Regulation of promoter occupancy during activation of cryptobiotic embryos from the crustacean Artemia franciscana
Instituto de Investigaciones Biomédicas CSIC/UAM, C/ Arturo Duperier No. 4, 28029 Madrid, Spain
* Author for correspondence (e-mail: lsastre{at}iib.uam.es)
Accepted 10 February 2003
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Summary |
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Key words: actin, Artemia franciscana, Ca2+-ATPase, cryptobiosis, development, gene expression, Na+/K+-ATPase, promoter, transcription
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Introduction |
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The branchiopodan crustacean Artemia franciscana is a model system
that has frequently been used to study this phenomenon
(Browne et al., 1991). Embryos
of these animals can enter cryptobiosis at the gastrula stage and remain
viable for years. The process involves complete dehydration of the embryos
(anhydrobiosis), which are surrounded by a hard shell and are known as cysts
(Drinkwater and Clegg, 1991
).
Reversion of cryptobiosis requires hydration of the embryos but also
additional activation steps. For example, hydrated embryos can continue under
cryptobiosis for several years under anoxia (anoxybiosis;
Clegg, 1997
). Subsequently,
development continues through the formation of a swimming nauplius, which
hatches from the cyst shell in approximately 24 h.
Artemia cysts present undetectable metabolic activity under
anhydrobiosis. Even in anoxybiosis, only a metabolic rate 50 000 times slower
than the aerobic rate could be detected
(Clegg, 1997). Several studies
have demonstrated that intermediary metabolism and protein synthesis are
reinitiated within minutes of cyst activation
(Tate and Marshall, 1991
). In
these early stages of reactivation, protein synthesis is dependent on the
existence of a large pool of stored mRNA
(Amaldi et al., 1977
).
Reinitiation of protein synthesis is also possible because of the existence of
large pools of stored ribosomes and accessory molecules, such as initiation
and elongation factors (Sierra et al.,
1974
). In vitro experiments have shown that cyst extracts
are not competent for protein synthesis, despite the presence of most of the
necessary components, as mentioned above, and several activation mechanisms
have been proposed (Wahba and Woodley,
1984
; Moreno et al.,
1991
). One of the signals involved seems to be a rapid increase in
intracellular pH, which could be determinant in the processes that mediate
cyst activation (Busa and Crowe,
1983
).
Transcription activation during cyst re-activation has been studied in less
detail than has protein synthesis. Studies of steady-state mRNA levels have
shown that some RNAs accumulate as soon as 2 h after cyst activation but most
of the mRNAs studied only start to accumulate after 4 h of development
(Díaz-Guerra et al.,
1989; Escalante et al.,
1994
). These results indicate a relatively late onset of gene
transcription after cyst activation compared with the rapid resumption of
intermediate metabolism and protein synthesis. The mechanisms involved in
transcriptional activation are largely unknown. One possibility is that
transcription is repressed in the cyst and activation is the consequence of
releasing repression. Alternatively, factors required for transcription could
be limiting in the cyst, and their expression/activation may be induced after
exit from cyst cryptobiosis. Studies of two transcription factors from
Artemia corroborate the latter hypothesis. The expression of the
TATA-binding protein (TBP) general transcription factor doubled between the
cyst and developing embryo (nauplius) stages
(Sastre, 1999
). DNA-binding
activity of the transcription factor SRF (serum response factor) increased
very significantly between cyst and nauplius stages
(Casero and Sastre, 2000
).
Moreover, van Breukelen et al.
(2000
) have reported
activation of transcription through the increase in intracellular pH that
occurs after cyst activation.
In this article, we confirm and extend previous results by analyzing the
mechanisms involved in the activation of three A. franciscana genes
whose expression is induced during development: those coding for the
Na+/K+-ATPase 1 subunit
(García-Sáez et al.,
1997
), the actin 302 isoform
(Ortega et al., 1996
) and the
sarco/endoplasmic reticulum Ca2+-ATPase (SERCA; Escalante and
Sastre, 1994
,
1995
). The intron/exon
structure, promoter regions and transcription initiation sites of these genes
have previously been characterized. Transcription regulatory regions of these
promoters were identified by means of analysis of in vitro
proteinDNA interactions and by functional analysis in cultured
mammalian cells. The presence of protein factors that bind to the determined
promoter regulatory regions in cyst and nauplius extracts was analyzed by
electrophoretic mobility shift assays. The results obtained indicate that
protein factors binding to these regulatory regions are expressed after cyst
activation. The existence of repressor transcription factors specifically
expressed in cyst nuclei also cannot be excluded for some of the promoter
regions.
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Materials and methods |
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Cell culture and transfection
Monkey kidney Bsc40 cells were cultured in Dulbecco's modified Eagle's
medium (Dulbecco and Freeman,
1959) supplemented with 10% newborn bovine serum and 2 mmol
l1 glutamine. Cells were transfected with 5 µg of the
luciferase reporter vectors and 1 µg of the ß-galactosidase expression
vector pCMVß (Clontech Laboratory Inc., Palo Alto, CA, USA) by the
calcium phosphate precipitation method
(Chen and Okayama, 1987
). Cells
were harvested 48 h after transfection, lyzed and the luciferase and
ß-galactosidase activities determined
(Murguía et al., 1995
).
Luciferase activity was determined with a commercial kit (Promega, Madison,
USA) according to the manufacturer's instructions. The
luciferase/ß-galactosidase ratio was determined for each sample to
correct for transfection efficiency. Each experiment was repeated at least
three times with duplicate samples and means ± S.D. are represented in
the figures.
Electrophoretic mobility shift assays
Nuclear extracts from Artemia franciscana cysts and nauplii,
obtained after 20 h of cyst culture, were prepared as previously described
(Sastre, 1999). 1020
µg of each extract (as indicated in each experiment) were incubated with
13 ng of 32P-labeled double-stranded oligonucleotide at
4°C for 20 min in 20 mmol l1 Hepes, pH 7.0, 70 mmol
l1 NaCl, 2 mmol l1 dithiothreitol (DTT),
0.005% NP40, 50 µg ml1 bovine serum albumin (BSA), 2%
Ficoll and 200 µg ml1 Poly(dI-dC) and analyzed in 6%
polyacrylamide gels. Fifty times excess of unlabeled oligonucleotides were
added to the indicated incubation mixtures 10 min before the labeled
oligonucleotide.
DNase I protection assays
DNase I protection assays were performed according to Ausubel et al.
(1994). Double-stranded DNA
probes were generated by PCR reactions, where one of the oligonucleotide
primers was labeled using [
-32P]ATP and polynucleotide
kinase. Aliquots consisting of 100 000 c.p.m. of the probe were incubated with
7.5 µg or 15 µg of nuclear extracts from A. franciscana cysts,
nauplii or both, as indicated in each sample. After 60 min at room
temperature, the samples were incubated with 0.02 mg ml1 of
DNase I for 1 min, phenol/chloroform extracted, ethanol precipitated and
analyzed in 6% polyacrylamide7 mol l1urea gels.
Sequencing reactions of the same DNA fragment, using the labeled
oligonucleotide as primer, were carried out with the AmpliCycle sequencing kit
(Perkin Elmer, Norwalk, USA) and run in parallel to the DNase I protection
assays to identify the nucleotide sequence of protected regions.
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Results |
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By contrast, analysis of the Actin302 promoter showed that a
fragment containing the more proximal 354 nt, upstream of the transcription
start site, had transcriptional activity that was similar to that of the
longest genomic fragment available (1063 nt). The 354-nt fragment was further
deleted from its 5' end to generate deletions 1 to
8,
whose 5' ends are indicated in Fig.
1A. Deleted fragments were cloned into the pXP2 reporter vector
and the constructs were transfected into Bsc40 cells. Luciferase activity
obtained for each construct is shown in
Fig. 1B. A significant
difference in activity was observed between the 354-nt fragment and the
longest deletion fragment (
1), indicative of the existence of an
activator region between nucleotides 375 and 409 of the
Actin302 promoter. The rest of the deletions analyzed gave more
similar promoter activities, which were significantly higher than that of the
empty reporter vector. These results are indicative of the presence of a
proximal basal promoter in the Actin302 gene.
|
In order to identify the possible relevance of this finding in Artemia, rather than in transfected mammalian cells, we studied the existence of Artemia proteins from cryptobiotic cysts and developing nauplii, which could specifically bind to the putative regulatory regions in vitro, by means of electrophoretic mobility shift assays (EMSAs). The results of these assays for the 409/388 Actin302 promoter region are shown in Fig. 2. Similar prominent complexes could be observed after incubation with cyst and nauplius nuclear extracts (lanes 2, 5). These complexes are specific for this DNA region, since their formation was inhibited by a 50-fold excess of the same (S) oligonucleotide (lanes 3, 6) but not with a non-related (N) oligonucleotide (lanes 4, 7). In addition to these complexes, three other slower-migrating, specific complexes were observed when the probe was incubated with nauplius nuclear extracts but not with cyst extracts (labeled with arrows in Fig. 2). The formation of these nauplius-specific complexes was significantly reduced when cyst nuclear extracts were added together with the nauplius extracts (lanes 8, 9). These results indicate that the activator region identified in mammalian cells is also a protein-binding region in Artemia and that some of the corresponding binding proteins are present in nauplius but not in cyst nuclear extracts.
|
Promoter region 2 of the SERCA-encoding gene, which directs expression in non-muscle tissues, was similarly analyzed. The analysis of a 1376-nt fragment and of several deletions originated from its 5' end is shown in Fig. 3A. The deletion of 166 nt from the 5' end of this fragment reduced promoter activity by 85%. These data suggest the presence of an activator region between nucleotides 1311 and 1477 of this promoter. A second activator region was detected between nucleotides 837 and 1120. Deletion of both regions almost completely abolished promoter activity. The 1311/1477 region was analyzed in more detail through its subdivision into smaller fragments. To this end, the pT109 reporter vector, which contains the luciferase reporter gene under control of the minimal thymidine kinase promoter, was employed. A diagram of the fragments analyzed is shown in Fig. 3B, together with the luciferase activities obtained in Bsc40 cells. These data suggest the existence of both activator and repressor elements in this promoter region. Deletion of the more 3' region (fragments F1F4) increased promoter activity, suggesting the presence of a promoter region that represses transcription in mammalian cells. There is also evidence of the presence of additional activator sequences, which are present in fragments F2F4 but only partially in fragments F1 and F6. The activity of this element is, at least partially, orientation independent, since fragment F5, which contains the complete analyzed region inverted with respect to the reporter gene, showed significant activity. This characteristic is typical of eukaryotic enhancer elements.
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An oligonucleotide probe was designed from the identified activator region (nucleotides 1397 to 1349) and used in EMSAs to check for the presence of proteins in Artemia nuclear extracts, which would bind to this DNA region. A specific retardation complex was observed after incubation of the probe with nauplius nuclear extracts (Fig. 4; lanes 25). The formation of this complex was reduced by a 50-fold excess of the same unlabeled oligonucleotide but not by two non-related oligonucleotides. This specific complex was also observed, but in much smaller amounts, when the probe was incubated with the same amount of cyst nuclear extract, (Fig. 4; lanes 69). An additional, slower-migrating retardation complex was observed with cyst extracts but not with nauplius nuclear extracts.
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Analysis of protein-binding regions in A. franciscana promoters
Analysis of A. franciscana promoters in cultured mammalian cells
is limited by the fact that only those regions recognized by mammalian
transcription factors are detectable in these assays. To circumvent this
limitation, we employed DNA footprinting in which protein-binding regions of
the promoters were identified by DNase protection after their incubation with
cyst or nauplius nuclear extracts. These experiments also allowed a comparison
of DNA-binding proteins present in nuclear extracts from cysts or nauplii.
An end-labeled fragment of the proximal promoter region of the
Na+/K+-ATPase 1-subunit-encoding gene promoter
was incubated with different amounts of cyst or nauplius nuclear extracts, or
with mixtures of both of them, and submitted to partial digestion with DNase I
(Fig. 5A). Some nucleotide
bonds were protected from DNase digestion after incubation with nauplius
extracts but not with cyst nuclear extracts. Addition of cyst nuclear extracts
together with nauplius extracts did not alter the protections observed with
nauplius extracts alone. Some of the additional bands observed after
incubation with nauplius nuclear extracts could be due to the activity of an
A. franciscana DNase whose expression is induced during development
(Domingo et al., 1986
). These
results indicate the presence of nauplius-specific DNA-binding proteins that
are not detected in cyst nuclear extracts. To confirm these results,
oligonucleotide probes were designed from one of the protected regions
(nucleotides 207 to 188, indicated by an open box in
Fig. 5A) and used in EMSAs. The
results (Fig. 5B) indicate the
formation of specific DNAprotein complexes when the probe is incubated
with nauplius nuclear extracts but not when incubated with cyst nuclear
extracts. The addition of cyst nuclear extracts together with nauplius
extracts did not affect the formation of DNAprotein complexes
(Fig. 5B; lane 5).
|
The proximal region of the Actin302 promoter was also analyzed by footprinting. Protection from DNase I digestion was observed after incubation with nauplius nuclear extracts but not with cyst nuclear extracts (Fig. 6A). Simultaneous incubation with both extracts did not alter the protection observed with nauplius extracts. An oligonucleotide probe that includes the protected region shown in Fig. 6A (open box) was used for EMSAs. A specific proteinDNA complex was detected after incubation with nauplius nuclear extracts, indicated by an arrow in Fig. 6B. No specific complexes were observed after incubation of the probe with cyst nuclear extracts. Incubation with cyst and nauplius nuclear extracts slightly decreased the intensity of the proteinDNA complex obtained with nauplius extracts.
|
Finally, similar footprinting experiments were carried out with a fragment of the proximal region of promoter 2 of the SERCA-encoding gene. A representative region of the footprint is shown in Fig. 7A. DNase I protection was once again observed after incubation of the DNA with nauplius nuclear extracts but not with cyst nuclear extracts. Simultaneous incubation with both extracts produced the same protection as with nauplius extracts alone. An oligonucleotide probe was designed for the protected region shown in Fig. 7A (indicated by the open box) and used for EMSAs. A specific proteinDNA complex was observed after incubation with cyst extracts (Fig. 7B). A similarly migrating complex was also observed with nauplius nuclear extracts. In addition, a faster-migrating complex was specifically observed after incubation with nauplius extracts.
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Discussion |
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To help clarify this issue, we examined protein binding to different gene promoters in cryptobiotic embryos and in embryos that have resumed development. We analyzed the promoters of three genes whose expression is known to be induced a few hours after cyst activation. We identified putative regulatory regions of these promoters and subsequently analyzed the presence of proteins from cryptobiotic (cysts) or developing (nauplii) embryos that would bind to these regions.
Regulatory regions were initially identified by means of a functional
analysis of the promoters in cultured mammalian cell lines. It is likely that
some promoter regions that play an important regulatory role in
Artemia are not recognized in mammalian cells, since the
functionality of Artemia promoters in this heterologous system
depends on the conservation of DNAtranscription factor interactions.
Nevertheless, Artemia cell lines are not currently available. One
example of the utility of this assay came from the study of the
Actin403 gene promoter. Functional analysis in mammalian cells
allowed the identification of an activator element homologous to mammalian
serum response element (Casero and Sastre,
2001). Subsequent studies led to the identification of cDNA clones
coding for an A. franciscana protein homologous to the serum response
factor, the transcription factor that binds to the serum response element
(Casero and Sastre, 2000
).
Our second approach consisted of identifying protein-binding regions in the promoters through footprinting analysis. This is a homologous system in which Artemia proteins are assayed on Artemia DNA, but the system does not provide information about the functionality of the interactions. Putative protein-binding regions identified by either of these two criteria were confirmed by EMSAs. This confirmation is particularly relevant when the regions were previously identified by functional assays in mammalian cells, since it shows that these regions also have the capacity to specifically bind proteins from Artemia extracts, suggesting the conservation of this interaction. Because of these limitations, the regions identified by these techniques may not be the most physiologically relevant in terms of gene regulation. Nevertheless, both techniques can detect regulatory regions that are useful for a comparative study of transcriptional regulation during cyst activation.
The presence of DNA-binding proteins in cyst and nauplius nuclear extracts
was compared by EMSAs for those regions identified by functional assays. EMSAs
and footprinting assays were used for the remaining regions. In all the cases
analyzed, specific proteinDNA complexes were detected using nauplius
nuclear extracts. These complexes were not detected using cyst nuclear
extracts or were present in much smaller amounts. These results indicate that
the expression of these DNA-binding proteins is induced after activation of
cryptobiotic cysts. Footprinting analyses also detected other promoter regions
that were protected when incubated with nauplius extracts and not when
incubated with cyst extracts, although these data were not confirmed by EMSA
(not shown). Analysis of the expression of the basal transcription factor TBP
(Sastre, 1999) and of the
DNA-binding activity of the sequence-specific transcription factor SRF
(Casero and Sastre, 2000
) also
showed a significant increase between the cyst and nauplius stages. These
results are consistent with the model that proposes the induction of
expression and/or activity of transcription factors after cyst activation.
EMSA experiments using the SERCA promoter 2 and Actin302 promoter probes detected the existence of DNA-binding proteins in cyst extracts. The binding of these proteins was not detected in footprinting experiments. At present, the functional significance of these proteinDNA interactions remains unknown. These proteins might exert an inhibitory function in the cysts, as predicted by one of the two hypotheses initially considered. In any case, the existence of these putative repressive interactions was not general for all the promoter regions analyzed.
The differences observed between cyst and nauplius extracts in the
formation of DNAprotein complexes could also be due to the presence of
some inhibitory factors in the cysts. This possibility was studied through the
addition of cyst nuclear extracts to nauplius extracts to see if there was any
interference with the formation of nauplius-specific DNAprotein
complexes. No interference was detected in any of the DNase I protection
experiments analyzed. Similarly, EMSAs with the
Na+/K+-ATPase 1 subunit and Actin302
proximal promoter probes showed no variation in nauplius-specific complexes
after addition of cyst extracts. Only EMSAs using the Actin302 distal
promoter probe showed inhibition of the nauplius-specific retardation
complexes after addition of cyst nuclear extracts. This probe formed a very
abundant retardation complex with cyst extracts
(Fig. 2). Simultaneous addition
of cyst and nauplius extracts could produce either inhibition of the
nauplius-specific complexes or a competition between cyst and nauplius
DNA-binding proteins. The higher concentration or affinity of the cyst
proteins may reduce the formation of the nauplius-specific complexes.
The results obtained in this study favor a model in which transcription
factors are absent, or are present in very limited amounts, in the nuclei of
cryptobiotic embryos. Activation of the cysts would lead to the induction of
transcription factor expression and/or activity. Increases in activity could
be due to post-translational modifications of existing transcription factors.
Nuclear translocation of transcription factors may also occur, in a manner
similar to the rapid translocation of the crystalline homologue p26 during
cyst activation (Clegg et al.,
1994,
1995
). Increases in expression
would be dependent on protein synthesis, which is known to be induced a few
minutes after cyst activation (Tate and
Marshall, 1991
). The synthesis of some of these transcription
factors could be directed by mRNAs stored in the cyst, which would make this
process independent of transcription reactivation. In agreement with this
idea, mRNAs coding for the SRF transcription factor have been found stored in
cryptobiotic cysts at levels similar to those found in developing nauplii
(Casero and Sastre, 2000
).
The existence of transcription factor induction does not rule out the participation of other regulatory mechanisms. Some of the data obtained are compatible with the presence of repressor molecules in the cyst, as mentioned above. The existence of a repressive chromatin environment is another possibility that cannot be discarded. The DNA-binding proteins involved in these interactions may not have been detected by the footprinting and electrophoretic mobility assays utilized in this study. Despite these considerations, the data presented strongly support the idea that induction of transcription factor expression and/or activity is an important mechanism of activation of gene expression in Artemia embryos exiting cryptobiosis.
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Acknowledgments |
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References |
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