Reciprocal activation of HSF1 and HSF3 in brain and blood tissues:

Is redundancy developmentally related?

Ariel Shabtay1,2 and Zeev Arad1,*

1Department of Biology, Technion-Israel Institute of Technology,

Haifa 32000, Israel

2Department of Cattle and Genetic Sciences, Institute of Animal Science,

Agricultural Research Organization, Newe-Ya’ar Research Center, P.O.B

1021, Ramat Yishay 30095, Israel.

*Corresponding author: Zeev Arad, Department of Biology, Technion, Haifa 32000,

Israel. Tel: 972-4-8293416; Fax: 972-4-8225153; e-mail: zarad@tx.technion.ac.il

Running head: Reciprocal Activation of Heat Shock Factors

Copyright © 2006 by the American Physiological Society.

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Abstract

Transcriptional induction of heat shock genes in response to temperature elevation

and other stresses is mediated by heat shock transcription factors (HSFs). Avian

cells express two redundant heat-shock responsive factors, HSF1 and HSF3, which

differ in their activation kinetics and threshold induction temperature. Unlike the

ubiquitous activation of HSF1, the DNA-binding activity of HSF3 is restricted to

undifferentiated avian cells and embryonic tissues. Herein we report a reciprocal

activation of HSF1 and HSF3 in vivo. Whereas HSF1 mediates transcriptional

activity only in the brain upon severe heat shock, HSF3 is exclusively activated in

blood cells upon light, moderate and severe heat shock, promoting induction of heat

shock genes. Although not activated, HSF1 is expressed in blood cells nuclei in a

granular appearance, suggesting regulation of genes other than heat shock genes.

Intraspecific comparison of heat sensitive and heat resistant fowl strains indicates

that the unique activation pattern of HSF3 in blood tissue is a general phenomenon,

non-related to thermal history. Taken together, HSF1 and HSF3 mediate

transcriptional activity of adult tissues and differentiated cells in a non-redundant

manner. Instead, an exclusive, tissue-specific activation is observed, implying that

redundancy may be developmentally related. The physiological and developmental

implications are discussed.

Keywords: HSF, reciprocal activation, redundancy

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Introduction

The heat shock response is characterized by induced synthesis of heat shock proteins

(HSPs), the accumulation of which confers cellular thermotolerance (15). The expression

of HSPs is primarily regulated at the level of transcription by a family of heat shock

transcription factors (37). To date, four members of the HSF gene family have been

isolated and characterized in vertebrates (42, 44, 46, 47, 51). The existence of multiple

family members suggests that different HSFs mediate the responses to various forms of

physiological and environmental stimuli (45). Accordingly, HSF1 acts as a classical

stress-responsive factor, inducing transcriptional activity of heat shock genes in response

to diverse forms of stress. HSF2 is not activated in response to classical stress stimuli, but

is rather active under developmentally related conditions (14, 54-55) and mediates

bookmarking of inducible HSPs (61). The unique avian-specific factor HSF3 (19), like

HSF1, is a heat responsive transcription factor but is considered as a high temperature

activator (43, 56). The mammalian HSF, HSF4, is restricted to certain tissues, and has the

properties of a negative regulator of heat shock gene expression (44, 58). Among the

three HSF genes that are expressed in avian cells, HSF1 and HSF3 are considered

redundant heat shock responsive factors (42-43). Consistent with this, HSF3 exhibits a

delayed response compared to the relatively rapid activation of HSF1, and is detected

predominantly upon severe heat shock (43, 56).

The activation of HSF1, the predominant stress-responsive factor, involves multiple

steps, including translocation to the nuclear compartment, oligomerization from the latent

monomer to a trimer, acquisition of DNA-binding activity, inducible serine

phosphorylation, and subsequent transcriptional activity (9, 11, 18, 27, 48, 59). Non-

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active HSF3 is a dimer which is exclusively cytosolic (43). Upon heat stress, HSF3 is

translocated into the nucleus, using the nuclear localization signal to be converted to an

active trimer (40, 43).

In contrast to the ubiquitous activation of HSF1 in vertebrates (48), acquisition of HSF3

DNA-binding activity in response to heat shock appears to be a cell-specific event, and

active HSF3 has been detected only in avian undifferentiated cells and embryonic tissues

(28, 43, 56). Most interestingly, disruption of HSF3 in HSF1-expressing chicken

B-lymphoblast cells results in impaired heat shock response and in loss of

thermotolerance (57). In light of the above reported findings we sought to gain insight

into the relative contribution of HSF1 and HSF3 to the transcriptional induction of heat

shock genes in mature tissues and differentiated cells, in vivo. For this purpose we chose

brain and blood of mature hen, two unique tissues that are differentially confronted with

thermal challenges. Our results show that in each tissue, only one transcription factor is

activated upon heat stress. Whereas HSF1 mediates transcriptional activity in the brain,

HSF3 is exclusively activated in blood cells upon moderate and severe heat shock to

induce the expression of heat shock genes.

Materials and Methods

Real time in vivo measurements of the heat shock response

Two different groups of mature chickens, Leghorn and Bedouin (n=3 in each group), that

differ genetically in their resistance to heat were examined in this setup. The setup was

recently described in details (52). Briefly, Twenty hours prior to experiment, a

polyethylene cannula was implanted in the brachial vein of mature fowl under local

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anaesthesia, and a 5cm-long, custom-made polyethylene cannula was implanted dorsal to

the rectum and fastened to the skin. Experiments were carried out within a temperature-

controlled room (±0.3oC; 12L:12D light cycle, lights on at 6 am). The birds had free

access to food and water and could freely move in their individual cages. Each

experiment started at 8-9 am at an ambient temperature (Ta) of 24oC (RH=50±5%) which

is within the thermoneutral zone of chickens (5). A copper-constantan thermocouple was

introduced into the rectal cannula, locked at a pre-determined depth of 5cm and

connected to a digital thermometer (±0.1oC). All temperature probes were calibrated

against a mercury-in-glass thermometer having accuracy (0.1 oC) traceable to the U.S.

Bureau of Standards. After approximately 10min, body temperature (Tb) stabilized

around 41oC (the normal resting Tb for this species), after which blood was remotely

sampled through extended tubing. Ta was then elevated to 38oC (RH=50±5%). Body

temperature was followed continuously and blood samples were taken at each 1oC

increase in Tb up to 45oC. At this time, ambient temperature was lowered back to 24oC

and blood samples taken at each 1oC decrease in Tb down to 41oC. These samples were

analyzed for HSF DNA-binding activity, transcriptional and translational activity and

HSF translocation. After each blood sample the cannula was flushed with heparinized

saline.

Protein extraction

For the purpose of protein extraction, brain tissues were sampled from Tb=45oC heat

shocked mature hens. The dissected tissues were homogenized in ice cold buffer,

containing 0.1M NaCl, 20mM Tris pH 7.4, 0.2mM EDTA, 20% glycerol (v/v), 0.5mM

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dithiothreitol (DTT), 15µg/ml leupeptin, 1mM phenylmethylsulfonylfluoride (PMSF).

Samples were centrifuged for 30 min (4oC, 12000 rpm) and supernatants were collected,

frozen in liquid nitrogen and stored at -70oC. To extract proteins from blood cells, blood

samples were washed once with ice cold PBS and centrifuged for 2 min (4oC, 1500 rpm).

The cells were re-suspended in ice cold TMP buffer (containing 10mM Tris pH 7.4, 1mM

EDTA, 5mM MgCl2, 0.5mM DTT, 15µg/ml leupeptin, 1mM PMSF), and were frozen

(liquid nitrogen) and thawed (37oC) in 4 cycles. Samples were centrifuged for 30 min

(4oC, 12000 rpm) and supernatants were collected, frozen in liquid nitrogen and stored at

-70oC. Blood cell nuclear proteins were extracted as previously described by Dyer and

Herzog (13).

SDS PAGE and Western Blot

Blood cell nuclear proteins lysates were boiled in sample application buffer containing

2-mercaptoethanol. Equal amounts of proteins (in each lane) were loaded and separated

by SDS-polyacrylamide gel (10%), and transferred onto nitrocellulose membrane

(Schleicher&Schuell). The membranes were probed with polyclonal anti HSF1β (a

generous gift from Professor Akira Nakai), followed by appropriate secondary antibodies.

The proteins were visualized by enhanced chemiluminescence. Quantification of HSF1

expression was carried out by a densitometry software (TINA).

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Electromobility shift assay (EMSA)

Electromobility shift assay was performed as previously described (38). Briefly, equal

amounts of cellular proteins (20µg for brain extraction and 5µg for blood nuclei proteins)

were incubated with a 32P-labeled double-stranded oligonucleotide

(5’- CTAGAAGCTTCTAGAAGCTTCTAG-3’). The protein-bound and free

oligonucleotides were electrophoretically separated by 4% native polyacrylamide gels.

The gels were dried and auto-radiographed.

Supershift experiments

The antibody supershift experiments were performed by incubating 2µl of 1:10 diluted

antisera (unless otherwise stated in the figure legend) of anti HSF1γ and anti HSF3 γ (a

kind gift from Professor Akira Nakai) in PBS, with 1–2 µl of cell lysates in a total

volume of 10 µl. These antibodies recognize the carboxyl-terminal region of each HSF.

After incubation on ice for 20 min, a nucleotide probe mixture containing 0.1ng of 32P-

labeled self-complementary ideal HSE oligonucleotides and 0.5µg of poly(dI-dC)

(Pharmacia Fine Chemicals, Piscataway, NJ) in 10mM Tris-HCl (pH 7.8) and 1 mM

EDTA was added to a final volume of 25µl. The binding reactions were incubated on ice

for 20 min, mixed with 2µl of a dye solution (0.2% bromphenol blue, 0.2% xylene

cyanol, and 50% glycerol) and then separated on 4% native polyacrylamide gels. Gels

were run in 0.5xTBE buffer at room temperature for 1.7 h at 140 V, dried, and exposed to

x-ray film at -80°C with intensifying screens.

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RNA isolation and Northern blot

Total RNA was isolated from blood cells by TRI REAGENT-BD (MRC) according to

the manufacturer's instructions. RNA (5µg) was separated in formaldehyde-agarose gel

and transferred onto nylon membrane (Zeta-Probe, Bio-Rad). Chicken HSP90α cDNA

(a kind gift from Professor Akira Nakai) was labeled by the extension priming method

using [α-32P]dATP. Membrane was hybridized for 16 hours at 55oC, washed with

0.1%SDS in 1 X SSC at 45oC, 50oC and 55oC, and exposed to x-ray film (Kodak BioMax

MS) at –70oC in the presence of intensifying screen.

Immunocytochemistry

Blood samples from control and Tb=45oC heat shocked mature hens were prepared as

smears. The smears were air dried and fixed with 4% paraformaldehyde (in PBS). After

blocking with 10% normal goat serum in PBS, cells were incubated with 1:500 diluted

polyclonal αHSF1 (a kind gift from Professor Akira Nakai). The HSF1 signal was

detected by a secondary goat anti-rabbit antibody conjugated to FITC. Confocal

reconstructions were made by using an MRC-1024, laser confocal scanning microscope

(Bio-Rad, Hempstead, UK) with the objective Nikon Plan Apo 603/1.40. DNA was

counterstained with 250ng/ml propidium iodide (PI).

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Results

HSF3 is exclusively activated in chicken blood tissue

We have recently described the in vivo, real-time heat shock response of fowl from

strains that differ in their resistance to heat. Our data revealed a differential DNA-binding

kinetics of total HSF in each of the groups that was followed by transcriptional and

translational induction of heat shock genes (52). To elucidate the redundancy in the

activation of both, HSF1 and HSF3, in response to elevated temperature, blood of mature

hen was sampled, at 1oC increments of Tb. We determined the relative levels of HSF1 and

HSF3 DNA-binding activity by supershift experiments. At Tb = 45oC, HSF3-bound DNA

was entirely supershifted by each antisera concentration, while the DNA-binding activity

of HSF1 was undetected (Fig. 1A). It has been previously reported that HSF3 is the

dominant heat shock transcription mediator during severe heat shock conditions. It was

thus reasonable to assume that the activation of HSF1 may be abolished at Tb = 45oC, but

is likely to occur at lower body temperatures. To check the activation pattern of both

HSFs at moderate heat shock conditions, we performed supershift experiments of blood

sampled at Tb = 43oC, a well-documented temperature for the activation of the ubiquitous

factor HSF1. In this experiment we used the same HSF1 antisera concentrations, but 10-

fold more diluted concentrations of HSF3 antisera, to allow the examination of the anti-

HSF3 antibody specificity. As seen in figure 1B, HSF3 was the exclusive DNA-bound

heat shock transcription factor, also upon moderate heat shock conditions.

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HSF1 and HSF3 DNA-binding states are not affected by intraspecific variations in

thermal resistance

Our previous comparisons of the Leghorn chicken with the desert-origin, genetically heat

resistant Bedouin fowl identified various thermoregulatory mechanisms that contribute to

the superiority of the Bedouin fowl with respect to heat resistance (2, 4, 32). This

superiority was accompanied by a considerable delayed HSP response, both in relation to

time scale and to Tb level (52). In light of the above-mentioned observations it was

intriguing to examine the DNA-binding state of both HSFs in heat sensitive and heat

resistant strains of the same species. Consistent with the findings shown in Figure 1,

HSF3 was solely in a DNA-binding form in both fowl strains, and along the entire range

of heat shock conditions (Tb = 42oC to Tb = 45oC), without any redundant acquisition of

HSF1 DNA-binding activity in response to heat shock (Fig. 2).

The HSF3 DNA-binding activity in both the Leghorn and the Bedouin strains is slightly

detected in control, non-stress conditions. However, the intraspecific differences in the

cellular heat shock response are manifested at heat shock conditions, with Leghorn

showing a profound HSF3 activation at Tb = 42oC, in comparison with Tb = 44oC of the

Bedouin strain (Fig. 2).

HSF1 is expressed in blood cells and forms nuclear granules, but is not activated

Following the surprising findings that HSF1 may not participate in mediating in vivo the

cellular heat shock response of avian blood tissue, we next examined the expression of

HSF1 in chicken blood cells. We extracted nuclear proteins from blood samples of hens

that were exposed to heat shock and allowed to recover, and analyzed the expression of

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HSF1 by Western blot analysis, using polyclonal anti HSF1 antibody. HSF1 was revealed

in the nuclear fraction of the blood cells in control, during heat shock and during recovery

from heat shock. However, the amount of expressed HSF1 was not uniform in all

samples; its concentration tended to decrease around the peak of heat shock (Tb = 45oC;

Tb = 44oC, at recovery. These values were 2 and 3.3 fold lower, respectively, than the

control values) and tended to increase during recovery from heat shock while Tb was

decreasing (Fig. 3A). Despite its expression in blood cells nuclei in non-stress conditions

(Fig. 3A), immunohistochemical preparates from control and from heat shocked cells

(Tb = 44oC) indicate that HSF1 tranlocates from a cytosolic distribution in control to a

nuclear granular distribution upon heat shock (Fig. 3B).

Activated HSF1 exclusively mediated transcriptional activity of HSPs in chicken

brain tissue

Is the exclusive HSF3 DNA-binding pattern unique to the blood tissue, or may it reflect a

general in vivo phenomenon in adult fowl? To address this issue we conducted supershift

experiments in brain extracts from heat shocked (Tb = 45oC) mature hen, using polyclonal

anti HSF1 and anti HSF3 antibodies. A reciprocal mode of HSF activation to that of the

blood tissue was revealed in the brain, indicating that HSF1 was exclusively in a DNA-

binding state (Fig. 4A).

To elucidate whether the DNA-binding state of HSF1 in the brain induces the activation

of heat shock genes, we performed a Northern blot analysis with the entire chicken

HSP90α cDNA as a probe. A burst induction of HSP90 was observed in response to

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Tb = 45oC heat shock, implying that HSF1 is capable of mediating the transcriptional heat

shock response in the brain in a non-HSF3 redundant manner (Fig. 4B).

Discussion

Stress-induced transcription of heat shock genes in avian cells is mediated by HSF1 and

HSF3 (39). The co-expression and co-activation of these factors has led to the discovery

of the HSF functional redundancy (37). Indeed, when undifferentiated avian cells and

avian embryonic tissues are subjected to thermal challenges, HSF1 and HSF3 are

activated with different kinetic properties and threshold temperatures, to induce

transcription of heat shock genes (28, 43, 56). Surprisingly, as demonstrated in the

present study, circulating blood cells lose their functional redundancy and activate only

HSF3, also upon light and moderate heat shocks. These results are of developmental and

physiological significance, since they imply that during maturation of erythroblasts,

HSF1 activity is degenerated. As maturating erythroblasts go through morphological,

physiological and biochemical changes (26), loss of redundancy due to diminished

expression of HSF1 may be a reasonable explanation. Supported by the nuclear

expression pattern of HSF1 in control and heat shocked blood cells, it is more likely that

this degenerative event has occurred along the pathway that regulates DNA binding

activity. Transcriptional induction of heat shock genes may be a multiple step process.

Under normal conditions of cell growth, the non-DNA binding state of HSF1 is regulated

by two distinct mechanisms, involving both negative regulatory domains and constitutive

phosphorylation at serine residues (16, 29-30, 53). To induce transcriptional activity of

heat shock genes, HSF1 must acquire DNA binding activity, preceded by oligomerization

and nuclear localization (48). However, the trimer state of HSF1 does not assure binding

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interactions with the DNA, as interactions of the HSF1 oligomerization domain with

regulatory proteins may negatively affect HSF1 DNA-binding activity (50). Consistent

with the findings that DNA binding and transcriptional activities are uncoupled processes

(27, 31) HSF1 must be inducibly phosphorylated to promote transcription of heat shock

genes (11, 18).

In our current study, wing blood cells immunohistochemistry, show that apart from its

nuclear expression in control and heat shock conditions, HSF1 translocates from a

cytosolic distribution at control conditions to a nuclear granular distribution at Tb = 44oC.

Nuclear stress granules have been described as intra-nuclear sites at which HSF1 is

concentrating in response to stress conditions (48). The kinetics of appearance of HSF1

granules parallels the activation of HSF1 and the transient transcriptional activity of heat

shock genes (12). It has been suggested that these nuclear foci are locations at which

HSF1 is stored and regulated in its different activation states (22), thereby coordinating

the regulation of heat shock gene expression (23). As HSF1 stress granules are also

present in heat shocked mitotic cells that are devoid of transcription, it is alternatively

hypothesized that they are involved in chromatin organization and thus have a structural

role in protecting hypersensitive or fragile sites of the genome (23-25). The expression

levels of HSF1 in the present study tend to decrease as Tb reaches its experimental peak

and return to control levels upon recovery. This observation is in accordance with a

previous study (56), which reported, in vitro, the movement of HSF1 to the insoluble

fraction at high ambient temperature.

Nearly four decades ago the unique thermoregulatory capacities of the desert-origin

Bedouin fowl were described (1-2, 4-8, 32-33). Recently, we have monitored in real-time

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in vivo the heat shock response of this heat resistant fowl strain, and showed that its heat

resistance superiority was characterized by a considerable delayed activation of HSF,

compared with a heat sensitive, commercial strain (52). In the present study we

demonstrate that HSF3 is solely in a DNA-binding form in both fowl strains and along

the entire range of heat shock conditions, and that no redundant activity of HSF1 is taking

place. This intraspecific comparison of two evolutionary distinct strains, with respect to

thermoregulatory strategies, suggests that the unique activation pattern of HSF3 in the

blood tissue is a general phenomenon, non-related to thermal history. Together with the

above mentioned findings, these results imply that loss of HSF1 DNA-binding activity is

not caused by decreased levels of HSF1 message, nor because HSF1 had become

insoluble.

In chicken embryonic tissues, including red blood cells and brain tissue, HSF3 was the

dominant heat shock responsive factor in certain developmental stages, whereas in other

stages HSF1 and HSF3 showed functional redundancy (28). As opposed to the blood, in

which paralleled activation of HSF3 in embryos (28) and adult chicken (present study) is

sustained, the present findings in the brain tissue revealed an unexpected loss of HSF3

activation and the introduction of HSF1 as an exclusive heat shock responsive factor.

Chicken HSF3 remains as an inactive dimmer, exclusively in the cytoplasm of normally

growing cells and is translocated into the nucleus and converted to an active trimer upon

heat stress. Its DNA-binding properties, however, are regulated in a cell-specific manner

(43). As HSF3 activation in embryonic brain tissue is evident (28), and as HSF3 is

expressed in adult chicken brain tissue (present study, data not shown), we believe that

the unique regulation of avian brain temperature could provide a reasonable physiological

15

explanation for the non DNA-binding state of HSF3 in this tissue. Equipped with a

unique heat-exchange vasculature, the ophthalmic rete (35), the avian brain temperature

is maintained around 1oC below body temperature in a wide range of ambient

temperatures (see review by Arad; 3) and hydration conditions (8, 21, 36). In the context

of the present study this means that brain temperature probably did not exceed 44oC

during heat shock. Thus, although HSF3 has been previously shown to be activated at

44oC (56), we cannot exclude that the brain was not confronted with temperatures above

the threshold for HSF3 activation. It may alternatively be claimed that as the brain is

protected from reaching such high temperatures, it has lost part of its temperature induced

DNA-binding activity to promote transcription of heat shock genes, during ontogeny.

While chicken body temperature that exceeds 45oC is considered harmful and non-

reversible, this issue must be solved by a method of acclimation that would allow us

reach a body temperature of 46oC.

In mammals, where HSF1 is believed to be the only factor that mediates heat shock

signals (59), its disruption may not affect adulthood attainment, but may result in

multiple defective phenotypes, including “maternal effect” mutation (10, 60). Moreover,

mammalian HSF1-deficient cells are incapable of stress-induced transcription of heat

shock genes, and of acquirement of thermotolerance (34). Compared to the mammalian

HSF1, avian HSF1 has a little potency of activating heat shock genes in cells subjected to

heat stress (19). It is becoming clearer, however, that similar to the mammalian HSF1,

avian HSF1 also possesses thermal-protective traits, independent of heat shock genes

induction (19-20, 41). Also similar to HSF1 in mammalian cells, HSF3 is essential for the

heat shock response in avian cells, and its disruption decreases markedly the induction of

16

heat shock genes (57). This is further emphasized during chicken embryonic

development, where HSF3 that has nearly identical levels among most examined tissues

is the major heat shock responsive factor (28). These data are in accordance with the

“blood picture” but fail to explain the “brain picture” in the present study. The

physiological nature of both tissues in respect to thermoregulation may shed light on the

reciprocal activation pattern of both HSFs. In marked difference from the brain that

"escapes" the actual body temperature (review by Arad, 3), the blood tissue, among its

other roles, is responsible for heat transfer from the body core to the surface, and as such

is routinely confronted with temperature changes, and should have the ability to respond

to a broad range of temperatures. This function is shown here to be entirely fulfilled by

HSF3. In addition to the biochemical characteristics of HSF1 and HSF3 in regard to the

heat shock response (56), and as HSF3 lacks (so far, as we know) the characteristics of

non-stress responsive genes regulator, these physiological challenges make HSF3 a

reasonable candidate for taking over the transcriptional mediation of heat shock genes.

Indeed, HSF3 might be sufficient to activate transcription in chicken blood tissue, since

red blood cells of chicken embryos reveal a heat shock response in the absence of HSF1

(28). As to the functional redundancy observed in non-differentiated cells such as

erythroblasts, these cells are progenitors to circulating cells and are considered as the

bank of the differentiated cells. It is thus logical to have them protected from

physiological and environmental damage by a redundant system. In the brain, loss of

HSFs’ functional redundancy during ontogeny may be explained by the different thermal

states of the embryo and the mature chicken. The embryo, which is considered

ectothermic, undergoes a transition from ectothermy to endothermy, which is completed,

17

at the earliest, by hatching (17). While ectotherms are prone to changes in body

temperature in correlation with ambient temperature variations, the internal physiological

milieu of endotherms remains relatively stable despite acute external thermal

fluctuations. We have recently shown that embryos are the most heat sensitive stage in

the chicken's life cycle, and are tightly dependent on HSP synthesis for survival. On the

other hand, mature hen master additional mechanisms for heat dissipation and Tb

regulation during ontogeny (52). In light of the above, we suggest that functional

redundancy of HSFs in the brain is required in the thermal state that is most susceptible to

heat stress (i.e. embryos), but may be lost as the individual is capable of maintaining

homeostasis (i.e. adult).

It has been previously shown that the temperature set-point for HSF activation varies

among tissues of a single organism (49). Herein we present data that may confirm also

the exsitance of a tissue-specific activated HSF.

Taken together, the functional HSFs’ redundancy, previously described in avian

embryonic tissues and in undifferentiated cells, may be lost during ontogeny. As a result,

an exclusive reciprocal activation of HSF1 and HSF3 is observed in vivo in the brain and

in blood tissues, respectively. HSFs’ functional redundancy may be a mechanism unique

to undifferentiated embryonic cells, and is lost due to physiological alterations that

accompany maturation.

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Acknowledgements

We thank Professor Akira Nakai for kindly providing the anti-HSF1 and anti-HSF3

antibodies and the HSP90α cDNA. The technical assistance of Ms. Shoshana Goldenberg

and Mr. Udi Ron is deeply acknowledged. This research was supported by the Israel

Science Foundation. This study was approved by the Technion Review Board of Animal

Experimentation (No. B-93-6).

30

Figure legends

Fig. 1. The effect of heat exposure on HSF1 and HSF3 activation in blood cells from

mature hen at Tb=45oC (A) or at Tb=43oC (B). Samples were prepared from blood cell’

nuclei extracts for electro mobility shift assay (EMSA). After incubation of nuclei lysates

(5µg) with αHSF1γ or αHSF3γ (0.5, 1, 2µl of 1:10 diluted serum in PBS, panel A or 0.5,

1, 2µl of 1:10 diluted serum in PBS for αHSF1 and 0.5, 1, 2µl of 1:100 diluted serum in

PBS for αHSF3, panel B) at room temperature for 15 min, [32P]oligonucleotide,

containing four inverted nGAAn repeats (HSE) was added. The mixtures were loaded on

4% native polyacrylamide gel.

Fig. 2. The effect of heat exposure on HSF3 activation in blood cells from mature hens of

thermosensitive (Leghorn) and thermoresistant (Bedouin) fowl. Samples were prepared

from blood cell’ nuclei extracts for EMSA. After incubation of nuclei lysates (5µg) with

αHSF3γ (1µl of 1:10 diluted serum in PBS) at room temperature for 15 min,

[32P]oligonucleotide, containing four inverted nGAAn repeats (HSE) was added. The

mixtures were loaded on 4% native polyacrylamide gel.

Fig. 3. The effect of heat exposure on the nuclear expression (A) and translocation (B) of

HSF1 in blood cells. Blood was sampled from brachial vein of mature hen at Tb=45oC

and prepared as a smear for microscopic observation. The smear was fixed, treated with

RNase and stained with PI for DNA visualization (red) and with polyclonal αHSF1β. The

HSF1 signal was detected by a secondary antibody conjugated to FITC (green). The

31

yellow appearance is a result of merge between green and red and indicates the

translocation of HSF1 into the nucleus. Images were obtained by confocal microscopy.

Fig. 4. A. The effect of heat exposure at Tb=45oC on the activation of HSF1 and HSF3

(A) and on the transcription of HSP90 (B) in the brain tissue of mature hens.

A. Samples were prepared from whole brain extracts for electro mobility shift assay

(EMSA). After incubation of lysates (10 µg) with αHSF1γ or αHSF3γ (1 µl of 1:10

diluted serum in PBS) at room temperature for 15 minutes, [32P]oligonucleotide

containing four inverted nGAAn repeats (HSE) was added. The mixtures were loaded on

4% native polyacrylamide gel. NS – non-specific binding, PI – pre-immune serum. B.

HSP90 mRNA levels were detected by chicken HSP90α cDNA as a probe. Each lane

represents a different individual.

32

33

34

35

HSF1

Tb(oC) C 42 43 44 45 44r 43r 42r 41r

Fig 3A

HSF1

Tb(oC) C 42 43 44 45 44r 43r 42r 41r

Fig 3A

Control HS

Fig 3B

Control HS

Fig 3B

36

HSP90

rRNA

C HS

Fig 4B

HSP90

rRNA

C HS

HSP90

rRNA

C HS

Fig 4B

Activated HSF

NS

Supershift

Free DNA

- PI + - - PI + -- - - + - - - +

C HS

Fig 4AαHSF1αHSF3

Activated HSF

NS

Supershift

Free DNA

- PI + - - PI + -- - - + - - - +

C HS

Fig 4AαHSF1αHSF3