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First published online 4 October 2006
doi: 10.1242/dev.02610
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1 Department of Molecular Biology, University of Texas Southwestern Medical
Center, 6000 Harry Hines Blvd, Dallas, TX 75390, USA.
2 Department of Pathology, University of Texas Southwestern Medical Center, 6000
Harry Hines Blvd, Dallas, TX 75390, USA.
* Author for correspondence (e-mail: eric.olson{at}utsouthwestern.edu)
Accepted 5 September 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Mouse, Myocardin, Smooth muscle, Enhancer, Transcriptional regulation, Transgene
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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). Srf is expressed in a wide range of cell types and confers
cell type specificity and signal responsiveness to its target genes by
recruiting specific transcriptional cofactors.
Myocardin and myocardin-related transcription factors (MRTFs) comprise a
family of Srf coactivators that associate with the MADS (MCM1, agamous,
deficiens, Srf)-box of Srf and contribute their powerful transcription
activation domains to Srf target genes
(Pipes et al., 2006).
Myocardin is expressed specifically in cardiac and smooth muscle cells (SMCs)
and activates muscle genes associated with the differentiation of these cell
types (Wang et al., 2001;
Wang et al., 2003). In
transfected fibroblasts and stem cells, myocardin can activate a large array
of smooth muscle genes (Chen et al.,
2002; Du et al.,
2003; Pipes et al.,
2005; Wang et al.,
2003; Yoshida et al.,
2003), whereas ectopic expression of myocardin in Xenopus
embryos results in activation of cardiac muscle genes even in spinal cord
neurons (Small et al., 2005;
Wang et al., 2003). Knockout
mice lacking myocardin die during early embryogenesis with a lack of
differentiated SMCs (Li et al.,
2003). The myocardin-related transcription factors Mrtfa
(Mal/Mkl1) and Mrtfb (Mkl2) (Cen et al.,
2003; Du et al.,
2004; Ma et al.,
2001; Oh et al.,
2005; Wang et al.,
2002) are expressed in a broad range of cell types. Mrtfb has been
shown to play an essential role in the development of a specific subset of
vascular SMCs derived from the neural crest
(Li et al., 2005;
Oh et al., 2005).
Mrtfb knockout mice die by embryonic day (E) 14.5 and display severe
defects in the anatomy of branchial arch arteries, and cardiac outflow tract
accompanied by a defect in smooth muscle differentiation. Mice lacking Mrtfa
are viable and display a defect specific to lactating females in which
myoepithelial cells, which provide contractility required for secretion of
milk from the mammary gland, fail to differentiate and undergo apoptosis
(Li et al., 2006; Sun et al.,
2006).
During embryogenesis, the onset of myocardin expression coincides with
specification of the cardiac lineage in the cardiac crescent, a specialized
region of the developing mesoderm that adopts a cardiac fate in response to
inductive cues from adjacent tissues
(Fishman and Chien, 1997;
McFadden and Olson, 2002).
Expression of myocardin in early cardiac progenitors coincides with that of
Nkx2-5, the earliest known marker of the cardiac lineage
(Lints et al., 1993).
Thereafter, myocardin is expressed throughout the atrial and ventricular
chambers of the heart, as well as in a subset of SMCs in the dorsal aorta and
outflow tract of the heart, the esophagus, gut, lung and a subset of head
mesenchyme cells (Wang et al.,
2001).
While the downstream target genes of myocardin have been described in detail, little is known of the mechanisms that regulate this early regulator of cardiovascular development and gene expression. Here we describe an enhancer upstream of the mouse myocardin gene that is sufficient to direct cardiovascular expression of myocardin during mouse embryogenesis. This enhancer serves as a direct transcriptional target of Mef2, Tead and Foxo transcription factors. In contrast to nearly every known smooth muscle gene, the myocardin enhancer is independent of Srf. Intriguingly, however, myocardin activates its own enhancer via Mef2. These findings reveal a unique transcriptional strategy for the activation of smooth and cardiac-muscle-specific transcription and suggest the existence of a positive feedback loop, through which myocardin regulates its own expression in collaboration with Mef2.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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For the generation of transgenic mice, constructs were digested with
SalI to remove vector sequences. DNA fragments were purified using a
QiaQuick spin column (Qiagen, MD), injected into fertilized eggs from B6C3F1
female mice, and implanted into pseudopregnant ICR mice as previously
described (Lien et al., 1999).
Embryos were collected and stained for ß-galactosidase activity.
Sectioning, histology and Nuclear Fast Red staining were performed on the
embryos as previously described (McFadden
et al., 2000).
Primer sequences
MyE1, 5'-TTCCCACTAACATTGAGTACCAGGG-3' (forward) and
5'-AGGCCTCTTGATTATGTTTAAAATTGG-3' (reverse);
MyE2, 5'-TTCCCACTAACATTGAGTACCAGGG-3' (forward) and 5'-AGATACAGTGTCCACAACATACAC-3' (reverse);
MyE3, 5'-AGTTGGTTTCCATCAACAGGAG-3' (forward) and 5'-AGGCCTCTTGATTATGTTTAAAATTGGG-3' (reverse); MyE4, 5'-TTCCCACTAACATTGAGTACC-3' (forward) and 5'-TTTCTATGCATCATGGGTCCATGC-3' (reverse);
MyE5, 5'-TCAATTTTTCGAAGAGTTCCAGTG-3' (forward) and 5'-AGATACAGTGTCCACAACATAC-3' (reverse);
MyE6, 5'-AGTTGGTTTCCATCAACAGG-3' (forward) and 5'-TCCAAGCACCTCTATTTAAACC-3' (reverse);
MyE7, 5'-TATCTACCATTCACACTGAGACC-3' (forward) and 5'-AGGCCTCTTGATTATGTTTAAAATTGG-3' (reverse); and
MyE8, 5'-TTCTGACTGGGTCCCTTACCACTGTCTTTGCA-3' (forward) and 5'-TGCAGTAAAAAACAAATAGAACATTTGG-3' (reverse).
Constructs, transfections and luciferase assays
Mef2c, Myocardin and Foxo4 murine expression constructs have been described
(Liu et al., 2005;
Martin et al., 1993;
Wang et al., 2001). Expression
vectors encoding rat Tead1 were kindly provided by Dr Ian Farrance (Baltimore,
MD) (Zuzarte et al., 2000).
The 350 bp, evolutionarily conserved, myocardin enhancer (MyE8) was cloned
into the pGL2-E1b-luciferase reporter and the various deletion mutants were
constructed through PCR-based mutagenesis and sequence-verified. Cells were
transfected using Fugene 6 (Roche Molecular Biochemicals) with 250 ng of cDNA
expression library together with 100 ng of MyE8-luciferase reporter and 30 ng
of pCMV-lacZ. Forty-eight hours after transfection, cell extracts
were assayed for luciferase expression using the luciferase assay kit
(Promega). Relative promoter activities are expressed as luminescence relative
units normalized for ß-galactosidase expression in cell extracts. All
transfection assays were performed at least three times.
cDNA expression screening
A mouse E10.5 cDNA expression library was purchased from Invitrogen. cDNA
pools were prepared by using the PerfectPrep Plasmid 96 Vac Direct Bind kit
(Eppendorf). Each cDNA pool used in the screening contained 50-100 single cDNA
clones. In each well of a 24-well plate, 5x104 COS cells were
cultured in DMEM with fetal bovine serum (10%), L-glutamine (2 mmol/l), and
penicillin-streptomycin and transfected as described previously
(Chang et al., 2005). For
sib-selection, positive pools of cDNA expression plasmids were transformed
into competent cells and plated on LB agar dishes. For each positive pool, 96
single clones were picked from a dish and grown in LB liquid media. Twelve
single clones were combined as sub-pools, and plasmids were prepared and
transfected for the reporter assays as described above. Single clones from the
positive sub-pools were prepared and tested for their ability to activate
MyE8-luciferase reporter.
Electrophoretic mobility shift assays
Oligonucleotides corresponding to the conserved Mef2, Foxo and Tead-binding
and mutated binding sites were synthesized (Integrated DNA Technology) and
used in gel mobility shift assay. Oligonucleotide sequences were as follows
(the transcription factor binding sites in bold): Mef2 oligo,
5'-GGCCAAGATTGACAGCCACTATTTTAAGAAGTGCTTTTAA-3'; Foxo oligo
site 1, 5'-GGGCATTTCCATTAACATTTCTATTTCTGG-3'; Foxo oligo
site 2, 5'-GGGCAAGTTCAAAATAACATTCCAGAAGGAGTCA-3'; Foxo
oligo site 3, 5'-GGGAGTCACTAGAAAAACATTCAAGGGAAGAG-3'; Foxo
oligo site 4, 5'-GGAAGAGAAAAAAAT-TGTTTTCGTTTCGTAGCAGA-3';
Foxo oligo site 5,
5'-GGCAAATGTTCTATTTGTTTTTTACTGCAGAAAGCC-3'; Tead oligo,
5'-GGTGCAAGTTCAAAATAACATTCCAGAAGGAGTCA 3'.
Annealed oligonucleotides were radiolabeled with [32P]dCTP using
the Klenow fragment of DNA polymerase and purified using G50 spin columns
(Roche). Proteins were translated in vitro in a coupled
transcription-translation T7 reticulocyte lysate system (Promega). Reaction
conditions of the gel mobility shift assays were previously described
(McFadden et al., 2000). Mouse
anti-TEF1 (BD biosciences Pharmingen) was used for Tead1 supershift, anti-AFX
(goat polyclonal, Santa Cruz) for Foxo4 supershift and anti-Mef2c (rabbit
polyclonal, Santa Cruz) for Mef2c supershift experiments. Unlabeled
oligonucleotides used as competitors were annealed as described above and
added to the reactions at the indicated concentrations. DNA-protein complexes
were resolved on 5% polyacrylamide native gels and the gels were exposed to
BioMax X-ray film (Kodak).
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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The temporal and spatial expression pattern of the myocardin enhancer was
determined using a stable transgenic line bearing MyE-HsplacZ
(Fig. 1B). Enhancer activity
marked cardiomyocytes in the cardiac crescent beginning at E7.5
(Fig. 1B, parts a,b).
Transverse and sagittal sections through transgenic embryos showed intense
staining of cells within the cardiogenic plate
(Fig. 2A). By E7.75, the
enhancer directed high levels of expression in the bilateral cardiogenic
precursors and in no other regions of the embryo, an expression pattern very
similar to markers of the primary heart field (atrial myosin light chain 2v
and 
-cardiac actin) at this stage
(Cai et al., 2003;
Kelly et al., 2001).
Recent studies have revealed a population of cardiac precursor cells,
referred to as secondary heart field, which is derived from a region of the
splanchnic mesoderm medial to and distinct from the primary heart field that
makes up the cardiac crescent (Cai et al.,
2003; Kelly et al.,
2001; Kelly and Buckingham,
2002; Mjaatvedt et al.,
2001; Waldo et al.,
2001). Cells from the secondary heart field are added to the
anterior region of the heart tube at the onset of looping and contribute to
the formation of the outflow tract (OFT) and right ventricle (RV). The
myocardin enhancer did not direct lacZ expression to cells in the
splanchnic mesoderm (Fig.
2B,C), suggesting that myocardin is not expressed in the anterior
heart field at these stages.
Between E8.0 and 9.5, high levels of lacZ expression were seen throughout the linear heart tube, and expression remained homogeneous throughout the heart during looping (Fig. 1B, parts d,f and Fig. 2B,C). lacZ continued to be expressed at high levels throughout the developing heart (with the exception of the cardiac cushions) until E13.5 (Fig. 2D,E). Thereafter, expression gradually diminished to lower but detectable levels and persisted in adult hearts (Fig. 1B, parts e,l-n and Fig. 2J). We conclude that the MyE is sufficient to direct the complete cardiac expression pattern of the endogenous myocardin gene during mouse embryogenesis.
Like the endogenous myocardin gene
(Wang et al., 2001), the
MyE-HsplacZ transgene also directed lacZ expression in SMCs
of the developing dorsal aorta as early as E9.0
(Fig. 1B, parts f and g), which
coincides with the time when SMCs populate and differentiate in large
developing vessels (Owens,
1998). Smooth muscle markers, such as SM22 and
SM-actin, which are regulated by myocardin
(Wang et al., 2001;
Yoshida et al., 2003), become
expressed at E9.5-10.5 (Owens,
1998), supporting previous findings that myocardin is required for
the expression of these and other smooth muscle markers in vivo
(Li et al., 2003). During the
course of development, lacZ expression was apparent in developing
large vessels such as the dorsal aorta and branchial arch arteries
(Fig. 1B, parts g,h,k,l and
Fig. 2F) and carotid arteries
(Fig. 1B, part k). Expression
in vessels in the head region, pulmonary outflow tract and intersomitic
arteries became apparent after E13.5. In addition to its expression in the
cardiovascular system, myocardin is expressed in SMCs within visceral organs,
including lung, bladder, intestine and stomach
(Wang et al., 2001). Transgene
expression was not detectable in SMCs of the bladder and stomach, nor in the
vascular plexus of the yolk sac or head mesenchyme, also sites where the
endogenous gene is expressed (not shown). Thus the lacZ expression
pattern recapitulates most, but not all of the expression pattern of the
endogenous myocardin gene. In adult vascular tissues, lacZ expression
was detected in SMCs of the aorta (Fig.
1B, part o and Fig.
2H, coronary arteries, lung arteries
(Fig. 1B, part j and
Fig. 2G) and other small
arteries (Fig. 2I). Notably, in
adult tissues, lacZ was expressed in a mosaic manner
(Fig. 1B, parts e,o and
Fig. 2G,H,J). It is not clear
whether this reflects a heterogenous expression pattern of myocardin in adult
heart and vascular SMCs or whether it is intrinsic to the usage of randomly
integrated transgenes in which mosaic expression is not uncommon
(Liu et al., 2006).
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Identification of cardiac and smooth muscle regulatory regions of the myocardin gene
To further delineate the regulatory elements within the 10 kb MyE fragment,
we compared the mouse sequence with rat, human, dog and chicken sequences for
evolutionary conservation (UCSC genome informatics:
www.genome.ucsc.edu/).
Based on the conservation pattern depicted in
Fig. 3B, we tested a series of
MyE deletion fragments for the ability to direct cardiac- and
smooth-muscle-specific expression in transgenic embryos at E12.5. First, we
divided the 10 kb MyE fragment into two equal, non-overlapping fragments of 5
kb (MyE2 and MyE3 in Fig. 3B).
Interestingly, both fragments contained transcriptional enhancers sufficient
to direct lacZ expression in the heart. Whereas MyE3 directed
lacZ expression mainly to the heart, the MyE2 fragment showed strong
expression in the myocardial layer of the heart and smooth muscle
cells of the dorsal aorta and head vessels at E12.5. The MyE2 and MyE3 regions
were further subdivided and all subfragments were tested with
hsp68-lacZ (Fig.
3B).
Based on evolutionary conservation, a 350 bp subfragment (MyE8) of MyE2 was identified and found to be sufficient to direct lacZ expression in the heart and a distinct subset of SMCs (Fig. 3A,B). Deletion of the highly conserved 350 bp region in the context of the 3 kb MyE4 enhancer fragment (fragment MyE9) abolished lacZ expression completely, indicating that elements responsible for myocardin expression in differentiating SMCs and heart are present within the 350 bp sequence of MyE8. Fragment MyE3 was further delimited to a minimal enhancer of 2 kb (MyE6), which was sufficient to direct lacZ expression in the heart but not in vascular SMCs (Fig. 3A,B). As MyE8 contained cis-regulatory elements involved in the expression of both heart and smooth muscle, and could be narrowed down to a relatively small, highly conserved fragment, we chose to focus on this fragment for further studies. A summary of the expression patterns of all generated transgenic embryos is shown in Table S1 (in the supplementary material). Because the expression of active transgenes was lower in the vasculature than in the heart, animals with lower levels of transgene expression often did not show expression in the vasculature. Thus, the frequency of vascular versus cardiac expression was slightly reduced (see Table S1 in the supplementary material).
The myocardin enhancer is dependent on an essential Mef2 site
Inspection of the sequence of the MyE8 region revealed a highly conserved A
+ T-rich DNA sequence (CTATTTTAAG) (Fig.
3C) that resembled the binding site of Mef2 transcription factors,
which bind the consensus sequence CTA(A/T)4TAG
(Black and Olson, 1998). In
light of the expression of Mef2c in the cardiac crescent, developing heart and
vascular system (Black and Olson,
1998; Edmondson et al.,
1994), and the essential role of Mef2c in cardiac and smooth
muscle development (Lin et al.,
1998; Lin et al.,
1997), we tested whether Mef2c could bind this sequence in the
myocardin enhancer.
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To further define the potential role of the Mef2 site for cardiac and smooth muscle activity of the myocardin enhancer, we mutated this site in the context of the 3 kb enhancer (fragment MyE4) and tested this mutant enhancer for its ability to direct lacZ expression in combination with the hsp68 basal promoter in vivo. Examination of ten F0 transgenic embryos at E12.5 failed to show lacZ expression in heart and vessels with the mutant enhancer (Fig. 4B). In some embryos, ectopic expression of lacZ was observed, reflecting transcriptionally active sites of transgene integration. These results demonstrate that the Mef2 site in the myocardin enhancer is essential for expression in heart and smooth muscle in vivo.
Myocardin activates its own enhancer through an Srf-independent mechanism
In light of the recent discovery of a cardiac enriched isoform of myocardin
containing a unique N-terminal peptide sequence that confers the ability to
stimulate the transcriptional activity of Mef2
(Creemers et al., 2006), we
tested whether myocardin could activate its own enhancer through the
identified Mef2 site. Indeed, as shown in
Fig. 4C, the cardiac isoform of
myocardin (myocardin-956) and Mef2c synergistically activated the MyE8
enhancer linked to a luciferase reporter, whereas the enhancer in which the
Mef2 site was mutated was non-responsive. Responsiveness of the myocardin
enhancer to myocardin and Mef2c required the N-terminal domain of myocardin
that associates with Mef2, as the smooth muscle isoform of myocardin lacking
this domain (myocardin-856) failed to activate the enhancer. Notably, the
myocardin enhancer does not contain an Srf-binding site. These findings
suggest that myocardin cooperates with Mef2 to activate its own enhancer
through an Srf-independent mechanism.
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100 cDNA clones each from
a mouse E10.5 expression library. We screened 2000 cDNA pools,
representing 200,000 individual cDNA clones and identified a total of
four positive pools. Sib-selections of the positive pools resulted in the
identification of the transcription factors Tead2 and Foxo4 as activators of
the MyE8 enhancer (Fig. 5A,
Fig. 6A).
Four mammalian Foxo proteins have been described to date - Foxo1, Foxo3a,
Foxo4 and Foxo6 (Van Der Heide et al.,
2004) - with Foxo4 being most abundant in myocyte-containing
tissues (Furuyama et al.,
2000). Foxo proteins recognize and bind DNA sequences containing
the core nucleotide sequence AAACA (Arden
and Biggs, 3rd, 2002; Biggs,
3rd et al., 2001). We inspected the myocardin enhancer for
evolutionarily conserved Foxo-binding sites and noted five potential sites in
the MyE8 sequence (Fig. 3C). As
shown in Fig. 5B, full-length
Foxo4 potently activated the MyE8 reporter, but not the MyE8 reporter in which
the Foxo sites were mutated. Gel mobility shift assays showed that Foxo4 bound
to four of the five Foxo sites and could be supershifted using a
Foxo4-specific antibody (Fig.
5C).
To test the function of the Foxo sites in vivo, we introduced mutations into each site in the context of the MyE4-lacZ transgene and determined the effect of those mutations on enhancer function at E10.5 (Fig. 5D; see Table S1 in the supplementary material). Mutation of all five Foxo sites resulted in a dramatic reduction in lacZ expression in heart and developing vessels, but the overall pattern of lacZ expression was unchanged, indicating that these sites have a quantitative effect on expression in vivo.
The Tead family of transcription factors consists of four members
(Tead1-4), which share homology in a DNA-binding domain called a TEA domain.
Tead genes are expressed widely in embryonic and adult tissues, and display
distinct but overlapping expression patterns
(Jacquemin et al., 1998;
Kaneko et al., 1997). Tead1
mutant embryos die between E11 and 12 from heart defects
(Chen et al., 1994). Analysis
of the sequence of MyE8 revealed a consensus Tead-binding motif, CATTCCA
(Davidson et al., 1988;
Jacquemin et al., 1998;
Xiao et al., 1991), within
this enhancer (Fig. 3C).
To confirm the results of the expression screen, in which we identified Tead2 as a potential activator of the myocardin enhancer, we expressed Tead2 in COS cells along with the MyE8-luciferase reporter. Tead2 activated the MyE8 reporter approximately eightfold over the empty reporter and a mutation in the Tead-binding site reduced this activation approximately twofold. As an antibody for supershift experiments was only available for Tead1, we tested for binding of Tead1 to the putative binding site in the MyE8 using Tead1 translated in vitro. As shown in Fig. 6C, Tead1 was able to bind the radiolabeled Tead site, and the DNA-Tead1 complex could be supershifted by the addition of Tead1-specific antibody. Specificity of the interaction was further demonstrated by competition with excess unlabeled probe but not with a mutated probe (Fig. 6C).
As mutation of the Tead site in the MyE8 reporter did not abolish Tead
activity completely (Fig. 6B),
we searched for other possible Tead-binding sites within MyE8. We noticed a
sequence 20 bp downstream of the Tead-binding site that resembled, but
did not completely fit, the consensus sequence for a Tead site (CATTCA instead
of the CATTCCA) and gel shift analysis with probes for this site showed almost
no Tead1 binding (not shown). Therefore, we do not consider this second site
to be a Tead site, although we cannot rule out that it contributes to the
residual luciferase activity seen in the MyE8-Tead mutant in
Fig. 6B. Perhaps the residual
activity of Tead2 on the mutated MyE8 reporter is indirect, via interaction
with Mef2, or another factor. As Mef2 and Tead2 can physically interact
(Maeda et al., 2002), we
co-transfected Mef2c and Tead2 expression plasmids in COS cells, but saw no
synergistic activation of the MyE8 reporter.
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). We
conclude that Tead proteins act specifically to control myocardin expression
and smooth muscle differentiation in branchial arch arteries and dorsal
aorta. In an attempt to identify potential molecular interactions in the regulation of the myocardin enhancer, we transfected all possible combinations of Mef2c, Foxo4 and Tead2 expression plasmids in COS cells along with the MyE8 reporter. Luciferase assays failed to show a combinatorial effect (synergistical activation or repression) of Mef2c, Foxo4 and Tead2 on the MyE8 reporter.
In addition to the Mef2, Tead and Foxo sites, we also tested candidate NKE- and E-box-binding sites to determine if these putative sites in the myocardin enhancer might be targets for Nkx2.5 and HAND proteins, respectively (Fig. 3C). Mutations introduced in those sites in the context of the MyE4-lacZ transgene demonstrated that these sites were dispensable for enhancer function in vivo (see Table S1 in the supplementary material). Together, the results of the in vivo and in vitro assays demonstrate that the binding sites for Mef2, Tead and Foxo proteins in the myocardin enhancer are required for complete enhancer function during cardiac and smooth muscle development in vivo.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), and in SMCs
within large vessels such as the branchial arch arteries (E10.5-11.5) and
carotid arteries (E12.5). The myocardin enhancer is a direct target of Mef2
and Foxo in cardiac and smooth muscle cells and is regulated by Tead factors
in dorsal aorta and neural-crest-derived SMCs. Notably, myocardin can also
activate its own enhancer, but in contrast to the majority of myocardin target
genes, myocardin acts not through Srf, but through Mef2, to regulate its
transcription. To our knowledge, the myocardin enhancer is the only
cis-regulatory region described to date that relies on this
combination of transcription factors for cardiovascular expression.
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; Sun et
al., 2006). By contrast, our results show that expression of myocardin in
smooth muscle does not require CArG boxes, reflecting its unique mode of
regulation as an upstream regulator of subordinate CArG-independent genes. The
histidine-rich calcium binding protein (HRCBP) gene is another rare
example of a smooth muscle gene that is not controlled by CArG boxes
(Anderson et al., 2004).
Notably, Mef2 also serves as an essential regulator of HRCBP
expression, but Tead and Foxo have not been shown to regulate the
HRCBP enhancer.
Multiple, distant myocardin enhancers specify expression during development
Modular control of gene transcription, in which multiple independent
regulatory elements combine to direct the complete expression pattern of a
gene, has emerged as a common theme in the control of cardiac gene expression
(Firulli and Olson, 1997).
Through analysis of evolutionarily conserved DNA sequences and the testing of
several deletion fragments in transgenic embryos, we were able to identify two
independent enhancers (MyE6 and MyE8) that control myocardin expression. Both
regions were sufficient to direct lacZ expression to the heart, but
only one of those fragments (MyE8) was able to direct lacZ expression
to SMCs. In addition to its expression in the cardiovascular system, myocardin
is expressed in SMCs within visceral organs, including lung, bladder,
intestine and stomach. The MyE8 enhancer is active in lung SMCs, but not in
SMCs within these other organs, suggesting that expression in these tissues is
controlled by additional enhancers, which either lie outside the 90 kb of
genomic sequence we surveyed in transgenic mice or are influenced by negative
regulatory elements that preclude their identification in the constructs we
generated.
We note that Nkx2-5 has been reported to activate the proximal myocardin
promoter in transfection assays via an Nkx-binding element (NKE), although the
potential involvement of this DNA sequence in myocardin expression in vivo was
not examined in that study (Ueyama et al.,
2003). The distal upstream enhancer we identified is approximately
10 kb away from the region described by Ueyama et al.
(Ueyama et al., 2003) and it
does not appear to require Nkx2-5 for activity. Our results also indicate that
the region they described is unable to direct expression in vivo.
Transcriptional regulation of myocardin by Mef2
The myocardin enhancer MyE8 contains a conserved Mef2 site, which is
required for enhancer activity in vivo. Among the four members of the Mef2
family, Mef2c is the earliest marker of the cardiac lineage, and is required
for morphogenesis and maturation of the heart and vascular system
(Lin et al., 1998;
Lin et al., 1997;
Naya et al., 2002). Thus,
Mef2c is likely to act through this enhancer element. As myocardin and Mef2
are co-expressed in the early cardiac crescent, we think Mef2c is most likely
to participate in amplification and maintenance of myocardin expression rather
than the initial activation of the gene.
Recently, we discovered that a cardiac isoform of myocardin, containing a
unique N-terminal Mef2-binding motif, is able to interact with Mef2 and
activate transcription through a subset of Mef2-dependent regulatory elements
in the heart (Creemers et al.,
2006). This cardiac isoform of myocardin was able to coactivate
the myocardin enhancer with Mef2, and mutation of the Mef2 site abolished
myocardin responsiveness of the enhancer. The dependence of the myocardin
enhancer on Mef2 suggests the existence of a positive autoregulatory loop,
through which myocardin may amplify and maintain its expression in the heart
via this site. The predominant myocardin splice variant expressed in SMCs
lacks the Mef2-binding peptide and is incapable of coactivating Mef2
(Creemers et al., 2006). Thus,
although the Mef2 site in the myocardin enhancer is required for enhancer
activity in cardiac and smooth muscle cells, this putative positive feedback
loop would only be operative in the heart, suggesting that Mef2 cooperates
with other cofactors to control myocardin transcription in SMCs. In this
regard, Mef2 has been shown to cooperate with members of the Tead family
(Maeda et al., 2002).
The existence of a possible autoregulatory loop to maintain myocardin
expression specifically in cardiac muscle but not in SMCs may reflect one of
the intrinsic differences between these two muscle cell types. In this regard,
cardiomyocytes, like skeletal muscle cells, differentiate irreversibly.
Positive autoregulation of the myocardin gene may serve to stabilize and
maintain the cardiac phenotype in a manner similar to the autoregulation of
the MyoD family in skeletal muscle (Thayer
et al., 1989). By contrast, SMCs are highly plastic and modify
their phenotype in response to developmental, physiological and pathological
cues. The potential regulation of myocardin transcription by Mef2 and the
resulting reinforcement of myocardin expression are schematized in
Fig. 7.
Regulation of the myocardin enhancer by Foxo
The myocardin enhancer depends on multiple Foxo-binding sites for activity
in vivo. The Foxo family of forkhead-type transcription factors regulates the
transcription of genes involved in various cellular processes, including
proliferation, differentiation, metabolism and survival
(Accili and Arden, 2004). All
four Foxo family members, Foxo1, Foxo3a, Foxo4 and Foxo6, are able to bind the
myocardin enhancer (data not shown), but we can only speculate as to which
specific family members regulate the enhancer in vivo. In this regard,
abundant expression of Foxo4 in myocyte-containing tissues
(Furuyama et al., 2000) and
recent evidence for the involvement of Foxo4 in phenotypic modulation of SMCs
(Hayashi et al., 1998;
Liu et al., 2005) support a
possible role for Foxo4 as an upstream regulator of myocardin transcription.
Interestingly, Foxo4 appears to trigger dedifferentiation of SMCs by
interacting with and repressing the transcriptional activity of myocardin
(Liu et al., 2005). This may
counterbalance the actions of Foxo4 as an activator of myocardin transcription
to finetune the potent transcriptional activity of myocardin.
Foxo1 expression overlaps that of myocardin in the heart, dorsal aorta and
small vessels such as intersomitic and head vessels during early embryogenesis
(Furuyama et al., 2004;
Hosaka et al., 2004).
Moreover, Foxo1 null embryos die around E11 with defects in branchial
arches and vascular remodeling (Hosaka et
al., 2004). Whether these abnormalities reflect an essential role
for Foxo1 solely in endothelial cells or whether there is an additional defect
in SMC differentiation, possibly mediated by myocardin, is an interesting
question for future investigation.
Regulation of the myocardin enhancer by Tead proteins
Through a eukaryotic cDNA expression screen we identified the transcription
factor Tead2 as an activator of the myocardin enhancer. The Tead (TEA domain)
family of proteins contains a highly conserved 72 amino acid DNA-binding
domain, which is evolutionarily conserved among yeast, Drosophila,
rat, chick, mouse and human (Jacquemin et
al., 1996; Kaneko et al.,
1997; Kaneko and DePamphilis,
1998). Tead proteins recognize the canonical M-CAT motif
(CATTCCT/A) (Farrance and Ordahl,
1996; Larkin et al.,
1996) found in promoters of muscle specific genes such as those
encoding smooth muscle -actin, skeletal -actin, cardiac and
skeletal troponin T and - and ß-MHC
(Larkin et al., 1996;
Swartz et al., 1998).
Moreover, disruption of the Tead1 gene disrupts heart development
(Chen et al., 1994). Our
results show that transcriptional activity of the myocardin enhancer crucially
depends on a Tead-binding site, specifically in the branchial arch arteries
and dorsal aorta. Previously reported expression data suggest that among
members of the Tead family, Tead2 is most strongly expressed in regions of
neural crest and branchial arches.
The requirement of a Tead-binding site for activation of the myocardin
enhancer in neural-crest-derived smooth muscle cells is consistent with the
recent identification of Tead2 as a neural-crest-specific regulator of the
transcription of Pax3, which is one of the earliest markers of neural
crest induction (Milewski et al.,
2004). Interestingly, Pax3-expressing neural crest precursors
become the SMCs of the aortic arch artery and major cranial vessels.
Implications
In addition to its important roles in the control of cardiac and smooth
muscle development, myocardin has also been implicated in remodeling of the
adult cardiovascular system during disease. In this regard, myocardin and
CArG-dependent muscle genes are upregulated in the failing heart
(Torrado et al., 2003;
Xing et al., 2006).
Overexpression of myocardin increases cardiomyocyte size and activates the
expression of atrial natriuretic factor
(Badorff et al., 2005;
Xing et al., 2006), a
sensitive Srf-dependent marker of cardiac hypertrophy. In addition,
pathological remodeling of the vessel wall during atherosclerosis and
restenosis involves a switch in SMC phenotype from a differentiated
contractile to a proliferative `synthetic' state
(Owens et al., 2004). Signals
that perturb myocardin activity in turn block transcription of SMC contractile
genes and promote phenotypic switching
(Liu et al., 2005).
Given the signal responsiveness of Mef2, Foxo and Tead factors
(Accili and Arden, 2004;
McKinsey et al., 2002;
McLean et al., 2003), it is
likely that different extracellular signals modulate myocardin expression via
their effects on these activators. Thus, the discovery of the enhancer that
controls myocardin expression in the cardiac and smooth muscle cell lineages
provides insights, not only into the mechanisms governing the establishment of
these cell types during embryogenesis, but also offers possibilities for
manipulating myocardin expression in various therapeutic settings.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/21/4245/DC1
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ACKNOWLEDGMENTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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M. J. Potthoff and E. N. Olson MEF2: a central regulator of diverse developmental programs Development, December 1, 2007; 134(23): 4131 - 4140. [Abstract] [Full Text] [PDF]
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X. Long and J. M. Miano Remote Control of Gene Expression J. Biol. Chem., June 1, 2007; 282(22): 15941 - 15945. [Abstract] [Full Text] [PDF]
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