Tài liệu Báo cáo Y học: Transactivation domains are not functionally conserved between vertebrate and invertebrate serum response factors potx

Transactivation domains are not functionally conserved between
vertebrate and invertebrate serum response factors
Sonia Avila, Marie-Carmen Casero, Rocı
´
o Fernandez-Canto
´
n and Leandro Sastre
Insitituto de Investigaciones Biome
´
dicas CSIC/UAM, C/Arturo Duperier, Madrid, Spain
The transcription factor serum response factor (SRF) regu-
lates expression of growth factor-dependent genes and
muscle-specific genes in vertebrates. Homologous factors
regulate differentiation of some ectodermic tissues in inver-
tebrates. To explore the molecular basis of these different
physiological functions, the functionality of human, Droso-
phila melanogaster and Artemia franciscana SRFs in mam-
malian cells has been compared in this article.
D. melanogaster and, to a lesser extend, A. franciscana SRF
co-expression represses the activity of strong SRF-depen-
dent promoters, such as those of the mouse c-fos and
A. franciscana actin 403 genes. Domain-exchange experi-
ments showed that these results can be explained by the
absence of a transactivation domain, functional in mam-
malian cells, in D. melanogaster and A. franciscana SRFs.
Both invertebrate SRFs can dimerize with endogenous
mouse SRF through the conserved DNA-binding and
dimerization domain. Co-expression of human and
A. franciscana SRFs activate expression of weaker SRF-
dependent promoters, such as those of the human cardiac
a-actin gene or an A. franciscana actin 403 promoter where

the SRF-binding site has been mutated. Mapping of
A. franciscana SRF domains involved in transcriptional
activation has shown that the conserved DNA-binding and
dimerization domain is neccessary, but not sufficient, for
promoter activation in mammalian cells.
Keywords:SRF;Artemia; Drosophila; transcription; evolu-
tion.
The serum response factor (SRF) is a transcription factor
initially isolated as responsible for activation of several
immediate early genes, such as c-fos, in the response of
quiescent cells to serum [1]. It is characterized by its binding
to the minor grove of the DNA through a conserved
domain, the MADS (MCM1-Agamous-Deficiens-SRF)
box [2]. This domain is shared by an extensive family of
transcription factors that also include the animal MEF-2
factor and a large number of plant transcription factors
[3,4]. Immediately C-terminal to the MADS box there is a
region of amino acids (SAM-domain) conserved between
SRFs from different species but not with other members of
the MADS box family [3]. SRF is assembled as a
homodimer and the dimerization domain has been also
mapped to the MADS box [1]. SRF does not seem to be
active by itself and requires association with other tran-
scription factors bound to the same promoter or directly
interacting with SRF [5]. Many of these interactions also
occur through the MADS box [6]. In addition to this
functional domain, a transactivation domain has been
located in the C-terminal region of vertebrate SRF [7,8].
This transcription factor has been also involved in the
activation of several muscle-specific genes in vertebrates [9].

Besides, the generation of SRF-null mice showed that this
factor is necessary for mesoderm induction and for the
proper differentiation of several mesodermal tissues [10,11].
SRF binding sites found in the promoter of serum-induced
and muscle-specific genes are very similar and contain
consensus CArG boxes: CC(A/T)
6
GG [12]. Despite the
similarity in their SRF-binding sites, some genes, such as
c-fos, are activated in response to serum and others, such as
a-actin genes, are induced during muscle differentiation
that, in cell culture, usually implies serum withdrawal.
Moreover, SRF-dependent immediately early genes are
repressed after muscle differentiation [13]. Recent experi-
ments have shown that this complex regulation is promoter-
context dependent [14]. The transcriptional regulation of
each SRF-dependent promoter seems to be determined by
the binding of SRF cofactors. Some of these cofactors are
tissue-specific, as the muscular Nkx2.5 [15], GATA-4 [16] or
myocardin [17] transcription factors. Other cofactors are
regulated by growth-factor transduction pathways, such as
the classical ternary complex factors, that are activated by
MAP-kinase pathways [18].
Several nonvertebrate SRF homologues have been
described that are mainly involved in differentiation
processes. Drosophila melanogaster SRF (DmSRF) is neces-
sary for differentiation of terminal tracheal cells and cells of
the wing’s intervein regions [19,20]. Artemia franciscana
SRF (AfSRF) is specifically expressed in ectodermal tissues
[21]. The gene srfA is necessary for several morphogenetic

processes and terminal spore differentiation in the social
amoeba Dictyostelium discoideum [22,23]. Therefore, it
seems that, although vertebrate and invertebrate SRFs have
in common the participation in differentiation processes, the
tissues involved are different, suggesting that SRF must
Correspondence to L. Sastre, Insitituto de Investigaciones Biome
´
dicas
CSIC/UAM, C/Arturo Duperier, 428029, Madrid, Spain.
Fax: + 34 91 5854587, Tel.: + 34 91 5854626,
E-mail:
Abbreviations: AEBSF, 4-(2-aminoethyl) benzene sulfonyl fluoride;
DMEM, Dulbecco’s modified Eagle’s medium; SRE, serum response
element; SRF, serum response factor.
(Received 13 March 2002, revised 4 June 2002, accepted 18 June 2002)
Eur. J. Biochem. 269, 3669–3677 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03077.x
regulate different sets of genes in these species. In accord
with this idea, no evidence was found for the involvement of
SRF in the regulation of muscle actin genes in D. melano-
gaster, as would be expected from the data obtained in
vertebrates. The bases for these differences, despite the high
similarity of the SRF amino-acid sequences in the MADS
box and SAM-domain sequences, are not known. SRF
regulatory pathways could have diverged in the different
species, responding to different tissue-differentiation signals.
Alternatively, SRF structure and regulation could be
conserved but SRF expression patterns and SRF-dependent
promoters could have changed during evolution.
Further understanding of the evolution of SRF and SRF-
dependent genes would require a better characterization of

functional domains from this transcription factor in differ-
ent species. MADS boxes and SAM domains have been
very conserved during evolution. MADS-box regions are
98% identical and SAM-domains over 79% identical
between vertebrate and invertebrate SRFs. No similarity
has been detected outside these domains, except for a short
region around vertebrate Serine 103 [21]. However, some
important transcription factor functional domains, such as
transactivation domains, are very variable in their amino-
acid sequence and their location could not be predictable
from amino-acid sequence comparisons. As an approach to
the comparative study of these domains, the functionality of
SRF proteins from an insect (D. melanogaster), a crustacean
(A. franciscana) and humans, in mammalian cells, has been
studied in the present article. The evidence obtained suggests
that functions associated with the MADS box, such as
DNA binding and dimerization, are well preserved in SRFs
from different species. However, the transactivation domain
is not conserved. D. melanogaster and A. franciscana
C-terminal regions are devoid of transactivation activity in
mammalian cells. A weaker transcriptional activation
domain has been found in the N-terminal and MADS
box regions from A. franciscana SRF.
EXPERIMENTAL PROCEDURES
Cell culture and transfection
Myoblastoid C
2
C
12
[24] and monkey kidney epithelial cells,

Bsc1 [25] and Bsc40 (a Bsc1 subline) were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supple-
mented with 10% fetal bovine serum and 2 m
M
glutamine.
Embryonic fibroblastoid cells NIH3T3 were cultured in
DMEM supplemented with 10% neonatal bovine serum
and 2 m
M
glutamine. Cells were transfected by the calcium
phosphate precipitation method [26,27]. Five micrograms of
the reporter vector, 1 lg of the SRF expression vector and
1 lgoftheb-galactosidase expression vector pCMV-bgal
(Clontech Laboratory Inc, Palo Alto, CA, USA) were used
for each transfection, except in some experiments, that are
specified in each case. Cells were harvested 72 h after
transfection, and b-galactosidase activities were determined
[28]. Luciferase activity was determined with a commercial
kit (Promega) according to the manufacturer’s instructions.
The luciferase/b-galactosidase ratio was determined for each
sample and the relative activity was calculated in relation to
samples transfected with the reporter vector alone or
together with the pcDNA3-myc vector without any SRF,
that were given the value of 100. Each experiment was
repeated at least three times with duplicated samples.
Media ± SD are represented in the figures. The statistical
significance of the differences observed, in relation with the
sample transfected with the reporter vector alone, was
analyzed with the
PRISM

2.0 program, using the one-way
ANOVA
and Turkey tests (*p < 0.05; **p <0.01;
***p < 0.001).
Cells lines permanently expressing SRFs from the three
different species were generated by transfecting 10
6
C
2
C
12
cells with 5 lg of each pcDNA3-myc-SRF vector by
calcium phosphate precipitation. Sixteen hours after
transfection, cells were changed to the same medium
containing 1 mgÁmL
)1
of geneticin for 24 h. Cells were
washed and cultured in the same medium containing
0.5 mgÁmL
)1
of geneticin for 15 days before collection
and further culture.
Generation of reporter and expression vectors
The reporter vector containing the human cardiac a-actin
promoter was generated by inserting a fragment of this
promoter, generated by PCR, between SacIandKpnIsites
of the pXP2 reporter vector [29]. The oligonucleotides 5¢-
GGTACCCTGGCTGATCCTCTCCCC-3¢ and 5¢-GAG
CTCGGGTGGCTGGCTCCAGGAGG-3¢, that amplify
the region between nucleotides )170 and +1 of the cardiac

a-actin gene [9] were used as primers in the PCR reaction.
Reporter vectors containing the wild-type A. franciscana
actin 403 promoter )176 to )38 region (Act403)andthe
same region with the CArG box mutated (Act403mut) have
been described previously [30]. The reporter vector contain-
ing the c-fos promoter was kindly provided by U. Moe
¨
ns
(University of Tromso, Norway) [31].
The expression vector pcDNA3-myc was generated by
inserting a BamHI–EcoRI fragment that codes for six copies
of myc epitope, obtained from pCS2+MT [32], in the
plasmid pcDNA3 (Invitrogen). cDNA molecules coding for
human [33], D. melanogaster [34] and A. franciscana [21]
SRFs were cloned in the pcDNA3-myc vector, in the same
reading frame as the myc epitope. An EcoRI site was
inserted in front of the initiating methionine of each SRF by
PCR for these constructions. Besides, each cDNA was
isolated as an EcoRI–EcoRI fragment and cloned in the
plasmid expression vector pSG5 [35]. All the constructions
generated were sequenced to confirm that no mutation had
been introduced in PCR reactions.
Hybrid SRF molecules were generated using a BclIsite
conserved in the same position, relative to the MADS box,
in the cDNA clones from the three species. The N-terminal
coding region from each species, including the MADS box,
was isolated as an EcoRI–BclI fragment and ligated to the
C-terminal coding region from the other species in the
pcDNA3-myc vector.
A. franciscana SRF N-terminal deletions were generated

by PCR between a series of oligonucleotides containing a
EcoRI site, designed to conserve the myc epitope reading
frame, and a common oligonucleotide downstream of the
MADS box coding region. The different deleted fragments
were isolated as EcoRI–SnaIfragmentsandusedtoreplace
the full-length EcoRI–SnaI fragment in the original
pcDNA3-myc-AfSRF vector. The oligonucleotides uti-
lized as PCR primers were N1: 5¢-GGGAATTCGGGT
GGTCTTGAACCCGATATT-3¢;N2:5¢-GGGAATTCG
3670 S. Avila et al. (Eur. J. Biochem. 269) Ó FEBS 2002
TCTTATATGAATGCAGTTCTG-3¢;N3:5¢-GGGAAT
TCGCTAGGCCACAGTTTGAATTTG-3¢;N4:5¢-GGG
AATTCGACCTCTGAAAATGTAAAACAG-3¢;N5:
5¢-GGGAATTCGGATCCTCTAACTGGGTTAGAT-3¢;
N6: 5¢-GGGAATTCGTCCCCGGACGAGGACAGG
TCA-3¢;N7:5¢-GGGAATTCGCCTGCCAATGGTAAA
AAGACA-3¢.
TheC1deletionwasgeneratedbyPCRusingthe
oligonucleotide containing the methionine initiation codon
and an oligonucleotide (C1: 5¢-GCGGCCGCCTAAACG
TTATATGTGAGTTCCG-3¢) where the codon of amino
acid 220 was changed to a stop codon. The fragments
generated by PCR were cloned in pcDNA3-myc. Fragment
M was generated by PCR, using oligonucleotides N7 and
C1asprimers,andinsertedinpcDNA3-myc.Allthe
fragments generated by PCR were sequenced to check for
possible mutations.
Western blots and co-immunoprecipitations
Expression of SRF-myc molecules in transiently transfected
cells was analyzed by Western blot. After electrophoresis of

10 lg of the extracts, the samples were transferred to
poly(vinylidene difluoride) membranes and incubated with
polyclonal rabbit anti-(a-myc) Ig (A14, Santa Cruz Bio-
technologies). Horseradish peroxidase-conjugated goat
anti-(rabbit IgG) Ig (Santa Cruz Biotechnologies) was used
as secondary antibody, and detected by chemiluminiscence
(ECL, Amersham Pharmacia Biotech).
Co-immunoprecipitation experiments were performed
with nuclear extracts obtained as described previously [36].
Protein samples (50–100 lg) from nuclear extracts (200–
400 lgforAfSRF expressing cells) were immunoprecipitated
with 0.5 lL of anti-SRF Ig (G20, Santa Cruz Biotechnol-
ogies) or monoclonal anti-myc Ig (9E10, Santa Cruz
Biotechnologies) in 20 m
M
Hepes, pH 7.0, 70 m
M
NaCl,
0.005% NP40, 0.05 mgÁmL
)1
BSA, 2% Ficoll, 2 m
M
dithiothreitol, 0.5 m
M
phenylmethanesulfonyl fluoride, leu-
peptin, AEBSF and aproteinin. Immunoprecipitates were
analyzed by 10% SDS/PAGE and transferred to poly(viny-
lidene difluoride) membranes. Control samples containing
5–10 lg of nuclear extracts (10–20 lgforAfSRF expressing
cells) were also included in the electrophoresis as positive

controls. Membranes were incubated with anti-SRF Ig
(G20, Santa Cruz Biotechnologies) or anti-myc Ig (A14,
Santa Cruz Biotechnologies) rabbit polyclonal antibodies.
Horseradish peroxidase-conjugated goat anti-(rabbit IgG)
Ig (Santa Cruz Biotechnologies) was used as secondary
antibody and its binding was detected by chemiluminis-
cence, as described above.
RESULTS
Effect of the expression of SRFs from different species
on the activity of SRE-dependent promoters
Expression vectors containing cDNAs coding for human
SRF (HsSRF), DmSRF or AfSRF were transfected in
mammalian cells. The activity of the encoded proteins was
studied by cotransfecting reporter vectors where luciferase
gene expression was placed under control of serum response
element (SRE)-containing promoters. Four different pro-
moters were used in these studies, corresponding to the c-fos
genes from mice (c-fos) [18], human cardiac a-actin (CA)[9],
A. franciscana actin 403 (Act403) and a mutated actin 403
promoter where two nucleotides of the SRF-binding site
had been changed to decrease SRF binding (Act403mut)
[30]. The reporter vectors containing c-fos and Act403
promoters presented similar high levels of expression in
cultured mammalian cells while CA and Act403mut pro-
moters presented much weaker activity, 10–20 times less
active in the cell lines tested (Fig. 1A).
Expression of the different SRFs was obtained by
transfection of pcDNA-3 and pSG5 vectors containing
the corresponding cDNA clones in Bsc40 cells. The
expression from the pcDNA-3 vectors could be analyzed

by Western blot as SRF molecules are fused to six repeats of
the myc epitope. The results are shown in Fig. 1B. HsSRF
and DmSRF were expressed at similar levels, higher than
those of A. franciscana SRF, probably due to the higher
A/T content of the cDNA coding for the later protein. An
additional, faster migrating, band was observed after
HsSRF transfection, that probably corresponds to a degra-
dation product as it was present in very variable proportions
in different experiments.
The effects of SRF co-expression on the activity of the
SRE-containing promoters are shown in Fig. 1C for
pcDNA-3 expression vectors, similar results were obtained
for pSG-5 vectors (data not shown). Co-expression of
HsSRF increased expression from c-fos and, specially, from
the weaker CA and Act403mut promoters. In the case of the
strong Act403 promoter there was no effect or a slight
inhibition (50%) by HsSRF co-expression. Co-expression
of AfSRF also produced an increase in the activity of the
weak CA and Act403mut promoters, but had no effect on
c-fos and produced a small inhibition on the Act403
promoter. Activation was always smaller with AfSRF than
with HsSRF. In contrast to these factors, co-expression of
DmSRF produced a marked inhibition of the more active
promoters, c-fos and Act403, and had no significant effects
on the weaker CA and Act403mut promoters. The activity
of the SRFs from these three species was also tested for c-fos
and CA promoters on two more mammalian cell lines: the
fibroblast cell line NIH3T3 and the mouse myoblastic cell
line C
2

C
12
. The effects on the activity of c-fos and CA
promoters, using the pcDNA-3 expression vectors, were
similar to those described previously for Bsc40 cells (data
not shown).
As the differences in expression levels shown in Fig. 1B
could affect the functional consequences of co-expression,
the effect of transfecting different amounts of each
expression vector was studied. The activity of HsSRF
and AfSRF was studied on the CA promoter (Fig. 2A,B).
The inhibitory effect on the c-fos promoter was studied for
DmSRF (Fig. 2C). The results obtained showed that the
effects observed are dose-dependent and were in agree-
ment with the activating capacity of HsSRF and AfSRF,
and with the inhibition obtained by D. melanogaster
co-expression.
Interaction between transfected and endogenous SRFs
The stimulatory effects observed for HsSRF and AfSRF
could be due to the increase of intracellular SRF concen-
tration that would raise the amount of SRF bound to the
promoters and therefore transcription of the reporter gene.
Ó FEBS 2002 Functionality of arthropod SRFs in mammalian cells (Eur. J. Biochem. 269) 3671
Inhibition by DmSRF and, in the case of Act403 promoter,
AfSRF could be due to a dominant negative effect where
exogenous SRF dimerizes with the endogenous mammalian
factor avoiding its productive interaction with activating
cofactors. Alternatively, inhibition could be explained by
diluting of these cofactors by an excess of nonDNA-bound
SRF. This would be the more likely mechanism of the

inhibition observed by co-expression of HsSRF with the
Act403 promoter, given the high similarity of human and
mouse SRFs.
The proposed dominant negative mechanism requires
dimerization between the endogenous SRF and the trans-
fected SRFs. To check for these possible interactions,
stably transformed C
2
C
12
cell lines that expressed HsSRF,
DmSRF or AfSRF were established. The interaction
between SRF molecules was assayed by co-immunopreci-
pitation experiments. Exogenous SRFs were specifically
recognized by antibodies directed against the c-myc epitope.
Endogenous SRF was recognized by a monoclonal anti-
body specific for vertebrate SRF. Extracts from C
2
C
12
cells,
either nontransfected (C
2
C
12
) or stably expressing human
(C
2
-Hs), D. melanogaster (C
2

-Dm)orA. franciscana (C
2
-Af)
SRFs were immunoprecipitated using anti-(c-myc) Ig
(aMyc) or with no antibody (–). Immunopreciptates were
analyzed by Western blot using an anti-(vertebrate-SRF) Ig
and the results are shown in Fig. 3A. The migration and
relative expression level of endogenous (mouse) and human
SRFs is shown in the lanes labeled as E, where smaller
amounts of cell lysates were subjected to Western blotting.
The complementary experiment is shown in Fig. 3B where
immunoprecipitation was carried with antivertebrate-SRF
antibodies and the immunoprecipitates analyzed by West-
ern blot with anti-(c-myc) Ig. The results obtained indicate
co-immunoprecipitation and, therefore, interaction between
Fig. 1. Effect of SRF co-expression on the activity of CArG-containing promoters. (A) Activity of CArG-containing promoters from the genes c-fos
from mice (c-fos), A. franciscana Actin 403 (Act403), an Actin 403 promoter with the CArG box mutated (Act403 mut) and human cardiac a-actin
(CA) in Bsc40 cells. Luciferase activity is expressed as times of induction over the activity of the reporter vector without promoter (pXP2). (B)
Expression of exogenous SRFs in Bsc40 cells. Cells were transfected with 1 lg of pcDNA3-myc vector containing cDNAs coding for human
(HsSRF), D. melanogaster (DmSRF) and A. franciscana (AfSRF) SRFs. Cellular extracts were analyzed by Western blotting using an antimyc
antibody. The migration of the fusion proteins containing the myc epitope: human (HsSRF-myc), D. melanogaster (DmSRF-myc) and A. fran-
ciscana (AfSRF-myc) SRFs, is indicated to the right. The migration of molecular weight markers is shown to the left. (C) Effects of SRF
co-expression, from the pcDNA3 expression vectors, on the activity of CArG containing promoters. Bsc40 cells were transfected with 5 lgofpXP2
reporter vectors containing the promoters indicated in the upper right corner of each graphic and 1 lg of the pcDNA3 expression vectors. The effect
of human (Hs), A. franciscana (Af) and D. melanogaster (Dm) SRFs is shown. The luciferase activity value 100 was assigned to the samples
transfected with the reporter vector alone (C). Asterisks indicate statistically significant variations with respect to control activity (C).
3672 S. Avila et al. (Eur. J. Biochem. 269) Ó FEBS 2002
HsSRF, DmSRF and AfSRF and the endogenous factor in
C
2

C
12
cells. These data also show similar levels of expression
of HsSRF and DmSRF and lower levels of AfSRF, as
previously observed in transient expression experiments
(Fig. 1B).
Functional analysis of SRF domains
The results obtained in the experiments shown above
indicate that HsSRF, AfSRF and DmSRF, expressed in
mouse cells lines, interact with the endogenous SRF. The
functional consequences of their expression are very differ-
ent, however. Human and AfSRFs can increase the activity
of SRE-containing promoters, while DmSRF co-expression
produced no activation but, instead, strong repression of the
more active promoters. The comparison between human
and D. melanogaster factors is especially significant as both
proteins are expressed at similar levels but produce opposing
effects. These results raised the possibility that important
functional regions of this transcription factor could be not
conserved between these species. As MADS boxes are over
90% identical in their amino-acid sequences, differences
could reside in other regions, possibly in the transactivation
domain, mapped to the C-terminal region of vertebrate
SRF [8]. A series of hybrid SRF molecules was constructed
to test for this hypothesis. The existence of a conserved BclI
site in equivalent positions in the cDNA clones of the three
species, immediately downstream of the MADS-box coding
region, was used for making these constructs. The hybrid
molecules coded for the N-terminal region, including the
MADS box, from one species and the C-terminal region

from another. The consequences of the co-expression of
hybrid SRFs on the expression of reporter vectors containing
Fig. 2. Dependence of promoter activities on
the amount of SRF expression vectors
cotransfected. (A) The indicated amounts of
the human SRF expression vector pcDNA3-
myc-HsSRF (0–5 lg) were transfected in
Bsc40 cells together with the reporter vector
containing the human cardiac a-actin pro-
moter. Luciferase activity value 100 was as-
signed to the sample without expression vector
(column 0). Statistical significance is referred
to differences with the activity obtained with-
out expression vector. (B) The indicated
amounts of A. franciscana SRF expression
vector (pcDNA3-myc-AfSRF) were cotrans-
fected together with 5 lg of the reporter vector
containing human cardiac a-actin promoter.
Relative luciferase activities and statistical
significances are as indicated in panel A.C The
amounts indicated of D. melanogaster
expression vector (pcDNA3-myc-DmSRF)
were cotransfected with 5 lg of the reporter
vector containing the c-fos promoter.
Luciferase activity and statistical significances
are expressed as indicated in (A.).
Fig. 3. Association of expressed SRFs with endogenous SRF. Nuclear
extracts were obtained from C
2
C

12
cells expressing A. franciscana
SRF-myc (C
2
Af), human SRF-myc (C
2
Hs) or D. melanogaster SRF-
myc (C
2
Dm), and from nontransfected C
2
C
12
cells. (A) Experiments
where nuclear extracts were immunoprecipitated with monoclonal
anti-myc Ig (a-myc) or without antibody (–) and the immunoprecipi-
tates analyzed by Western blot using anti-SRF Ig that recognize
human and mouse SRFs, as indicated to the right. One tenth of the
nuclear extracts used for immunoprecipitation was loaded on samples
E, as positive control of SRF expression. The experiment shown in (B)
was made using anti-SRF Ig for immunoprecipitation (a-SRF), or no
antibody (–), and anti-myc Ig for Western blotting. Anti-myc Ig rec-
ognized human (HsSRF-myc), D. melanogaster (DmSRF-myc) and
A. franciscana (AfSRF-myc) SRFs fused to the myc epitope whose
migration is indicated to the right. Samples labeled as E contain one
tenth of the nuclear extracts used for immunoprecipitation.
Ó FEBS 2002 Functionality of arthropod SRFs in mammalian cells (Eur. J. Biochem. 269) 3673
c-fos (Fig. 4A) or CA (Fig. 4B) promoters was determined.
Expression of the hybrid molecules was tested by Western
blot using anti-(myc epitope) Ig (Fig. 4C). Only hybrid

molecules containing the human C-terminal region (AH
and DH constructs) were able to increase CA promoter
activity, even if the N-terminal region and MADS box were
from D. melanogaster (DH) or A. franciscana (AH). How-
ever, the presence of A. franciscana or D. melanogaster
C-terminal regions produced inhibition of c-fos promoter
activity and did not activate CA promoter, even in the
presence of the human N-terminal region and MADS box
(HD and HA constructs). These results strongly suggest that
the transactivation domain present in the C-terminal region
of vertebrate SRFs is not functionally conserved in the
invertebrate D. melanogaster and A. franciscana factors.
Mapping of
A. franciscana SRF
transactivation domains
The lack of the C-terminal transactivation domain could
explain the results obtained for DmSRF. This protein would
inhibit the activity of the endogenous SRF through a
dominant negative effect, as previously described for
truncated vertebrate SRFs [37]. The activation observed
after cotransfection of AfSRF is, however, in apparent
contradiction with the absence of a transactivation domain
in the C-terminal region of this protein. One possible
explanation would be the existence of a transactivation
domain in a different region of this protein. This possibility
was tested through the deletion of several regions of AfSRF.
Seven progressive deletions of the region N-terminal to the
MADS box (N1 to N7), a construct lacking the region
C-terminal to the MADS box (C1), and a short construct
containing only the MADSbox and the SAM domain,

conserved between all known SRF proteins (M), were
generated (Fig. 5A). The constructs were cloned in the
pcDNA-3 vector, fused to a c-myc epitope, and cotrans-
fected in Bsc1 cells together with reporter vectors containing
CA and Act403mut promoters, that had been previously
showntobeactivatedbyAfSRF co-expression. The results
obtained, shown in Fig. 5B,C, confirmed that the
C-terminal region of AfSRF is not necessary for transcrip-
tional activity as the C1 construct has the same activity than
the complete SRF. However, deletion of the N-terminal
region did not abolish transcriptional activity completely,
either. A partial, although significant, decrease in transcrip-
tional activity could be observed between deletions N3 and
N4 on the Act403mut promoter only (Fig. 5B). These
results suggest that neither of the regions outside the MADS
box, N- or C-terminal, are essential for transactivation by
AfSRF. However, it appears that at least one of the regions
must be present in combination with the MADS-box to
achieve transcriptional activity, since co-expression of the
MADS box alone (M construct) does not activate
Act403mut promoter. The expression of the deleted mole-
cules was tested by Western blotting using anti-(c-myc) Ig
(Fig. 5D).
Fig. 4. Functionality of interspecies hybrid SRF molecules. SRF hybrid molecules were generated that contained the cDNA region coding for the
N-terminal and MADS-box domain from one species and the region coding for the C-terminal domain from another species. Hybrid molecules
were cloned in the pcDNA3 vector, fused to the myc epitope. Expression vectors coding for human (Hs), D. melanogaster (Dm), A. franciscana (Af)
and hybrids (H/D, D/H, H/A, A/H, D/A, A/D) were cotransfected in Bsc1 cells with reporter vectors containing c-fos (A) or human cardiac a-actin
(B) promoters. Hybrid SRF molecules have been named according to their components: the species contributing the N-terminal part of the protein
is indicated by the first letter and the species that provides the C-terminal region by the second letter (H, H. sapiens;D,D. melanogaster;
A, A. franciscana). Columns C show the activity obtained without SRF co-expression and were assigned the relative luciferase activity 100. (C) The

analyses of expression of human (Hs), D. melanogaster (Dm), A. franciscana (Af) and hybrid molecules (H/D, D/H, H/A, A/H, D/A, A/D) by
Western blot using anti-myc Ig. Ten micrograms of the cell extracts obtained in the transfection shown in (A) were analyzed. The migration of
molecular weight markers is indicated in the left margin.
3674 S. Avila et al. (Eur. J. Biochem. 269) Ó FEBS 2002
DISCUSSION
In this article, we have compared the function of SRF
molecules from a vertebrate, Homo sapiens,andtwo
invertebrates, the insect D. melanogaster and the crustacean
A. franciscana, in cultured mammalian cells. Complemen-
tary studies in invertebrate cells were hindered by the
absence of A. franciscana cultured cell lines. There are also
no available cell lines derived from D. melanogaster tissues
where SRF plays a relevant developmental function, such as
terminal tracheal cells and wing intervein cells.
SRF binds to DNA through SREs, whose consensus
nucleotide sequence, named CArG box, is CC(A/T)
6
GG
[38]. Promoters with very similar CArG boxes show very
different SRF-dependent regulation, probably due to the
presence of binding sites for other transcription factor
around the CArG box [14]. We have therefore assayed the
activity of the SRF molecules on two differently regulated
vertebrate promoters: the promoter of the c-fos gene,
stimulated by serum treatment of quiescent cells, and the
promoter of the muscle-specific cardiac a-actin (CA)gene.
Small fragments of both proximal promoter regions, that
had been shown to be sufficient for SRF-dependent regula-
tion, were used in these studies. The CA promoter contained
the two more proximal CArG boxes that have been identified

[9]. Besides, we have analyzed an invertebrate promoter, the
A. franciscana Act03 gene promoter, which is probably
regulated by SRF in this organism, as SRF and Act403 have
the same pattern of tissue-specific expression [21]. A mutated
Act403 promoter, with decreased affinity for SRF [30], was
also analyzed because preliminary studies had shown that
this promoter is activated by SRF co-expression.
The activity obtained for these promoters was markedly
different. C-fos and Act403 promoters showed high activity
in the transfected cells while CA and the Act403mut promoter
were much less active. The lower activity of the Act403mut
promoter is due to decreased affinity to SRF. In the case of
the cardiac actin promoter the reason for its lower activity is
not known, although it seems to depend on sequences outside
of the CArG box, as mentioned above. The lower activity
could be due to binding of transcriptional repressor mole-
cules to this promoter, to the absence of binding of SRF
coactivators or both. As the cell lines tested are not of cardiac
muscle origen, they possibly do not contain cardiac-specific
Fig. 5. Mapping domains of A. francisc ana SRF required for transcriptional activation. (A) Diagram of the deletions generated to map putative
transcriptional activation domains of A. franciscana SRF. The upper sketch shows the position of the conserved MADS-box and SAM domain in
A. franciscana SRF, with the first and last amino acids indicated on top. The name given to each construct is indicated to the right (N1 to N7, C1
and M). Numbers to the left indicate the N-terminal amino acid encoded by each construct. Constructs C1 and M terminated translation at amino
acid 219, as indicated to the right of the diagrams. All the constructs were cloned in pcDNA3-myc for cellular expression. (B) Luciferase activities
obtained after co-expression of full-length A. franciscana SRF (Af) or the deleted proteins (N1 to N7 and C1) with reporter vectors containing
human cardiac a-actin (CA) or Actin 403 mutated (Act403 mut) promoters in Bsc1 cells. Samples C show the activity obtained without any SRF
co-expression, that was given the value 100. Asterisks indicate statistically significant differences in relation with the activity obtained after full-
length SRF co-expression (Af). (C) Full-length A. franciscana SRF (Af) and deletions coding only for the conserved MADS-box and SAM domain
(M) or the conserved regions and the C-terminal region (C1) were cotransfected with reporter vectors containing the Act403 mutated promoter.
Sample C was transfected with the reporter vector alone and was assigned the relative activity 100. (D) Western blot analyses of full-length and

deleted proteins expression. Ten micrograms of each cellular extract were analyzed using an anti-myc Ig. Migration of molecular mass markers is
showntotheleft.
Ó FEBS 2002 Functionality of arthropod SRFs in mammalian cells (Eur. J. Biochem. 269) 3675
cofactors that could participate in the activation of this
promoter. The consequences of human SRF co-expression
on the activity of these promoters were also different. The less
active CA and Act403mut promoters were stimulated 12–20
times by human SRF co-expression in three different cell lines
and using two expression vectors, pcDNA3 and pSG5. The
levels of exogenous SRF expression were similar to those of
endogenous SRF, at least in the stably expressing C
2
C
12
cells.
Activation of cardiac a-actin promoter by SRF cotrans-
fection has been described previously [39]. Other cardiac-
and smooth muscle-specific gene promoters are also
activated by SRF cotransfection, such as those of smooth
muscle a-actin [40], atrial natriuretic factor [41] or SM22a
genes [42]. There are several possible mechanisms that could
explain promoter activation by an increase in SRF expres-
sion. Higher SRF concentration might increase the amount
of SRF bound to low affinity promoters, which could be the
case for the Act403mut promoter. The increase in SRF
concentration may also compete with inhibitory molecules
bound to the CArG box or to SRF itself, either bound to
DNA or in solution. Several inhibitory factors that bind to
SRF or compete with SRF-binding to the promoter have
been described [43]. This later mechanism could be respon-

sible for the activation of the CA promoter.
The repressor effect of DmSRF and, to a lesser extend,
AfSRF could be explained by the absence of transactivation
domains functional in mammalian cells. Vertebrate SRF
molecules without transactivation domain have been shown
to act as dominant negative transcription factors [37]. This
possible explanation would require interaction between
DmSRF and AfSRF, and the endogenous mouse SRF.
This interaction was demonstrated by co-immunoprecipita-
tion experiments. Hybrid proteins were expressed to further
test for this possibility. These molecules contained the
N-terminal region, including the MADS box and SRF-
conserved region, from one species and the C-terminal
region from another. The results obtained were in agreement
with the hypothesis that the C-terminal transactivation
domain is not conserved between species. Hybrid molecules
containing D. melanogaster or A. franciscana N-terminal
regions and human C-terminal region were able to activate
transcription from the CA and c-fos promoters. The activity
of these constructs on c-fos promoter was significantly higher
than that of human SRF, which could be due to the absence
of repressor domains localized in the N-terminal region of
this molecule [7]. These results suggest the capacity of the
N-terminal region of SRFs from these organisms to bind
DNA and dimerize with the mammalian SRF and of the
human C-terminal region to activate transcription. In
contrast, hybrid molecules that contained either D. mela-
nogaster or A. franciscana C-terminal regions did not acti-
vate CA promoter and strongly repressed the c-fos promoter,
independently of the origin of the N-terminal region.

The lack of activity of the A. franciscana C-terminal
region was unexpected as cotransfection of this SRF
molecule stimulated CA and Act403mut promoters. Dele-
tion experiments confirmed that AfSRF molecules lacking
the C-terminal region still activated CA-promoter-depen-
dent transcription. More extensive deletion experiments did
not allow to unequivocally locate a transactivation domain
in AfSRF. Molecules lacking the region N-terminal to the
MADS box also activated transcription, which would
suggest that the transcriptional activation domain is located
in the MADS box region. However, the conserved MADS
box and immediately C-terminal SAM domain, devoid of
the rest of N- and C-terminal regions, did not activate
transcription, suggesting that this region is necessary but not
sufficient for transcriptional activation. It is possible that
nonconserved N- or C-terminal regions could be necessary
for the proper structure of the MADS box. Alternatively,
cofactors implicated in activation could bind SRF through
the conserved MADS box and other nonconserved N- or
C-terminal Af SRF regions.
Despite the results discussed above, a significant decrease
in Act403mut promoter activation was observed in cells after
deletion of the region corresponding to amino acids 45–60.
These results suggest that this region can act as a transac-
tivation domain in a promoter-dependent manner. This
region includes a small evolutionary conserved domain that
has been reported to be phosphorylated by several protein
kinases in vertebrates [21]. Recently, Hanlon et al. [44] have
proposed that phosphorylation of Ser103 of vertebrate
SRF, located in this conserved region, promotes interaction

between SRF and C/EBPa to activate transcription.
In summary, the data obtained in this study indicate the
conservation of the SRF DNA-binding and dimerization
domains during evolution and the divergence of the
transactivation domain. These studies have been carried
out in mammalian cells and they might not be translated to
the cellular environment of the other species. There could be
transactivation domains functional in D. melanogaster and
A. franciscana that have not conserved the capacity to
interact with mammalian cofactors or with the basic
transcriptional machinery. This lack of conservation would
be in agreement with the very different patterns of expres-
sion and physiological functions of SRF in these organisms,
as described in the Introduction. It seems possible that each
SRF molecule could have evolved to interact with different
tissue-specific cofactors in the different species, which would
explain the divergence of their transactivation domains.
ACKNOWLEDGEMENTS
We are indebted to Dr Rosario Perona for donation of several vectors
and reagents and to Drs Rosario Perona and Ricardo Escalante for
critical reading of the manuscript. This work was supported by Grant
PB98-0517 from the Direccio
´
n General de Investigacio
´
n.
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