1 Department of Molecular Biology, University of Texas Southwestern Medical
Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9148, USA
2 Section of Molecular Genetics and Microbiology, Institute for Cellular and
Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA
3 Department of Pathology, University of Texas Southwestern Medical Center, 5323
Harry Hines Boulevard, Dallas, TX 75390-9148, USA
* Author for correspondence (e-mail: eric.olson{at}utsouthwestern.edu)
Accepted 4 April 2005
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SUMMARY |
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Key words: Cardiac gene expression, Skeletal muscle gene expression, MEF2 binding site, E-box, Cardiogenesis, Mouse, Anterior heart field
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Introduction |
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Recent studies have revealed a population of cardiac precursor cells,
referred to as the anterior or 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
(Abu-Issa et al., 2004;
Cai et al., 2003
;
Kelly et al., 2001
;
Kelly and Buckingham, 2002
;
Mjaatvedt et al., 2001
;
Waldo et al., 2001
). Cells
from the anterior heart field are added to the anterior region of the heart
tube at the onset of looping and give rise to the outflow tract (OFT) and
right ventricle (RV). By contrast, the primary heart field, which generates
the linear tube, serves as the source of precursors of the left ventricle (LV)
and atrial chambers. The existence of two populations of cardiac precursor
cells that contribute to different regions of the heart provides a potential
explanation for many cardiac abnormalities in mice and humans in which
specific cardiac structures are malformed or missing, leaving the remainder of
the heart unperturbed (Bruneau et al.,
2001
; Cai et al.,
2003
; Fishman and Olson,
1997
; Gottlieb et al.,
2002
; Lin et al.,
1997
; Srivastava et al.,
1997
; von Both et al.,
2004
). Further evidence for heterogeneity of cardiac precursors
has come from the analysis of cis-regulatory elements associated with cardiac
genes, which often direct expression in highly restricted regions of the
developing heart (Biben and Harvey,
1997
; Thomas et al.,
1998
).
Numerous transcription factors have been implicated in heart development
based on cardiac phenotypes of mutant mice, zebrafish and fruit flies, as well
as congenital heart defects in humans
(Fishman and Olson, 1997;
Hoffman and Kaplan, 2002
).
Deletion of the gene encoding the myocyte enhancer factor 2C (MEF2C)
transcription factor in mice results in severe abnormalities in the formation
of the right ventricle and outflow tract
(Lin et al., 1997
), which
mimic the cardiac defects observed in mice lacking islet 1 (ISL1), a LIM
homeodomain transcription factor expressed in the anterior heart field and its
derivatives (Cai et al., 2003
).
ISL1 was recently shown to bind a cardiac-specific enhancer that controls
Mef2c transcription in the anterior heart field, establishing a
direct transcriptional link between these cardiac control genes
(Dodou et al., 2004
). The
phenotype of Mef2c and Isl1 mutant embryos also resembles
that of embryos lacking the basic helix-loop-helix (bHLH) transcription factor
Hand2 (Srivastava et al.,
1997
). Similarly, mice lacking the Bop (Smyd1
Mouse Genome Informatics) gene, which encodes a muscle-restricted
transcriptional repressor and putative histone methyltransferase, die from
cardiac abnormalities similar to those of Isl1, Mef2c and
Hand2 mutant embryos (Gottlieb et
al., 2002
).
The intriguing similarity between the phenotypes of mice lacking Mef2c,
Hand2 and Bop raises the possibility that these transcription
factors act in a common developmental pathway. Indeed, prior studies have
shown that Hand2 is downregulated in the hearts of embryos lacking
Mef2c or Bop (Gottlieb
et al., 2002; Lin et al.,
1997
). However, it remains unclear whether the downregulation of
Hand2 expression in these mutant embryos reflects a direct influence
of MEF2C or BOP on the Hand2 gene, or a secondary consequence of the
loss of Hand2-expressing cells in these mutant embryos.
The Bop gene is expressed in cardiac precursor cells beginning at
E8.0. Thereafter, expression is maintained throughout the linear and
looping heart tube, as well as in the atrial and ventricular chambers of the
heart (Gottlieb et al., 2002
).
Bop is also expressed in the myotomal compartment of the somites and
in differentiated skeletal muscle. In an effort to further define the
potential regulatory relationship between Mef2c and Bop, we
investigated whether cardiac expression of Bop was dependent on
Mef2c. Here, we show that Bop expression in the developing
heart is downregulated in Mef2c mutant embryos, and we identify a
MEF2-response element that controls expression of Bop in the anterior
heart field during mouse embryogenesis. These findings identify Bop
as an essential downstream effector gene of MEF2C during formation of the
right ventricular chamber and OFT of the heart, and reveal a transcriptional
cascade involved in development of the anterior heart field and its
derivatives.
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Materials and methods |
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Mef2c mutant mice
Mef2c null mice have been described previously
(Lin et al., 1997). The
Mef2c mutant allele was maintained in a C57Bl6 x129SvEv hybrid
background.
Generation of transgenic mice
The reporter constructs containing hsp68-lacZ were digested with
SalI, whereas construct 4 was digested with XhoI and
NotI to remove the vector backbone. 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
(Cheng et al., 1993
).
Sectioning histology and Nuclear Fast Red staining were performed on the
embryos as previously described (McFadden
et al., 2000
).
Southern blot analysis of PCR-amplified cDNA
PCR-amplified cDNA was prepared from embryonic hearts as previously
described (Liu et al., 2001).
The membranes containing amplified cDNAs were hybridized in Rapid-hyb buffer
at 65°C with a Bop cDNA that was [32P]-labeled using
the Radprime DNA labeling system (Invitrogen). After overnight hybridization,
the membranes were washed in 0.1 xSSC, 0.1% SDS at 65°C for 10
minutes. Signals were visualized by autoradiography.
-enolase cDNA
probe was used as a loading control.
In situ hybridization of embryonic mouse tissue sections
In situ hybridization was performed on mouse sections at embryonic day (E)
9.0 using 35S-UTP-labeled Bop riboprobes (Maxiscript,
Ambion), as previously described (Shelton
et al., 2000).
Electrophoretic mobility shift assays
Oligonucleotides corresponding to the conserved MEF2-binding site in the
Bop muscle regulatory region, the mutated MEF2-binding site, and a
muscle creatine kinase (MCK) MEF2-binding site
(Gossett et al., 1989) were
synthesized (Integrated DNA Technology) as follows (+ strand sequences are
shown with the MEF2 site in bold and the mutation underlined):
Annealed oligonucleotides were radiolabeled with [32P]dCTP using
the Klenow fragment of DNA polymerase and purified using G50 spin columns
(Roche). Nuclear cell extracts were isolated from Cos-1 cells that were
transfected with pcDNAMYC-MEF2C. Reaction conditions of the gel mobility-shift
assays were previously described (McFadden
et al., 2000). 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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Identification of cardiac and skeletal muscle regulatory regions of the Bop gene
To determine whether the reduction in Bop expression in
Mef2c mutant embryos reflected a role for MEF2C in the control of
Bop expression, we sought to identify the cis-regulatory elements
responsible for cardiac expression of Bop. The Bop gene is
located on mouse chromosome 6 immediately 5' of the CD8b
(Cd8b1 Mouse Genome Informatics) gene and was originally
identified as a gene of unknown function transcribed in the opposite direction
to CD8b, hence the name CD8b opposite (Bop)
(Hwang and Gottlieb, 1995).
The Bop gene encodes protein products with distinct amino-terminal
sequences that are expressed specifically in T lymphocytes (referred to as
tBOP), and in cardiac and skeletal muscle (referred to as mBOP)
(Hwang and Gottlieb, 1997
).
The structure of the 5' region of the gene is shown in
Fig. 2A. The first exon for the
muscle-specific mBop transcript is located
70 kb 3' of the
first exon for the tBOP isoform. The DNA sequence immediately upstream of the
muscle-specific mBop first exon can be accessed at NCBI using
accession number AC115777 (with the Bop transcription start site
located at 54866 bp and the 5' end-point of our construct 1, 3.3
kb at 51570 bp). A TATA box resides between 21 and 24 bp
relative to the transcription initiation site of the mBop first exon,
and the ATG codon for translation initiation is located at +91 bp. In this
report, we will refer to muscle-specific mBop as Bop.
|
A DNA fragment extending from 637 to +196 bp fused to hsp68-lacZ also directed expression specifically in skeletal muscle and the heart (Fig. 2B, construct 2; Fig. 3E-H). However, the pattern of ß-galactosidase expression produced in the heart by this regulatory region was different from that of the larger genomic fragment. Whereas the 3304/+196 region directed expression throughout both the ventricular and atrial chambers, the 637/+196 region was active only in the RV and OFT. The region from the 3304 to 637 bp upstream region showed no transcriptional activity when fused to hsp68-lacZ (Fig. 2B, construct 3; data not shown).
|
The temporospatial pattern of expression of construct 2 was further defined by generating stable transgenic lines and analyzing expression on successive days of embryogenesis. Robust lacZ expression was seen in the heart tube at E8.0 and thereafter with construct 2 (Fig. 4A-H). Between E8.0 and 9.0, lacZ expression was especially strong in the anterior region of the heart tube, including the OFT and conotruncus, with weaker expression extending to the posterior region of the heart tube and into the sinus venosus. After E9.0, lacZ expression became localized to the right ventricular region and showed a sharp demarcation at the interventricular groove (Fig. 4G,H). Construct 2 was also expressed in the anterior somite myotomes beginning around E8.75 and was clearly seen at E9.0 (Fig. 4E). Somitic expression progressed caudally in parallel with somite maturation.
To ensure that the hsp68 basal promoter had no influence on the timing or tissue-specificity of Bop regulatory sequences, we created a transgene in which the region from 986 to +75 bp relative to the transcriptional start site was fused to a promoterless lacZ reporter (Fig. 2B, construct 4; Fig. 4I-P). Stable transgenic lines harboring this transgene showed ß-galactosidase staining throughout the linear heart tube at E9.0 (data not shown), and in the RV and OFT, as well as in the somite myotomes (Fig. 4I-K). Construct 4 was also expressed in the RV and OFT at E11.5, E13.5 (Fig. 4L-N,P) and E15.5 (data not shown). Expression was most prominent along the outer curvature of the OFT and at the outlet region of the RV. Construct 4 also showed intense expression throughout developing skeletal muscle (Fig. 4I-P).
|
The MEF2 site is essential for Bop expression in the anterior heart field
To determine whether the MEF2-binding site was required for Bop
expression, the MEF2-binding site was mutated in the context of the
3304/+196 and 637/+196 fragments
(Fig. 2B, constructs 5 and 6,
respectively) by replacing four consecutive A residues with G residues in the
core of the consensus-binding site. Mutation of the MEF2-binding site
(Fig. 2B, constructs 5 and 6)
abolished lacZ expression in the anterior heart field
(Fig. 5D,E). Remarkably,
however, the MEF2 site mutation expression did not abolish expression in
skeletal muscle. We conclude that Bop transcription in the anterior
heart field is dependent on a single MEF2-binding site, whereas transcription
in the skeletal muscle lineage is independent of the MEF2 binding at
329 to 320 bp.
E-boxes are required for Bop expression in developing skeletal muscle
Members of the MYOD1 (previously MyoD) family of bHLH transcription factors
activate skeletal muscle gene expression by binding E-box consensus sequences
(CANNTG) (Olson and Klein,
1994). Within the 637 bp regulatory region of the
Bop gene, we identified three E-boxes surrounding the essential MEF2
site (Fig. 5A). To determine
whether myogenic bHLH proteins bind any of these E-boxes, we performed a gel
mobility shift assay using extracts from COS-1 cells transfected with
MYOD1/E12 expression plasmids and observed that the region of Bop
containing the E-boxes bound strongly to the MYOD1/E12 complex (data not
shown). Mutation of the E-boxes in the context of the 637/+196 region
and the hsp68-lacZ transgene (Fig.
2B, construct 7) abolished lacZ expression in skeletal
muscle, but did not affect cardiac expression
(Fig. 6). Collectively, these
findings show that the E-boxes within the Bop control region are
necessary for expression in skeletal muscle but are dispensable for cardiac
expression.
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Discussion |
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Control of cardiac Bop expression by MEF2C
There are four members of the MEF2 family (MEF2A, MEF2B, MEF2C and MEF2D)
in vertebrate organisms and a single MEF2 factor in Drosophila
(Black and Olson, 1998). MEF2
proteins bind a conserved A/T-rich consensus sequence found in the control
regions of the majority of cardiac and skeletal muscle-specific genes, and
play numerous roles in growth, differentiation, morphogenesis and remodeling
of striated muscles (Black and Olson,
1998
). Drosophila embryos homozygous for a Mef2
null allele die during embryogenesis and display a complete loss of
differentiation of cardiac, somatic and visceral muscle cells
(Bour et al., 1995
;
Lilly et al., 1995
;
Ranganayakulu et al., 1995
),
demonstrating the central role of MEF2 as a regulator of muscle
differentiation. Analysis of the functions of the mammalian Mef2
genes based on loss-of-function phenotypes has been more difficult because the
four Mef2 genes display overlapping expression patterns in developing
muscle cell lineages and in other cell types
(Edmondson et al., 1994
).
Mef2c is expressed in the cardiac crescent and anterior heart field
beginning at E7.75 and subsequently throughout the linear, looping and
multichambered heart (Dodou et al.,
2004
; Edmondson et al.,
1994
). The other Mef2 genes are also expressed in the
early heart, although their expression is delayed slightly relative to that of
Mef2c (Edmondson et al.,
1994
).
Mice lacking Mef2c die at E9.5 from severe cardiac defects that
include a failure of the RV and OFT to develop
(Gottlieb et al., 2002;
Lin et al., 1997
). These
defects resemble those of Bop mutant embryos, which die between E9.5
and E10.5 (Gottlieb et al.,
2002
; Lin et al.,
1997
). The similarities between the Mef2c and
Bop mutant phenotypes prompted us to investigate whether these genes
might act in a cascade of cardiac control genes. Indeed, our results show that
cardiac Bop expression is dramatically downregulated in
Mef2c mutant embryos at E9.0, although residual Bop
expression can be detected in these mutant hearts. The complete loss of
cardiac expression of Bop-lacZ transgenes lacking the MEF2-binding
site suggests that the Bop regulatory region we have identified is
absolutely dependent on the binding of MEF2. However, it is also possible that
other enhancers that are MEF2-independent might support residual expression of
the endogenous Bop gene in Mef2c mutant embryos.
Alternatively, or in addition, the residual cardiac expression of the
endogenous Bop gene in Mef2c mutants could reflect
functional redundancy between Mef2c and other Mef2 genes,
which continue to be expressed in the heart of the Mef2c mutant
(Lin et al., 1997
).
Modular control of Bop transcription in the developing heart
The 637 bp of DNA sequence immediately upstream of the muscle-specific
Bop exon 1 is sufficient and necessary for transcription in the
anterior heart field and its derivatives the RV and OFT. However, in
contrast to the larger 3.3-kb upstream region, this smaller region does not
direct expression in the LV or atrial chambers. In Mef2c mutant
embryos, Bop expression is reduced throughout the heart, not just in
the anterior heart field. Because mutation of the MEF2 site in the context of
the 3.3-kb region eliminates all cardiac expression, we conclude that this
site is required for expression of Bop throughout the heart, but
regulatory sequences between 637 bp and 3.3 kb must also be
required for left ventricular and atrial expression. This type of modularity
of cis-regulatory elements, in which transcription in different regions of the
heart depends on separate enhancers, is emerging as a common theme of cardiac
gene regulation (Firulli and Olson,
1997). Such modularity is likely to reflect combinatorial
interactions among positive and negative regulators expressed in different
anatomical regions of the developing heart. In this regard, it should be
emphasized that, while MEF2 is clearly an essential activator of Bop
transcription, there must be additional regulatory factors that cooperate with
MEF2 to control cardiac Bop expression because MEF2 is highly
expressed in other cell types (e.g. neurons) in which Bop is not
expressed. Whether there are repressors of Bop expression in other
tissues or additional co-activators that cooperate with MEF2C in cardiac
muscle are interesting questions for the future.
|
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|
A cascade of cardiac transcription factors in the anterior heart field
The results of the present and prior studies have begun to reveal a
transcriptional pathway involved in development of the anterior heart field
and its cardiac derivatives, as schematized in
Fig. 7. The LIM-homeodomain
transcription factor ISL1 is expressed in cells of the anterior heart field,
and is required for RV and OFT formation
(Cai et al., 2003). The
forkhead transcription factor FOXH1 is also expressed in the anterior heart
field, and Foxh1 mutant embryos, like embryos lacking Isl1
and Mef2c, display defects in the RV and OFT
(von Both et al., 2004
). ISL1
and FOXH1 directly activate transcription of Mef2c in the anterior
heart field by activating two independent cardiac enhancers in collaboration
with GATA factors and NKX2.5, respectively
(Arceci et al., 1993
;
Dodou et al., 2004
). Thus,
these factors appear to act at the top of a cascade of cardiac transcription
factors in the anterior heart field. Our data demonstrates that Bop
is a direct target of MEF2C during anterior heart field development, implying
that Bop is indirectly regulated by ISL1/GATA factors and
FOXH1/NKX2.5. It should be pointed out that the phenotype of Mef2c
mutant embryos is more severe than that of Bop mutants, suggesting
that Bop is not the sole downstream target of MEF2C in the developing
anterior heart field, and that it may act together with other MEF2C target
genes.
It is intriguing that, although both Mef2c and Bop are expressed throughout the developing heart, the phenotypes associated with null mutations in these genes are largely confined to the anterior heart field and its derivatives. This anatomic restriction of cardiac defects could reflect redundant transcriptional mechanisms that operate outside the anterior heart field. Alternatively, an arrest in anterior heart field development may be a general consequence of diverse cardiac anomalies at E9.5.
The Bop and Mef2c mutant phenotypes show an intriguing
similarity to that of Hand2 null mice, including the absence of the
right ventricular chamber and a defect in looping morphogenesis
(Lin et al., 1997;
Srivastava et al., 1997
).
However, thus far there is no evidence indicating that MEF2C directly
activates Hand2 transcription
(McFadden et al., 2000
). In
fact, Hand2 expression is eliminated in the heart of Bop
mutant mice (Gottlieb et al.,
2002
), suggesting that Hand2 expression is governed by
BOP. As our results indicate that Bop is a direct target of MEF2C, it
is likely that MEF2C regulates Hand2 expression indirectly via
Bop in a transcriptional cascade during chamber-specific heart
development (Fig. 7).
Although the essential role of Bop in development of the anterior
heart field has been clearly established based on the phenotype of
Bop mutant embryos (Gottlieb et
al., 2002), the precise mechanism of action of BOP and its
transcriptional targets remains unclear. BOP does not bind DNA directly and,
instead, acts together with other chromatin-remodeling factors and
transcriptional regulators. BOP has been shown to associate with the
transcriptional co-activator skNAC, which is expressed specifically in cardiac
and skeletal muscle (Sims et al.,
2002
). BOP contains a SET domain
(Gottlieb et al., 2002
), which
in other proteins has been shown to possess histone methyl transferase
activity (Lachner and Jenuwein,
2002
), and a MYND domain, shown to function as a protein-protein
interaction domain in other transcription factors
(Lutterbach et al., 1998
).
Previous in vitro data suggest that BOP functions as a histone
deacetylase-dependent transcriptional repressor by interacting with class I
HDACs (Gottlieb et al., 2002
).
Thus, BOP is likely to modulate the expression of key cardiac effector genes
via its association with other components of the transcriptional machinery
required for development of the anterior heart field.
Potential roles of MEF2 and BOP in the adult heart
In addition to the roles of MEF2 factors in myogenesis and morphogenesis in
the developing heart, MEF2 factors have been implicated in hypertrophic growth
of the adult heart in response to stress signaling
(Zhang et al., 2002), and in
the control of genes involved in cardiac energy metabolism
(Czubryt et al., 2003
;
Moore et al., 2003
).
Bop expression is maintained in the adult heart, although its
functions at that stage remain unknown. In the future, it will be of interest
to determine the extent to which the transcriptional circuits involved in the
development of the heart are redeployed during adulthood to maintain cardiac
function.
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ACKNOWLEDGMENTS |
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