Tài liệu Báo cáo khóa học: Cloning and characterization of two distinct isoforms of rainbow trout

Cloning and characterization of two distinct isoforms of rainbow

trout heat shock factor 1

Evidence for heterotrimer formation

Nobuhiko Ojima and Michiaki Yamashita

Cell Biology Section, Physiology and Molecular Biology Division, National Research Institute of Fisheries Science,

Fisheries Research Agency, Yokohama, Japan

To elucidate the molecular mechanism underlying the

heat shock response in cold-water fish species, genes enco￾ding heat shock transcription factors (HSFs) were cloned

from RTG-2cells of the rainbow troutOncorhynchus mykiss.

Consequently, two distinct HSF1 genes, named HSF1a and

HSF1b, were identified. The predicted amino acid sequence

of HSF1a shows 86.4% identity to that of HSF1b. The two

proteins contained the general structural motifs of HSF1, i.e.

a DNA-binding domain, hydrophobic heptad repeats and

nuclear localization signals. Southern blot analysis showed

that each HSF1 is encoded by a distinct gene. The two HSF1

mRNAs were coexpressed in unstressed rainbow trout

RTG-2cells and in various tissues. In an electrophoretic

mobility shift assay, each in vitro translated HSF1 bound to

the heat shock element. Chemical cross-linking and

immunoprecipitation analysis showed that HSF1a and

HSF1b form heterotrimers as well as homotrimers. Taken

together, these results demonstrate that in rainbow trout cells

there are two distinct HSF1 isoforms that can form

heterotrimers, suggesting that a unique molecular mech￾anism underlies the stress response in tetraploid and/or

cold-water fish species.

Keywords: heat shock factor; HSF1; isoform; rainbow

trout; trimerization.

Heat shock proteins (HSPs) are highly conserved among a

wide range of animals and are induced by environmental

stressors such as elevated temperature, heavy metals, and

oxidants. Many kinds of HSP have been reported to act as

molecular chaperones that aid in the folding, assembly,

degradation and translocation of intracellular proteins [1].

The expression of HSP genes is regulated by heat shock

transcription factors (HSFs) that bind to a specific cis-acting

element, namely, the heat shock element (HSE) [2–4]. In

vertebrates, genes encoding four types of HSFs, HSF1–

HSF4, have been cloned. Among the HSF family members,

HSF1 is the principal transcriptional factor activated by

exposure to stresses such as heat shock, and this protein is

known to form homotrimers that bind DNA [2–4].

Fish are ideal models in which to study the cellular heat

shock response because they are poikilotherms and are

subjected to daily and seasonal temperature fluctuations.

Moreover, during evolution fish have adapted to live in

various ambient temperatures. Reflecting such adaptations,

the threshold temperature for HSP induction differs

between cold- and warm-adapted fishes. For example,

HSPs are induced in the 26–30
C range in rainbow trout

RTG-2cells [5], whereas HSP70 is induced in the 35–37
C

range in zebrafish tissues [6]. However, little is known about

the molecular mechanisms underlying the difference in HSP

induction temperatures in fish species. To date, one HSF1

cDNA has been isolated from zebrafish, which is a warm￾adapted fish [6]. Although Ra˚bergh et al. [6] have also

cloned a cDNA fragment encoding an HSF from bluegill

sunfish, a full-length HSF1 cDNA clone has not been

isolated from any fish other than zebrafish. Some authors

[7,8] have reported the presence of a protein that possesses

HSF1-like activity in rainbow trout; however, an HSF1

gene itself has not been identified in this cold-adapted fish.

In the present study, we have identified and characterized

a rainbow trout HSF in order to clarify the molecular

mechanism underlying the stress response in cold-water fish

species. Here, we present evidence for existence of two

distinct HSF1 isoforms in rainbow trout and the formation

of heterotrimers of these isoforms in vitro.

Materials and methods

Cell culture and animals

Rainbow trout gonadal fibroblast cell line RTG-2cells

[9] were cultured at 15
C in Leibovitz’s L-15 medium

Correspondence to N. Ojima, National Research Institute of Fisheries

Science, Fisheries Research Agency, Fukuura, Kanazawa-ku,

Yokohama 236-8648, Japan.

Fax: + 81 45 7885001, Tel.: + 81 45 7887643,

E-mail: [email protected]

Abbreviations: HSF, heat shock factor; HSP, heat shock protein; HSE,

heat shock element; HSC, heat shock cognate; DIG, digoxigenin;

HA, hemagglutinin; EMSA, electrophoretic mobility shift assay;

EGS, ethylene glycol bis (succinimidyl succinate); DBD,

DNA binding domain; HR, hydrophobic heptad repeat;

NLS, nuclear localization signal.

(Received 1 September 2003, revised 15 October 2003,

accepted 18 December 2003)

Eur. J. Biochem. 271, 703–712(2004) FEBS 2004 doi:10.1111/j.1432-1033.2003.03972.x