Use of Suppression Subtractive Hybridization to Identify Osmotic Stress
Transcription Factors in Tilapia (Oreochromis mossambicus)
Diego F. Fiol, Ph.D. and Dietmar Kültz, Ph.D.
Physiological Genomics Group, Department of Animal
Science, University of California, Davis, One Shields
Avenue, Davis, CA 95616, USA
Gills of euryhaline teleosts are excellent models for
studying osmotic stress adaptation because they
directly contact the aquatic environment and are
an important effector tissue during osmotic stress.
We acclimated tilapia (Oreochromis mossambicus) from fresh water (FW) to seawater (SW);
performed suppression subtractive hybridization
(SSH) of gill mRNAs; and identified two transcription factors, osmotic stress transcription factor
1 (OSTF1) and the tilapia homolog of transcription factor II B (TFIIB), that are rapidly and
transiently induced during hyperosmotic stress.
We conclude that OSTF1 and TFIIB are critical
elements of osmosensory signal transduction in euryhaline teleosts that mediate osmotic adaptation
by means of transcriptional regulation.
Introduction
Euryhaline teleosts are osmoregulators
that maintain plasma osmotic homeostasis
largely by extrarenal NaCl transport. In
seawater (SW) they actively secrete salt
(NaCl), while in fresh water (FW) they
actively absorb salt mainly across gill epithelium. During acclimation of euryhaline
fish from FW to SW, gill epithelium is extensively remodeled to account for altered
requirements of ion transport and permeability. Remodeling includes changes in
turnover of gill epithelial cells, altered differentiation patterns of gill epithelial cells,
and modulation of expression and activity
of many ion transporters actively involved
in osmoregulation (1). Many adaptive
responses to salinity change are based on
transcriptional regulation (2–7). However,
no specific osmotic stress transcription
factors have been identified in teleosts.
In the current study we have performed
suppression subtractive hybridization
(SSH) of gill mRNAs isolated from gill
epithelium of euryhaline tilapia (Oreochromis mossambicus). Using this approach
we identified two novel transcription
factors: osmotic stress transcription factor
1 (OSTF1) and the tilapia homolog of
general transcription factor II B (TFIIB).
5' DPP
3' RACE
244 bp
650 bp
5' RACE
1,700 bp
810 bp
OSTF full-length clone
ORF (410–1,078)
3,300 pb
SSH clone 0
5' RACE
340 bp
B
3' RACE
1,000 bp
600 bp
TFIIB full-length clone
ORF (29–981)
C
FW
OSTF1
4H SW
1,831 pb
D
FW
4H SW
TFIIB
Figure 1. Isolation of OSTF1 and TFIIB by
suppression subtractive hybridization
(SSH). SSH clone 2 (Panel A) and SSH
clone 10 (Panel B) were fully extended
using PCR-based methods such as RACE
(SMART RACE cDNA Amplification Kit)
and DPP (degenerated primer PCR). Semiquantitative RT-PCR from samples of FW
fish (control) and fish acclimated to SW for
4 hr are shown in Panels C and D.
Cloning of two transcriptional
factors that are regulated by
osmolality
Total RNA was isolated from gills of fish
transferred for 4 hr from freshwater (FW)
to seawater (SW) or from FW to FW
(handling controls) using Stratagene’s
RNA isolation kit. mRNA was purified
using the NucleoTrap mRNA Purification
Kit (Clontech). A subtracted library of
cDNAs enriched in the SW condition was
obtained using the SSH-PCR technique
via the Clontech PCR-Select™ cDNA
Subtraction Kit (Cat. No. 637401). Fulllength coding sequences of the original
SSH clones were obtained by using degenerate primers and Clontech’s SMART™
RACE cDNA Amplification Kit (Cat.
No. 634914) (Figure 1, Panels A and B).
Semi-quantitative PCR using gill epithelial mRNA samples from FW- and 4 hr
SW-acclimated fish confirms that OSTF1
and TFIIB transcripts are upregulated
during hypertonic stress (Figure 1, Panels
B and C). A more detailed description of
this study is available (8).
Clontech Laboratories, Inc. • www.clontech.com
CR692118
OSTF1 and TFIIB are immediate
early genes during hyperosmotic stress
SSH clone A
The tilapia clones enriched by SSH were
short and located mainly in the 3’ UTR.
Despite the short sequences identified
by SSH and tilapia being a non-model
species, we were able to rapidly obtain
the full-length sequences and identify
the corresponding clones as OSTF1 and
TFIIB by using Smart-RACE technology. Quantitative real-time PCR (qPCR)
showed that osmotic induction of both
transcripts is transient and follows a
similar time course (Figure 2). Thus,
OSTF1 and TFIIB are coinduced during
hyperosmotic stress with induction kinetics characteristic of immediate early genes
(IEGs). Kinetics of OSTF1 and TFIIB
protein abundance following hyperosmotic stress as assessed by immunoblotting with specific antibodies is similar to
that of mRNA induction with a slight
delay (Figure 3). This manifestation of
hyperosmotic induction of OSTF1 and
TFIIB at the protein level indicates that it
is functionally significant.
The induction of both transcription
factors clearly depends on the degree of
increase in environmental salinity (Figure
4). On the contrary, exposure of fish to
oxidative stress (1 mM H2O2) or heat
shock (36°C) does not increase mRNA
abundance of OSTF1 or TFIIB (Figure
4). To confirm that oxidative stress and
heat shock represented stressful conditions
to the fish we also assayed induction of
Hsp70 in the same samples. Our data indicate that OSTF1 and TFIIB induction
is highly specific for osmotic stress.
OSTF1 belongs to the TSC-22/GILZ
(Transforming Growth Factor beta-Stimulated Clone-22/Glucocorticoid-Induced
Leucine Zipper) protein family of leucine
zipper-containing transcription factors.
These proteins form homo- and heterodimers with other family members (at least
7 in mammals and 5–10 in teleosts).
Based on what is known about functions of TSC-22/GILZ family members,
Reprinted from Clontechniques April 2006 Use of Suppression Subtractive Hybridization to Identify Osmotic Stress
Transcription Factors in Tilapia (Oreochromis mossambicus)…continued
7
6
5
4
3
2
1
0
B
**
*
0
**
1
2
4
6
12
24
72
TFIIB
Relative mRNA
abundance
OSTF
Relative mRNA
abundance
A
7
4
3
2
1
0
**
*
0
1
2
4
Time (hr)
6
12
24
72
Time (hr)
Figure 2. Kinetics of hyperosmotic induction of OSTF1 and TFIIB mRNAs. OSTF1 (Panel A)
and TFIIB (Panel B) transcript abundance was analyzed using quantitative RT-PCR. Experiments were performed 4 times (n=4). Data are shown as means ± SEM, p<0.05 (*) and
p<0.01 (**) compared with control sample (0 hr = FW).
10
8
6
**
*
B
hr 0 2 4 8 152472
**
*
4
0
0
2
4
8
15
24
72
TFIIB
Relative mRNA
abundance
OSTF
Relative mRNA
abundance
A
12
10
8
6
4
0
**
hr 0 2 4 8 152472
**
0
2
4
Time (hr)
*
8
15
24
72
Time (hr)
Figure 3. Time course analysis of hyperosmotic induction of OSTF1 and TFIIB protein
abundance. OSTF1 (Panel A) and TFIIB (Panel B) protein levels were determined by Western
blot and further quantified by densitometry. A typical Western blot is shown as an insert.
Experiments were performed 4 times (n=4). Data are shown as means ± SEM, p<0.05 (*)
and p<0.01 (**) compared with control sample (0 hr = FW).
300 600 9001,000
mOsm/kg
OTSF
Relative mRNA
abundance
D
1.50
1.25
1.00
0.75
0.50
0.25
0
Control
Oxid.
stress
5.00
4.00
3.00
2.00
1.00
0
**
*
0
300 600 900 1,000
mOsm/kg
TFIIB
E
Heat
shock
1.25
1.00
0.75
0.50
0.25
0
Control
Oxid.
stress
HSP70
C
Relative mRNA
abundance
0
**
Relative mRNA
abundance
*
TFIIB
B
**
Relative mRNA
abundance
Relative mRNA
abundance
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0
300
250
200
150
100
50
0
**
**
**
0
300 600
mOsm/kg
Heat
shock
900
HSP70
F
Relative mRNA
abundance
OSTF
A
tion of TFIIB transcripts during stress has
been observed before in archaea and yeast
(10, 11). Because of its coinduction with
TFIIB in tilapia gills, OSTF1 may recruit
TFIIB preferentially to osmoprotective
genes. Consistent with this hypothesis,
TFIIB interacts with other transcription
factors during stress, including NF-kB, cjun, and p53, suggesting that it is targeted
to stress-response genes to facilitate their
transcription (12).
1,000
800
600
400
200
0
**
**
Control
Oxid.
stress
Heat
shock
Figure 4. Analysis of OSTF1, TFIIB, and Hsp70 mRNA expression during hyperosmotic,
oxidative (1 mM H2O2), and heat (+10°C) stresses. OSTF1 (Panels A and D), TFIIB (Panels B
and E), and Hsp70 (Panels C and F) transcript abundance of fish exposed to different
salinities (Panels A, B, and C), and oxidative stress or heat shock (Panels D, E, and F)
for 2 hr were measured using quantitative RT-PCR. Experiments were performed 4 times
(n = 6). Data are shown as means ± SEM, p<0.05 (*) and p<0.01 (**) compared with control
sample (FW, 26°C).
OSTF1 likely plays a dominant role in
the immediate response to hyperosmotic
stress.
to compensate for chromatin compaction
and decreased efficiency of transcription
during hyperosmotic stress (9).
Unlike OSTF1, TFIIB is a general
transcription factor and its hyperosmotic
induction was unexpected. The reason for
hyperosmotic TFIIB induction is currently unknown, although it may be required
Conclusion
References
1. Evans, D. H. (2002) J. Exp. Zool. 293(3):336–347.
2. Tipsmark, C. K., et al. (2002) J. Exp. Zool.
293(2):106–118.
3. Mistry, A. C., et al. (2001) Am. J. Physiol. Regul.
Integr. Comp Physiol 281(5):R1594–R1604.
4. Takeuchi, K., et al. (2000) Fish Physiol. Biochem.
23(2):173–182.
5. Cutler, C. P. & Cramb, G. (2002) J. Exp. Biol.
205(Pt 17):2643–2651.
6. Kültz, D., et al. (2001) J. Exp. Biol.
204(Pt 17):2975–2985.
7. Mistry, A. C., et al. (2001) Biochem. J.
360(Pt 1):107–115.
8. Fiol, D. F. & Kültz, D. (2005) Proc. Natl. Acad.
Sci. USA 102(3):927–932.
9. Kültz, D. (2000) Environmental Stressors and
Gene Responses, eds. Storey, K. B. & Storey, J.
(Elsevier, pp. 157–179.)
10.Thompson, D. K., et al. (1999) Mol. Microbiol.
33(5):1081–1092.
11.Hoopes, B. C., et al. (2000) Nucleic Acids Res.
28(22):4435–4443.
12.Espinosa, J. M., et al. (2003) Mol. Cell
12(4):1015–1027.
For Research Use Only. Not for use in diagnostic
or therapeutic procedures. Not for resale. Clontech,
Clontech logo, and all other trademarks are the property
of Clontech Laboratories, Inc. Clontech is a Takara Bio
Company. Copyright 2006.
Notice to Purchaser
For all licensing information, visit www.clontech.com
SSH methodology enabled us to identify
two new transcription factors involved
in osmotic regulation in tilapia. Induc-
Clontech Laboratories, Inc. • www.clontech.com
Reprinted from Clontechniques April 2006