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 S
Trang 1Transactivation 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 melanogasterand Artemia franciscana SRFs in
mam-malian cells has been compared in this article
D melanogasterand, 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)6GG [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: lsastre@iib.uam.es
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)
Trang 2regulate 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
E X P E R I M E N T A L P R O C E D U R E S
Cell culture and transfection
Myoblastoid C2C12[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 mMglutamine
Embryonic fibroblastoid cells NIH3T3 were cultured in
DMEM supplemented with 10% neonatal bovine serum
and 2 mMglutamine 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 lg of the b-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 thePRISM2.0 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 106C2C12 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 mgmL)1 of geneticin for 24 h Cells were washed and cultured in the same medium containing 0.5 mgmL)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 SacI and KpnI sites
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) and the 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 BclI site 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 franciscanaSRF 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–SnaI fragments and used to replace 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
Trang 3TCTTATATGAATGCAGTTCTG-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¢
The C1 deletion was generated by PCR using the
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
C1 as primers, and inserted in pcDNA3-myc All the
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 lg for AfSRF 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 mM Hepes, pH 7.0, 70 mM NaCl,
0.005% NP40, 0.05 mgmL)1 BSA, 2% Ficoll, 2 mM
dithiothreitol, 0.5 mMphenylmethanesulfonyl 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 lg for AfSRF 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
R E S U L T S
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 franciscanaactin 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 C2C12 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
Trang 4Inhibition 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
Act403promoter, 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 C2C12cell 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 C2C12cells, either nontransfected (C2C12) or stably expressing human (C2-Hs), D melanogaster (C2-Dm) or A franciscana (C2-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 lg of pXP2 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).
Trang 5HsSRF, DmSRF and AfSRF and the endogenous factor in
C2C12cells 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.
Trang 6c-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 ofA 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 shown to be activated by AfSRF 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 Act403mutpromoter 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.
Trang 7D I S C U S S I O N
In this article, we have compared the function of SRF
molecules from a vertebrate, Homo sapiens, and two
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)6GG
[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 Act403promoter, 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 franciscana 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-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 shown to the left.
Trang 8cofactors 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 C2C12cells
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-nogasteror 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
A C K N O W L E D G E M E N T S
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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