1996). Despite the fact that

the exact role and function of each HSF isoform is not well understood, it appears that they provide a means of differential control of transcription, and allow greater specialization of stress signals and interactions with other regulatory factors in the cell. In unstressed eukaryotic cells, HSF1 exists in the cytoplasm as an inactive monomer complexed with Hsp70 and Hsp90 (Ali et al., 1998; Baler et al., 1992, 1993; Rabindran et al., 1994; Zou et al., 1998). During stressful conditions, Hsp70 and Hsp90 dissociate from HSF1 to chaperone the increasing proportion of misfolded or damaged proteins, which in turn releases HSF1 (Morimoto et al., 1996; Shi et al., 1998; Winklhofer et al., 2001). HSF1 is then translocated into the nucleus where it trimerizes (Westwood et al., 1991; Westwood and Wu, 1993) and binds to the heat shock element (HSE), a domain of the promoter region of heat shock genes that consists of inverted adjacent arrays of the sequence 5′-nGAAn-3′ (Pelham, 1982; Xiao and Lis, 1988). Transcriptional activation is finally attained when HSF1 receives reversible serine phosphorylation by members of the mitogen-activated protein kinases (MAPKs) (Holmberg et al., 2002; Morimoto, 1998; Park and Liu, 2001). When the chaperoning ability of the heat shock proteins is no longer needed, free Hsp70 and Hsp90 re-associate with HSF1, releasing it from the HSE, thus terminating transcription. This multi-tiered gene regulatory response, combined with downstream mechanisms of mRNA processing and translation (Dellavalle et al., 1994; Laroia et al., 1999; Yost et al., 1990), provides the cell with multiple modes of Hsp gene regulation. In this study, we build on the current understanding of HSF1 activation and subsequent Hsp synthesis in the longjaw mudsucker by examining the kinetics of the regulation of the HSR in a more controlled environment that was free of other confounding environmental variables. This was done by effectively ‘erasing’ the recent natural thermal history of the fish through a month-long thermal acclimation at one of three environmentally relevant temperatures (13, 21 or 28 °C). This functioned to provide a new baseline of hsp70 expression from which to monitor the effects of environmentally relevant exposure temperatures on in vitro HSR kinetics in isolated liver sections, as the starting concentration of this protein should be instrumental in influencing the timing of HSF1 activation and hsp70 mRNA transcription. Uncovering the acclimation-induced modification of the kinetics of HSF1 activation and hsp70 mRNA synthesis in this eurythermal fish brings us closer to understanding the mechanisms involved in the plasticity of environmental gene regulation in non-model organisms. 2. Materials and methods 2.1. Animal collection and acclimation conditions Sixty male and female adult gobies, G. mirabilis, (13.1 ± 1.1 g; 109.6 ± 2.7 mm) were collected by baited trap from a coastal lagoon at Campus Point on the coast near the University of California, Santa Barbara (34.39 °N, 119.81°W) from July 6–8, 2003. Fish were transferred by cooler to the University of

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California aquatic facility where they were held for a minimum of 24 h in flow-through seawater aquaria set at a temperature of 20 °C. Twenty fish were then randomly transferred to one of three re-circulating saltwater (32%) acclimation tanks. Each acclimation tank was then slowly adjusted to its final temperature (∼ 1 °C every 4 h) of 13, 21 or 28 °C, where it was maintained for a period of 30 days. Water was changed weekly and fish were fed marine flake food (algae, krill, plankton) ad

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libitum. Survivorship for the 13 and 21 °C acclimated fish was 100% while the 28 °C acclimation group experienced 20% mortality. 2.2. In vitro thermal exposure conditions Following the 30-day acclimation period, fish from each temperature group were killed via cervical transection, and

Fig. 1. The effect of thermal acclimation on the kinetics of hsp70 mRNA induction in liver tissue from G. mirabilis. Fish were collected in July 2003 and acclimated for 1 month at 13, 21 or 28 °C (shaded bars from light to dark, respectively). Following acclimation, livers were excised, dissected and exposed to one of six exposure temperatures (13,17, 21, 25, 28 and 36 °C; A–F) for 0, 15, 30, 60 or 90 min. Hsp70 mRNA levels were measured using a Northern blotting approach. Band densities from Northern blots were corrected for loading differences using 18S RNA and were expressed relative to a reference sample that was loaded on each blot. All values are expressed as mean ± 1 SEM (N = 6, 13 and 21 °C; N = 4, 28 °C). Different letters within an acclimation group denote significant differences (p b 0.05).

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their livers were removed and sectioned into 25–35 mg pieces. One dissected liver section from every fish sampled was immediately frozen on liquid nitrogen for western analysis. Two dissected liver sections from each of six fish (four for the 28 °C acclimation group) were immediately frozen in a centrifuge tube on liquid nitrogen to serve as t = 0 controls, while two others were then held in 1 ml Eagles Minimum Essential Medium (MEM, Sigma-Aldrich Corp., St. Louis, MO), adjusted with NaCl to 335 mosM l− 1, (the plasma osmolarity of G. mirabilis) in microcentrifuge tubes in an aluminum heat block at a given temperature for 15, 30, 60 or 90 min. Liver sections were mixed and aerated with a Pasteur pipette at 15-min intervals. Temperatures used for all three acclimation groups were the same: 13, 17, 21, 25, 28 and 36 °C. Following thermal exposures, the MEM was removed and the tissue pieces were placed in separate microcentrifuge tubes and frozen on liquid nitrogen and stored at − 80 °C. 2.3. RNA extraction Total RNA was extracted from frozen samples in 500 μl TRIzol® according to manufacturer's instructions (TRIzol®, Invitrogen Life Technologies). RNA concentration and purity was determined by UV absorption at 260:280 using an Ultrospec 1100 Pro UV/Vis microspectrophotometer (Biochrom). RNA extracts were stored at − 80 °C prior to use in Northern blotting. 2.4. Probe development The hsp70 probe was generated from first strand cDNA synthesized from total RNA extracted from G. mirabilis isolated liver tissue, heat shocked in vitro at 36 °C. The constitutive hsc70 probe was generated from first strand cDNA synthesized from total RNA extracted from G. mirabilis non-heat shocked (17 °C) liver tissue. Hsp70 forward and reverse primers were designed by determining highly homologous regions from the alignment of hsp70 genes of 5 fish species (O. tschawytscha, D. rerio, X. maculatus, O. latipes and O. mossambicus) with the hsp70 genes of 3 mammals (M. musculus, R. norvegicus and H. sapiens), all obtained from GenBank and aligned using Clustal W (version 1.8). The hsp70 probe was a 351 bp internal segment of the G. mirabilis hsp70 coding region resting between nucleotides 752 and 1101 of the O. mossambicus hsp70 gene (GenBank accession no. AJ001312; see Molina et al., 2000). This probe was amplified by PCR at an annealing temperature of 55 °C using the forward primer 5′-CAC AAG AAG CAC ATC AGC CAG and the reverse primer 5′-GGG TTG ATG CTC TTG TTC AG. Primers used to amplify the 242 bp heat shock cognate (hsc70) probe were as previously described for rainbow trout Hsc71 (Currie et al., 1999). The hsc70 probe was amplified by PCR at an annealing temperature of 50 °C, followed by a second PCR under the same conditions using 1 μl of the first reaction as a template. All PCR reactions involved an initial denaturation (94 °C × 30 s) followed by 30 cycles of: 94 °C, 30 s; annealing temperature, 1 min; 72 °C, 1.5 min, followed by a final extension for

15 min at 72 °C. The resulting PCR products were ligated into pCR 2.1 (Invitrogen) and sequenced. The identities of the probe sequences were confirmed through NCBI BLAST analysis and Northern blotting using RNA extracted from control and heat shocked liver tissue from G. mirabilis. 2.5. Northern analyses Ten micrograms total RNA was fractionated by glyoxal/ dimethylsulfoxide (DMSO) denaturing electrophoresis on a 1% agarose gel and vacuum transferred to Zeta probe nylon membrane (BioRad) under 5 in. Hg for 2 h using 10× standard saline citrate (SSC). The membranes were UV cross-linked once at the optimal setting using a CL-1000 UV cross-linker (UVP) prior to hybridization with probes. The probes were labeled with [32P]-dCTP [1 × 10− 6 Ci/ng DNA, specific activity 3000 Ci/mmol l− 1] using the Ready-toGo labeling system (Amersham Pharmacia BioTech). Membranes were prehybridized in 20 ml Church's buffer [O.5 M NaPO4, 10 mmol l− 1 EDTA, 7% sodium dodecylsulfate (SDS)] at 60 °C for 3 h, followed by hybridization with labeled probe at 60 °C for 18 h. Following hybridization, membranes were washed twice at room temperature for 15 min with a low stringency wash buffer [1 × SSC/0.1% SDS], and once at 60 °C for 20 min with a high stringency wash buffer [0.25 × SSC/0.1% SDS]. Following washing, membranes were wrapped in saran wrap and exposed to a phosphor screen for 4–18 h. Phosphor screens were scanned using the BioRad Personal FX imager and densitometry was performed with Quantity One software (BioRad). 2.6. Electrophoretic mobility shift assays For the electrophoretic mobility shift assays (EMSAs), frozen tissues were thawed on ice in 100 μL extract buffer containing 25% (v/v) glycerol, 20 mM Hepes, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF and 0.5 mM dithiothreitol. Tissue extracts were centrifuged at 21,000×g at 4 °C for 10 min. Pellets were discarded and the supernatants were frozen at − 80 °C, after a sample was taken for total protein content determination by Bradford assay (Pierce Table 1 Fold increase in hsp70 and hsc70 mRNA levels over t = 0 controls following 90 min of exposure to 13, 17, 21, 25, 28 or 36 °C for gobies acclimated at each of three acclimation temperatures (13, 21 and 28 °C)

Exposure temperature (°C)

13 °C Acclimated

21 °C Acclimated

28 °C Acclimated

hsp70

hsc70

hsp70

hsc70

hsp70

hsc70

13 17 21 25 28 36

1.13 0.32 9.81 15.42 97.52 398.53

1.25 1.16 1.38 1.21 1.66 1.37

1.79 0.70 3.18 20.33 117.26 816.95

1.28 1.19 1.23 1.18 1.46 1.79

1.10 0.93 0.27 1.31 12.18 289.10

1.17 0.96 1.00 1.10 1.57 2.19

Shading (from light to dark) indicates the exposure temperature at which hsp70 mRNA was first significantly induced over control levels for each acclimation group (13 °C, 21 °C and 28 °C, respectively).

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Chemical Co., Rockford, IL, USA). EMSAs were conducted using the Gel Shift Assay Core System (Promega Corp., Madison, WI) and 32P-dATP labeled HSE oligonucleotide probe (5′-GCCTCGAATGTTCGCGAAGTTT-3′; see Airaksinen et al., 1998). Samples (20 μg total protein) were incubated at 13 °C in the presence of 5× binding buffer containing 20% v/v glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris–HCl, pH 7.5 and 0.1 μg/μL poly dI/dC in a final volume of 10 μL for 10 min prior to the addition of radiolabelled HSE oligonucleotide. Incubation in the presence

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of radiolabelled HSE was then carried out for an additional 20 min at the same temperature. After incubation, HSF1– HSE complexes were separated from unincorporated HSE probe on 5% acrylamide non-denaturing gels and electrophoresed for approximately 1 h at 250 V. Gels were then fixed in a solution of 30% methanol, 10% acetic acid for 30 min, followed by re-hydration for 15 min in distilled water. Gels were then placed on Whatman filter paper (3 MM Chr) and dried using a Bio-Rad gel dryer (Model 583 and Hydrotech vacuum pump (BioRad) at 80 °C for approximately 1 h. Dried gels were

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Relative Hsc70 mRNA content

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Fig. 2. The effect of thermal acclimation on the kinetics of hsc70 mRNA induction in liver tissue from G. mirabilis. Fish were collected in July 2003 and acclimated for one-month at 13, 21 or 28 °C (shaded bars from light to dark, respectively). Following acclimation, livers were excised, dissected and exposed to one of six exposure temperatures (13,17, 21, 25, 28 and 36 °C; A–F) for 0, 15, 30, 60 or 90 min. Hsc70 mRNA levels were measured using a Northern blotting approach. Band densities from Northern blots were corrected for loading differences using 18S RNA and were expressed relative to a reference sample that was loaded on each blot. All values are expressed as mean ± 1 SEM (N = 6, 13 and 21 °C; N = 4, 28 °C). Different letters within an acclimation group denote significant differences (p b 0.05).

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exposed to a phosphor screen (Eastman Kodak Co.) and then visualized and quantified using a BioRad Personal FX Imager driven by Quantity One software (BioRad). To allow for intergel comparisons, a standard (30 °C heat-shock sample) was loaded in the ninth lane of every gel. Specificity of the HSE probe was confirmed in assays where unlabelled HSE probe was added in excess as a competitor. 2.7. Protein sample preparation, SDS-PAGE and western blotting Western blotting analysis was used to quantify Hsp70 in the livers of all gobies from each of the three acclimation groups. Frozen tissues were thawed on ice and homogenized by Teflon pestle in 100 μL of homogenization buffer containing 50 mM Tris–HCl, pH 6.8, 4% SDS, 1 mM EDTA and 1 mM PMSF. Homogenates were heated at 100 °C for 5 min and centrifuged at 12,000×g for 10 min. Pellets were discarded and total protein content of the supernatants was determined by Bradford assay (Pierce). Protein samples were diluted in a 1:1 ratio with Laemmli's buffer, boiled, and then loaded onto a one-dimensional SDS polyacrylamide gel (7.5%) and subjected to electrophoresis (10 μg protein.lane− 1). To allow for inter-gel comparisons, a standard (36 °C in vitro heat shock followed by a 2-h recovery at 20 °C) was loaded in the first lane of every gel. Proteins were then transferred (100 V for 30 min) onto nitrocellulose membranes with a mini trans-blot cell (Bio-Rad) using a transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol). Membranes were blocked for 1 h with a blocking solution consisting of 5% non-fat dry milk (NFDM) in phosphate buffered saline (PBS; 1.5 mM NaH2PO4, 8 mM Na2HPO4, 145 mM NaCl, pH 7.4). Membranes were then probed with an anti-Hsp70 rat monoclonal primary antibody (MA3-001, Affinity Bioreagents) diluted 1:2500 in PBS containing 5% (w/v) NFDM, 20% (v/v) fetal bovine serum, 1 mM PMSF and 0.02% (w/v) thimerosal for 1.5 h. This antibody recognizes both inducible and constitutive Hsp70 family members, appearing as a single band in goby westerns, and is therefore later described as Hsp/Hsc70 protein concentration. This was followed by a 30-min incubation in a rabbit-anti rat bridging antibody (A-4000, Vector) at a 1:2000 dilution in blocking solution, and a final exposure to a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (BioRad 170-6515) diluted 1:50,000 in PBS blocking solution for 1 h. Bands were visualized by chemiluminescence using SuperSignal West Dura Extended Duration Substrate (Pierce) and a VersaDoc Imaging System (BioRad) driven by Quantity One software (BioRad). Band size was confirmed using prestained low-range molecular markers (BioRad).

(P b 0.05) in hsp/hsc70 mRNA and protein band density and for the percent increase in HSF1 DNA-binding activity. 3. Results 3.1. Hsp70 and Hsc70 mRNA induction temperatures In vitro kinetics of hsp70 mRNA induction in G. mirabilis liver varied greatly depending upon the thermal history of the fish (Figs. 1 and 3). Specifically, the lower the acclimation temperature, the lower the exposure temperature (and duration) necessary to elicit a transcriptional response. Hsp70 mRNA levels in 13 °C acclimated fish exhibited a 9-fold induction over control levels after 90 min of a 21 °C thermal exposure (Fig. 1C; Table 1, light-gray shading) whereas a significant induction did not occur until 90 min of a 25 °C exposure in the 21 °C acclimated fish (Fig. 1D, Table 1, mid-gray shading) and until 60 min of a 28 °C exposure in the 28 °C acclimation group (Fig. 1E). Peak hsp70 mRNA induction occurred in all acclimation groups after 90 min of 36 °C exposure (Fig. 1F) at which point the levels of hsp70 mRNA ranged from 300to 800- fold higher than control levels in each acclimation group (Table 1). While hsp70 mRNA was most significantly induced in the 13 and 21 °C acclimation groups, respectively, throughout the 21–28 °C thermal exposures, this relationship was inverted in the 36 °C exposure where the highest hsp70 mRNA induction levels were seen in the 28 °C acclimated fish followed by the 21 and 13 °C acclimation groups, respectively. In contrast to hsp70, levels of hsc70 mRNA in goby liver were largely unaffected by acclimation history and intensity/ duration of in vitro thermal exposure (Figs. 2 and 3). Significant changes in hsc70 mRNA content occurred after 60 min at the lowest exposure temperature of 13 °C in both 13 and 21 °C acclimated fish (Fig. 2A), however even at the highest exposure temperature (Fig. 2F), the maximal hsc70 increase was only twofold higher than control levels in the 28 °C acclimated fish after 90 min at 36 °C (Table 1; dark-gray shading). 3.2. HSF1 activation kinetics Following the month-long acclimation period, the effect of thermal exposure on HSF1 activation kinetics in G. mirabilis Exposure time (min) 15

30

60

90

21 28 13

21 28 13

21 28

Hsp70

Hsc70

2.8. Statistical analysis Heat shock mRNA expression, HSF1 DNA-binding activity and protein data are presented as means ± 1 standard error of the mean (SEM). A one-way analysis of variance (ANOVA) followed by a Student-Newman-Keuls test for the pair-wise comparison of means was used to assess significant differences

13 21

28

13

Acclimation temperature (ºC) Fig. 3. Representative Northern blots for hsp70 and hsc70 as illustrated in Figs. 1 and 2. Blots are composed of RNA samples extracted from liver tissue excised from fish acclimated at 13, 21 and 28 °C and subsequently exposed to 36 °C (in vitro) for 15, 30, 60 and 90 min (N = 1).

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Exposure time (min) 15

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60

90

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60

90

15

30

60

90

HSE-HSF1 complexes

Free probe

21

13

28

Acclimation temperature (ºC) Fig. 4. Representative banding pattern of HSF1–HSE complexes visualized via electrophoretic mobility-shift assay (EMSA). HSF1–HSE complexes are from G. mirabilis liver tissue sections excised from fish acclimated at 13, 21 and 28 °C and subsequently exposed to 36 °C (in vitro) for 15, 30, 60 and 90 min (N = 1).

liver was determined by EMSA (Fig. 4) and was expressed as percent increase in HSF1 DNA-binding activity over control levels at time zero for each acclimation group (Fig. 5). Upon examination of the exposure temperatures and time-points where hsp70 mRNA levels first became significantly increased over control levels for each acclimation group, it is interesting to note that there was a corresponding peak or rise in HSF1 DNA-binding activity (Fig. 6). Percent increase in HSF1 DNAbinding activity was highest for both the 13 and 21 °C acclimated groups after 60 min at a 36 °C exposure temperature (Fig. 5F), which directly corresponded with the highest levels of hsp70 mRNA for both of these groups. While peak hsp70 mRNA levels also occurred at this temperature/time-point in the 28 °C acclimated fish, the highest percent increase in HSF1 DNA-binding activity occurred at 25 °C after 60 min. In general, HSF1 DNA-binding activity showed the greatest percent increase with increasing exposure temperature and duration in the 13 °C acclimation group. The 21 °C acclimation group was the only group that consistently exhibited a significant increase over control levels at each exposure temperature with the exception of 17 °C, while the 28 °C acclimated group exhibited the greatest percent increase in HSF1 DNA-binding activity at the more moderate exposure temperatures of 21 and 25 °C (Fig. 5C and D). 3.3. Hsp70/Hsc70 concentration The concentration of Hsp/Hsc70 was determined for each acclimation group following the one-month thermal exposure (Fig. 7). There was a visible increase in Hsp/Hsc70 concentration with increasing acclimation temperature. Hsp/Hsc70 levels in the livers of the 28 °C acclimated fish were significantly

higher than those in the 13 and 21 °C groups. Mean Hsp/Hsc70 concentrations in the 28 °C group were 55 and 40% higher than in the 13 and 21 °C acclimation groups, respectively. 4. Discussion Although the coding sequences of Hsp genes are highly conserved across a diverse range of taxa, the thermal threshold, magnitude, and kinetics of Hsp expression have been shown to be extremely plastic, covarying with the recent thermal history of the organism under examination (reviewed in Barua and Heckathorn, 2004; Feder and Hofmann, 1999). While a few studies have begun to examine the regulatory basis of this plasticity in poikilotherms (Buckley and Hofmann, 2002, 2004; Lerman and Feder, 2001; Zatsepina et al., 2000), additional research is required to develop a tractable model of environmentally controlled Hsp gene regulation. In this study we furthered our understanding of the molecular mechanisms of Hsp gene regulation in the eurythermal goby, G. mirabilis, by characterizing the activation kinetics of one of the key regulators involved in Hsp expression, HSF1, and the corresponding transcription of hsp/hsc70 mRNA, following a month-long laboratory acclimation at one of three environmentally relevant temperatures. Overall the salient findings of this study were three-fold: (1) the kinetics of HSF1 DNA-binding activity in laboratory acclimated gobies varied as a function of acclimation temperature, exposure temperature and duration of exposure with 21 °C acclimated individuals exhibiting the most robust response; (2) the induction kinetics of hsp70 mRNA largely paralleled the trends observed for HSF1 activity with 13 °C acclimated fish inducing most rapidly and robustly until the upper exposure temperature of 36 °C; (3) at higher exposure

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Fig. 5. The effect of thermal acclimation on the kinetics of HSF1 DNA-binding activity in liver tissue from G. mirabilis expressed as percent increase over control (exposure time of 0 min). Fish were collected in July 2003 and acclimated for one-month at 13, 21 or 28 °C (shaded bars from light to dark, respectively). Following acclimation, livers were excised, dissected and exposed to one of six exposure temperatures (13,17, 21, 25, 28 and 36 °C; A–F) for 0, 15, 30, 60 or 90 min. HSF1 DNA-binding activity was measured by electrophoretic mobility shift assay (EMSA). Band densities from EMSAs were expressed relative to a reference sample that was loaded on each blot and then expressed as a percent increase over control levels (t = 0) within each acclimation group. All values are expressed as mean ± 1 SEM (N = 6, 13 and 21 °C; N = 4, 28 °C). Different letters within an acclimation group denote significant differences (p b 0.05).

temperatures, the magnitude of the percent increase in HSF1 activity was much more pronounced in fish acclimated at 13 and 21 °C than in those acclimated at 28 °C. The present study clearly corroborates previous work (reviewed in Barua and Heckathorn, 2004) showing that organisms acclimated to warm environments constitutively express Hsps at higher levels (Fig. 2; Fig. 7) and have a higher thermal threshold for Hsp induction than do organisms inhabiting cool-

er environments (Fig. 1). This study is unique, however, in that it examines the regulatory basis for this difference in a eurythermal fish that routinely sees large temperature fluctuations in its natural environment. A plethora of regulatory mechanisms exist that may work alone or in concert to effectively control the expression of Hsps including transcriptional level regulation (Li et al., 1996; Lindquist, 1986; Lindquist and Craig, 1988; Mason and Lis, 1997; Morimoto et al., 1994), RNA processing

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Exposure time (min) Fig. 6. The effect of thermal acclimation on the kinetics of HSF1 DNA-binding activity and hsp70 mRNA induction in liver tissue from G. mirabilis at the exposure temperature where hsp70 mRNA was first significantly induced over time zero control levels for each acclimation group (A: 21 °C for the 13 °C acclimation group; B: 25 °C for the 21 °C acclimation group; C: 28 °C for the 28 °C acclimation group—see Fig. 1). HSF1 DNA-binding activity was measured by electrophoretic mobility shift assay (EMSA). Band densities from EMSAs were expressed relative to a reference sample that was loaded on each blot. Hsp70 mRNA levels were measured using a Northern blotting approach. Band densities from Northern blots were corrected for loading differences using 18S RNA and were expressed relative to a reference sample that was loaded on each blot. All values are expressed as mean ± 1 SEM (N = 6, 13 and 21 °C; N = 4, 28 °C). Crosses denote significant difference over time zero controls for HSF1 DNA-binding activity, while asterisks denote significant difference over time zero controls for hsp70 mRNA levels (p b 0.05).

(Lindquist, 1993; Yost et al., 1990), mRNA stability (Petersen and Lindquist, 1989) and translation (Hess and Duncan, 1996; Zapata et al., 1991) among others. The regulatory mechanism

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that has become the focus of the present study is transcriptional level regulation through DNA-binding activity of the heat shock transcription factor, HSF1. Understanding the kinetics of this response and the corresponding changes in Hsp transcription will shed new light in this largely unexamined area. The percent increase in HSF1 DNA-binding activity illustrated in Fig. 5 is a reflection of the increase in the number of HSF1 molecules in the cell that have trimerized and are capable of actively binding the heat shock element on the appropriate heat shock gene. HSF1 DNA-binding activity alone is not a direct measure of Hsp gene transactivation, but is a necessary step in the process and may thus play a role in the observed plasticity of the heat shock response in ectotherms. Hsp70 and other molecular chaperones are well-known negative regulators of HSF1 (Morimoto, 1998, 2002) and because a number of diverse experiments on a wide range of organisms have shown that the temperature threshold for HSF1 activation is highly malleable (Lerman and Feder, 2001; Zatsepina et al., 2000), and has previously been shown to vary with acclimation temperature and seasonal acclimatization in the long-jaw mudsucker (Buckley and Hofmann, 2002, 2004), it is thus a potential source of thermal sensitivity in the heat shock response. In general, the liver tissue from gobies acclimated at 13 °C showed the lowest level of HSF1 activation of any acclimation group at the lower exposure temperatures (Fig. 5A–D). This pattern changed at 28 and 36 °C when the HSF1 activation in this cooler acclimation group became much more robust (48% and 90% increase in HSF1 activation after 60 min of exposure in comparison to time zero controls, respectively; Fig. 5E and F). The liver tissue from gobies acclimated at 21 °C exhibited the largest overall percent increase in HSF1 DNA-binding activity at each of the six exposure temperatures, reaching a peak 110% increase over control levels after 60 min of exposure at 36 °C. This acclimation group was also the only one to consistently show a statistically significant percent increase in HSF1 DNA-binding activity, generally after 60–90 min of exposure (Fig. 5C–F). The pattern of percent increase in HSF1 DNA-binding activity with duration of exposure in the 28 °C acclimation group was fairly consistent with that observed in the 21 °C group for the lower exposure temperatures (Fig. 5A–D), however, at an exposure temperature of 28 °C and especially at 36 °C, the percent increase in HSF1 DNA-binding activity noticeably decreased in comparison to the two other acclimation groups (Fig. 5E–F). Interestingly, this marked decline in HSF1 DNA-binding activity at 28 and 36 °C was paralleled by the largest increases in Hsp70 mRNA seen in this warmest acclimation group, and in the entire study (Fig. 1E and F). Because HSF1 DNA-binding precedes the induction of hsp70 mRNA synthesis in the cell, one would assume that the changes in HSF1 DNA-binding activity would predict, or at least parallel, the induction pattern of hsp70 mRNA. Although this was not the case for the 28 °C acclimated fish at the two highest exposure temperatures (Figs. 1 and 5), it appears to be the trend for all groups, at least during the first 60 min of thermal exposure. As can be seen in Figs. 1 and 5, and more

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Hsp/Hsc70 Fig. 7. (A) Concentration of Hsp/Hsc70 in liver tissue from G. mirabilis. Fish were collected in July 2003 and acclimated for one-month at 13, 21 or 28 °C (shaded bars from light to dark, respectively). Following acclimation, livers were excised, dissected and immediately flash-frozen on liquid nitrogen. Levels of Hsp/Hsc70 were determined by western blotting using an antibody that recognizes both the inducible and constitutive isoform of the protein, and appears as one band on goby westerns. Bands were visualized by chemiluminescence and their densities were determined using scanning densitometry. All values are expressed as mean ± 1 SEM (N = 20, 13 °C; N = 19, 21 °C; N = 16, 28 °C). Different letters denote significant differences between acclimation groups (p b 0.05). (B) Representative western blot for Hsp/Hsc70 levels in G. mirabilis liver tissue following a 1-month exposure to one of three acclimation temperatures as described in (A).

specifically in Fig. 6 which gives a synthesis of HSF DNAbinding activity and hsp70 mRNA content for each acclimation group at the exposure temperature where hsp70 was first significantly increased over control levels, the changes in both molecular factors appear to occur in tandem. This general trend was also observed in a recent study by Buckley and Hofmann (2004) who examined the kinetics of the response in Juneacclimatized gobies; however, these authors observed an attenuation of the hsp70 mRNA response which was not observed here. In the present study attenuation occurred, not in the Hsp70 mRNA response which continued to rise, but in the HSF1 DNA-binding activity after 90 min in each group at all exposure temperatures. This may be explained, at least in part, by a delay in mRNA synthesis following transcriptional activation of the gene by HSF1. Fig. 6 also shows that the DNA-binding activity in goby liver tissue at time zero (prior to thermal exposure) is not zero, and is higher, although not significantly, in the 21 and 28 °C acclimated fish, respectively. Since several putative HSEs have been found in the promoter region of hsc71 in rainbow trout (Zafarullah et al., 1992), it is likely that at least a small fraction of them are functional and would be activated through HSF1 binding during non-stress conditions. A more recent study by Ojima et al. (2005) identified, through quantitative RT-PCR, differential accumulation of hsc70 mRNA from two separate hsc70 genes in rainbow trout upon heat shock, suggesting that the two genes may also possess promoters with different activities. In the present study we made use of a hsc70 cDNA probe that consistently binds only to the constitutive, albeit perhaps more than one, isoform (Ojima et al., 2005), to examine the kinetics of expression of this chaperone under highly controlled temperature conditions in vitro. Hsc70 mRNA was slightly induced with increasing duration of exposure at the higher

heat block temperatures in the liver tissue of gobies from each of the three acclimation groups (Fig. 2). Upon examination of the fold increase in hsp70 and hsc70 mRNA over time zero controls after 90 min of exposure to each of the six heat block temperatures (Table 1), it became clear that the small but significant increase in the transcription of hsc70 mRNA is likely just a Q10 effect. The increase in hsc70 mRNA levels over time zero controls after 90 min of thermal exposure varied between 0.3 and 2.2X while the increase of hsp70 mRNA exceeded 800-fold in the 21 °C acclimated fish after 90 min at 36 °C. The smallest fold induction in hsp70 mRNA after 90 min at an exposure temperature of 36 °C was observed in the 28 °C acclimation group (Table 1). This could perhaps be explained by the fact that this group had the highest starting levels of Hsp/Hsc70 protein (∼ 2X those in the 13 and 21 °C acclimation groups), and the highest levels of Hsp and Hsc70 mRNA immediately following the 4-week acclimation period, prior to the in vitro thermal exposures (Fig. 7; t = 0, Figs. 1 and 2). While the presence of higher baseline levels of Hsp70 in the 28 °C acclimated fish can be viewed as a pre-adaptation for further high temperature exposure events, it may also indicate that significant protein damage has already occurred in the liver tissue of these fish and that their growth and/or reproductive potential may be compromised by their increased allocation of cellular resources to Hsp synthesis. Further studies are necessary to help explain the observed discrepancy between the HSF1-DNA binding activity data and hsp70 mRNA expression levels in the 28 °C acclimation group at an exposure temperature of 36 °C. While the decline in HSF1 DNA-binding activity suggests that the liver tissue from these warm acclimated fish may be ‘failing’ after 30 min at 36 °C, the robust Hsp70 mRNA response (Fig. 1F) suggests otherwise. Future studies examining the kinetics of the heat shock

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response in vivo may also help to explain the observed Hsp70 mRNA induction after 60 min of 28 °C exposure in the 28 °C acclimated fish. This unexpected finding suggests that the stress level imposed on the isolated tissue heat shocked in vitro could potentially be greater than that experienced by the whole fish. HSF activation is one of many possible targets on which selection may act to modify Hsp expression. Others include promoter sequences as well as those affecting Hsp message degradation, translation and other traits. In addition, other cellular targets, including membrane structure and protein stability may be affected by acclimation in ectotherms. Previous studies using exogenous hsp70 promoters inserted into zebrafish and mobility shift assays in Xenopus have shown that it is not the promoter that determines the threshold temperature of hsp70 activation but HSF1 and its regulation in the cell (Adam et al., 2000; Ovsenek and Heikkila, 1990). In order to more thoroughly differentiate the role that HSF1-DNA binding activity plays in the plasticity of heat shock protein expression in ectothermic organisms, future studies will need to more specifically characterize the full suite of Hsps induced by heat stress, their promoter sequences, and in-situ binding activity of HSF1 and other factors relevant to the transcriptional competency of heat-shock genes. In summary, this study shows that the kinetics of HSF1 DNA-binding activity and corresponding induction of hsp70 mRNA in goby liver tissue are significantly influenced by the thermal history of the fish and are highly dependent on subsequent exposure temperature and duration. The observed acclimation-induced variability in the timing of the transcriptional level regulation of one of the most highly conserved and thermal-responsive heat shock genes suggests that this may be a key component of the ‘cellular thermometer’ lending flexibility to organisms in highly variable thermal environments. Acknowledgments We would especially like to thank Dr. Bradley Buckley for sharing his wealth of knowledge about performing EMSAs with goby liver tissue. We would also like to express our appreciation to everyone who took part in the collection and monitoring of the gobies during their stay in the laboratory. Grants: This research was supported by National Science Foundation (NSF) grants IBN 0096100 and OPP 0301927 to GEH, and by Susan and Bruce Worster to GEH as the Worster Scholar at the University of California, Santa Barbara. SGL was supported by a Natural Sciences and Engineering Research Council (NSERC) Canada, Postdoctoral Fellowship. In addition, MRR was partially supported by a California Alliance for Minority Participation in Science, Engineering and Mathematics (CAMP) Fellowship at UC, Santa Barbara. References Adam, A., Bartfai, R., Lele, Z., Kron, P.H., Orban, L., 2000. Heat-inducible expression of a reporter gene detected by transient assay in zebrafish. Exp. Cell Res. 256, 282–290.

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