An intragenic MEF2-dependent enhancer directs
muscle-specific expression of microRNAs 1 and 133
Ning Liu*, Andrew H. Williams*, Yuri Kim*, John McAnally*, Svetlana Bezprozvannaya*, Lillian B. Sutherland*,
James A. Richardson†, Rhonda Bassel-Duby*, and Eric N. Olson*‡
Departments of *Molecular Biology and†Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148
Contributed by Eric N. Olson, November 7, 2007 (sent for review October 17, 2007)
The muscle-specific microRNAs, miR-1 and miR-133, play important
roles in muscle growth and differentiation. Here, we show that the
MEF2 transcription factor, an essential regulator of muscle devel-
opment, directly activates transcription of a bicistronic primary
transcript encoding miR-1-2 and 133a-1 via an intragenic muscle-
specific enhancer located between the miR-1-2 and 133a-1 coding
regions. This MEF2-dependent enhancer is activated in the linear
heart tube during mouse embryogenesis and thereafter controls
transcription throughout the atrial and ventricular chambers of the
heart. MEF2 together with MyoD also regulates the miR-1-2/-
133a-1 intragenic enhancer in the somite myotomes and in all
skeletal muscle fibers during embryogenesis and adulthood. A
similar muscle-specific intragenic enhancer controls transcription
of the miR-1-1/-133a-2 locus. These findings reveal a common
architecture of regulatory elements associated with the miR-1/-133
genes and underscore the central role of MEF2 as a regulator of the
transcriptional and posttranscriptional pathways that control car-
diac and skeletal muscle development.
striated muscle ? heart development ? transcriptional regulation
ponents of the contractile apparatus, enzymes, receptors, and
ion channels. The myocyte enhancer factor-2 (MEF2) transcrip-
tion factor is required for cardiac and skeletal muscle develop-
ment in organisms ranging from fruit flies to mammals (1).
MEF2 activates muscle gene expression in combination with
other transcription factors that are often cell type-restricted and
signal-responsive. In skeletal muscle, for example, MEF2 acti-
vates transcription synergistically with members of the MyoD
family of muscle-specific bHLH proteins (2).
Recent studies have revealed a previously unrecognized layer
of muscle gene regulation in which small, noncoding RNAs,
known as microRNAs (miRNAs), modulate growth, differenti-
ation, and morphogenesis of muscle cells (3). MiRNAs, which
are ?22 nt in length, act as negative regulators of gene expres-
sion by promoting mRNA degradation and/or inhibiting mRNA
translation through sequence-specific interactions with the 3?
UTRs of target mRNAs (4). MiRNAs have been implicated in
diverse biological processes including cell proliferation, apopto-
sis, tissue morphogenesis, tumorigenesis, and heart disease.
miRNA biogenesis is initiated when primary miRNA precur-
sors (pri-miRNAs), ranging in length from a few hundred to
thousands of nucleotides, are transcribed by RNA polymerase II.
Pri-miRNAs contain stem–loop structures that are recognized
and cleaved by a ‘‘microprocessor complex’’ composed of the
RNase III enzyme Drosha and its protein partner DGCR8 (5).
The resulting pre-miRNA stem–loop is then transported to the
cytoplasm by Exportin 5, where it is processed into an imperfect
RNA duplex by the RNase Dicer and its protein partners (5).
The mature miRNA in the duplex is assembled into an RNA-
induced silencing complex (RISC), which directs miRNA bind-
ing to the 3? UTR sequences of target mRNAs.
Three pairs of related muscle-specific miRNAs (miR-1-1/
133a-2, miR-1-2/133a-1, and miR-206/133b), which are tran-
uscle development is accompanied by the transcriptional
activation of large sets of structural genes encoding com-
scribed as bicistronic transcripts on different chromosomes, have
and differentiation. miR-1 inhibits cardiac growth by repressing
the expression of the Hand2 transcription factor, a positive
regulator of cardiac growth (6, 7), and promotes myoblast
differentiation as a consequence of its repressive influence on
histone deacetylase 4, a transcriptional repressor of myogenesis
(8). miR-133, in contrast, enhances myoblast proliferation by
repressing expression of serum response factor (SRF), an es-
sential activator of myogenesis (8), and miR-206 reinforces the
muscle differentiation program by inhibiting the expression of
DNA polymerase and the inhibitory HLH protein Id, which
functions as a negative regulator of MyoD (9).
In the course of analyzing miRNA functions in the adult heart
(10, 11), we noted that miR-1-1/133a-2 and miR-1-2/133a-1 were
down-regulated in the hearts of mice lacking MEF2 expression.
Consistent with these findings, a MEF2-dependent enhancer
upstream of the miR-1-1/133a-2 locus has been shown to regu-
late cardiac and skeletal muscle expression in vivo (7). Here, we
show that MEF2 also activates transcription of the bicistronic
precursor RNA encoding miR-1-2 and miR-133a-1 via an intra-
genic muscle-specific enhancer. A similar muscle-specific en-
hancer is located within the miR-1-1/133a-2 gene. We conclude
that MEF2 regulates muscle gene expression at transcriptional
and posttranscriptional levels by governing the expression of
muscle structural genes and miRNA genes, respectively.
Down-Regulation of miR-1/133 in Hearts from MEF2 Mutant Mice. In
adult heart (10, 11), we noted that the miR-1-1/133a-2 and
miR-1-2/133a-1 miRNA pairs were down-regulated in hearts of
mice lacking MEF2C and MEF2D (Fig. 1). miR-1-2 and miR-
133a-1 are transcribed from a bicistronic miRNA precursor on
the antisense strand of the Mindbomb (Mib) gene on mouse
chromosome 18. Similarly, miR-1-1 and miR-133a-2 are tran-
scribed together on mouse chromosome 2. However, little is
known about the structures of primary transcripts from which
these miRNAs are derived, and there are conflicting reports as
to whether miR-1-2 and miR-133a-1 are independently regu-
lated (8, 12).
Analysis of miR-1-2 and 133a-1 pri-miRNAs. Because MEF2 has not
been shown to regulate miR-1-2/133a-1, we focused on its
transcriptional regulation and began by mapping the 5? tran-
scriptional start site and 3? end of the bicistronic pri-miRNA by
5?- and 3?-RACE by using mouse embryonic (E10–E12.5)
Author contributions: N.L., R.B.-D., and E.N.O. designed research; N.L., A.H.W., Y.K., S.B.,
J.A.R., R.B.-D., and E.N.O. analyzed data; and N.L. and E.N.O. wrote the paper.
The authors declare no conflict of interest.
‡To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
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RACE-ready cDNA. In the RACE reactions, we used primers
specific to the miR-1-2 and 133a-1 stem–loop sequences for
primary amplification (p3, p5, and their reverse complements in
Fig. 2A). Cloning and sequencing of the RACE products re-
vealed three RACE products containing miR-1-2 stem–loop
sequences and two products containing miR-133a-1 stem–loop
sequences (Fig. 2B). Two of the miR-1-2 RACE products and
both of the miR-133a-1 products aligned discontinuously along
the genome, indicating that they were derived from spliced
primary transcripts (Fig. 2B). All of the RACE transcripts were
mapped to the same 5? start site, which is located ?2.1 kb
upstream of the genomic sequence predicted to encode the
miR-1-2 stem–loop and 4.4 kb upstream of the predicted miR-
133a-1 stem–loop sequence. Inspection of the genomic sequence
revealed a canonical TATA box (TATAAA) 30 nt upstream of
the 5? terminus and a canonical polyadenylation signal
(AATAAA) 20 nt upstream of the 3? terminus of pri-miR-
133a-1 transcripts [supporting information (SI) Fig. 7]. None of
the pri-miR-133a-1 transcripts that we were able to detect
contained miR-1-2 stem–loop sequences, suggesting that these
sequences are rapidly spliced out of the common pri-miR-1-2/
133a-1 precursor before processing by Drosha. The presence of
exon supports this conclusion (data not shown). Thus, it is likely
that RNA polymerase II transcribes a common pri-miRNA
precursor from the miR-1-2/miR-133a-1 locus (Fig. 2A), and
subsequent alternative splicing generates different primary tran-
scripts that serve as substrates of Drosha processing to generate
pre-miR-1-2 and pre-miR-133a-1 stem–loops (Fig. 2B).
Identification of an Intragenic miR-1-2/133a-1 Enhancer. Sequence
analysis of genomic DNA encompassing the miR-1-2/133a-1
locus revealed an evolutionarily conserved sequence (CTATTT-
TAG) resembling the MEF2 consensus binding site [CTA(A/
T)4TAR] in the genomic region between the miR-1-2 and 133a-1
coding regions (SI Fig. 8). To test whether this MEF2-like
binding site might direct muscle-specific expression, we fused the
2.5-kb intragenic region to the hsp68 basal promoter upstream
of a lacZ reporter gene and generated stable lines of transgenic
mice. Robust lacZ expression from this transgene was seen in the
heart as early as E8.5 and persisted throughout the heart from
embryogenesis to adulthood (Fig. 3A and SI Fig. 9). Within the
heart, lacZ was expressed in the outflow tract region and all four
cardiac chambers at all stages analyzed. At E12.5, lacZ was
in endocardial cushions (Fig. 3B). The expression pattern of the
intragenic enhancer in the embryonic heart differs from that of
the upstream enhancer (enhancer 1 in Fig. 2A), which directs
expression in the ventricular chambers and outflow tract but not
in the atria (7). In developing skeletal muscle cells, the miR-1-
2/133a-1 intragenic enhancer directed robust lacZ expression in
the somite myotomes and, later, throughout the skeletal mus-
culature irrespective of fiber type (Fig. 3 and SI Fig. 9).
Delineation of the miR-1-2/133a-1 Intragenic Enhancer. To delineate
the regulatory elements within the intragenic enhancer, we
created a series of deletions and assayed their expression in F0
transgenic embryos at E12.5 (Fig. 4). These studies showed that
a 330-bp fragment was sufficient to direct cardiac and skeletal
muscle expression (construct 5, Fig. 4 and SI Fig. 10). Deletion
of the 330-bp region in the context of the 2.5-kb enhancer
fragment abolished expression in both heart and somites (con-
struct 6, Fig. 4 and SI Fig. 10), indicating that this fragment is
necessary and sufficient for cardiac and skeletal muscle expres-
sion. This region shows high cross-species conservation in ver-
tebrates (Fig. 4).
A MEF2 Site Is Required for Activity of the miR-1-2/133a-1 Intragenic
Enhancer. The MEF2-like-binding site in the 330-bp minimal
intragenic enhancer could bind MEF2C in gel mobility shift
assays with extracts of COS cells transfected with myc-MEF2C
expression plasmid, but not with extracts from cells transfected
not form a DNA–protein complex with MEF2C protein. The
DNA–protein complex was competed with cognate unlabeled
oligonucleotide competitor in a dose-dependent manner, and
could be supershifted by an anti-MEF2C antibody.
A mutation of the MEF2 site in the context of the 2.5-kb
enhancer (construct 7 in Fig. 4) abolished skeletal muscle
expression in nine F0transgenic embryos analyzed at E12.5 (Fig.
5B). LacZ expression was also dramatically reduced in ventric-
ular myocardium but not in the atria of these embryos (Fig. 5C).
Skeletal Muscle Expression of the Intragenic Enhancer Requires an
E-Box. The 330-bp enhancer region also contained a conserved
E-box (CANNTG) (SI Fig. 8), the consensus binding site for the
Mef2c and Mef2d were deleted in the heart by breeding Mef2cloxP/?;
Mef2dloxP/loxPmice to Nkx2.5-Cre transgenic mice (KO). RNA was isolated from
of the indicated miRNAs was detected by real-time PCR.
Down-regulation of miR-1 and 133 in hearts of MEF2 mutant mice.
is shown. The premiR-1-2 stem–loop is 2.5 kb upstream of the premiR-133a-1.
Locations of enhancer 1 (E1) and the 330-bp intragenic enhancer (E2) are
shown. Primers used in the RACE and RT-PCR are also shown. The proposed
structure of the primary transcript containing stem–loop sequences of both
miRs is shown. This transcript was not detected in RT-PCR. (B) Structures of
primary transcripts of miR-1-2 and miR-133a-1 mapped by 5?- and 3?-RACE.
(Upper) Three transcripts were identified for pri-miR-1-2, and two of them
were spliced internally. (Lower) Two transcripts were identified for pri-miR-
133a-1. Both transcripts were spliced and did not contain premiR-1-2
The miR-1-2/133a-1 gene and primary transcripts encoding miR-1-2
Liu et al.
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MyoD family of bHLH transcription factors (2). Cell extracts
expressing myc-tagged MyoD and its dimerization partner E12
bound to the E-box probe but not to a probe in which the E-box
was mutated (Fig. 5D). Binding by MyoD/E12 was competed by
the cognate unlabeled sequence in a dose-dependent manner,
and the MyoD/E12-DNA complex was supershifted with anti-
A mutation of the E-box site abolished lacZ expression in the
somites and body wall musculature at E12.5 but did not affect
of the heart as well as in somites. Embryos from the stable transgenic line were stained for ?-galactosidase activity, sectioned, and counterstained with Nuclear
Fast red. h, heart; oft, outflow tract; ra, right atrium; la, left atrium; rv, right ventricle; lv, left ventricle; m, somite myotomes.
Muscle-specific expression of the intragenic miR-1-2/133a-1 enhancer. (A) ?-Galactosidase staining was performed on embryos from staged mating of
Fractions of F0transgenic embryos showing cardiac and skeletal muscle (SKM) expression at E12.5 are indicated in the right column. Construct 1 is identical to
the construct used in Fig. 3. Evolutionary conservation of the 2.5-kb fragment is shown at the bottom.
Delineation of a miR-1-2/133a-1 muscle-specific enhancer. Summary of transgenic constructs used to delineate the intragenic miR-1-2/133a-1 enhancer.
www.pnas.org?cgi?doi?10.1073?pnas.0710558105Liu et al.
in the miR-1-2/133a-1 intragenic enhancer is necessary for expres-
sion in skeletal muscle but dispensable for cardiac expression.
Locus. In light of the responsiveness of miR-1-1 and miR-133a-2
to MEF2 (Fig. 1) and the presence of a muscle-specific enhancer
within the miR-1-2/133a-1 locus, we analyzed the genomic DNA
between the miR-1-1 and miR-133a-2 coding regions for a
similar enhancer. We identified a MEF2-like site and an E-box
as well as a CArG box, the binding site for SRF, in this region
of the miR-1-2/133a-1 locus (Fig. 6A). Moreover, the 9.2 kb of
intragenic DNA separating the miR-1-1 and miR-133a-2 coding
regions directed robust expression specifically in the atrial and
ventricular chambers of the heart and skeletal muscle (Fig. 6 A
and B). Deletion mutations localized the enhancer activity to a
4.3-kb DNA fragment encompassing the MEF2-like site and
E-box, as well as a CArG box. We conclude that the miR-1-2/
regulatory elements that confer cardiac and skeletal muscle
The results of this study show that MEF2 directly regulates the
expression of muscle-specific miRNAs through a muscle-specific
enhancer in an intron separating the miR-1-2 and miR-133a-1
coding regions. These findings, together with those of previous
studies (7, 13, 14), demonstrate that MEF2 exerts its control over
the programs for muscle development through direct and indi-
rect mechanisms by coordinating the regulation of mRNAs and
miRNAs, which act through a plethora of downstream targets to
modulate cell phenotypes.
Control of the Intragenic miR-1-2/miR-133a-1 Enhancer by MEF2.
MEF2C expression is initiated in the cardiac crescent at E7.75,
mobility shift assay. A32P-labeled oligonucleotide probe containing the MEF2-binding site and total-cell extract from COS-1 cells transfected with a MEF2C
expression plasmid formed a DNA–protein complex in the assay. A32P-labeled oligonucleotide probe containing a mutated MEF2-binding site did not form a
the MEF2-binding site competed for binding. (B) Mutation of the MEF2-binding site in the 2.5-kb enhancer abolished expression in somites at E12.5. (C) Heart
atrium; la, left atrium; rv, right ventricle; lv, left ventricle. (D) Binding of MyoD and E12 complex to the E-box binding site in the minimal enhancer element by
and E12 expression plasmids formed a DNA–protein complex in the assay. Mutant E-box probe did not form a DNA–protein complex with MyoD and E12. The
complex was supershifted by using a Myc-specific antibody and unlabeled WT oligonucleotide containing the E-box binding site competed for binding. The
asterisk represents nonspecific binding. (E) Mutation of the E-box-binding site in the 2.5-kb enhancer abolished expression in somites and ventral myoblasts at
E12.5. Expression in the heart was not affected by the E-box mutation.
Analysis of the MEF2 site and E-box in the miR-1-2/133a-1 enhancer. (A) Binding of MEF2C to the MEF2-binding site in the minimal enhancer by gel
Liu et al.
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looping defects (14, 15). Therefore, it is likely that MEF2C
controls early cardiac expression of the miR-1-2/133a-1 intra-
genic enhancer. Although MEF2 factors are expressed in all four
chambers of the developing heart, activity of the miR-1-2/133a-1
intragenic enhancer in the atria was unaffected by mutation of
the MEF2 site in the enhancer, suggesting the existence of
additional atrial activators of the enhancer.
Within the skeletal muscle lineage, the miR-1-2/133a-1 en-
hancer is strongly activated in the somite myotomes by E9.5.
E-box-binding site for myogenic bHLH proteins, consistent with
the known cooperativity between MEF2 and MyoD in activation
of the skeletal muscle gene program (2). These findings are
consistent with ChIP experiments, which showed binding of
MyoD and myogenin in the region of the miR-1-2/133a-1
intragenic enhancer, although the functional significance of
these sites was not addressed (12).
Multiple Muscle-Specific Enhancers for the miR-1-2/miR-133a-1 Locus.
Muscle genes are commonly controlled by multiple, independent
cis-regulatory elements that cooperate to generate the complete
developmental expression pattern of the gene (16, 17). Multiple
enhancers expand the regulatory potential and allow for fine-
tuning of temporal-spatial control of gene expression. Redun-
dancy of enhancer activity may also provide a means of rein-
forcing the expression patterns of critical genes during
Zhao et al. (7) described an enhancer upstream of the miR-
1-2/133a-1 locus that also required MyoD for activity in the
skeletal muscle lineage. However, in contrast to the requisite
role of MEF2 in activation of the intragenic enhancer within the
heart, this upstream enhancer relies on SRF for cardiac expres-
sion. The upstream and intragenic miR-1-2/133a-1 enhancers
also differ with respect to their expression patterns in the heart.
Whereas the intragenic enhancer is highly active in the atrial and
ventricular chambers, the upstream enhancer is active only in
ventricular myocardium (7). Another enhancer located ?50 kb
upstream of the gene was reported to direct cardiac and skeletal
muscle expression in transgenic Xenopus laevis (8), but its
temporal-spatial expression pattern during mouse embryogen-
esis has not been reported.
Like the miR-1-2/133a-1 locus, the miR-1-1/miR-133a-2 locus
is controlled by multiple muscle-specific regulatory elements.
Our results show that a cardiac and skeletal muscle-specific
enhancer, containing putative binding sites for MEF2 and
myogenic bHLH proteins, lies between the miR-1-1 and miR-
133a-2 coding regions. Although we have not analyzed the
functions of these sites through mutagenesis, a MyoD and
myogenin-binding site was identified in the genomic region of
this enhancer by ChIP analyses (12). Similarly, a cardiac and
skeletal muscle-specific enhancer upstream of the gene serves as
a direct target of SRF in the heart and MEF2 in skeletal muscle
(7), and another enhancer was identified ?50 kb upstream of
the miR-1-1 gene (8). Thus, the miR-1-1/miR-133a-2 and miR-
1-2/miR-133a-1 genes employ a common architecture of enhanc-
ers and upstream regulators to control muscle-specific gene
Implications. miR-1 and miR-133 play opposing roles in the
control of muscle cell proliferation and differentiation (8).
differentiation pathway, whereas miR-133 promotes myoblast
growth, which suppresses differentiation. It seems paradoxical
that two miRNAs expressed from the same locus under control
of common cis-regulatory elements would counteract each
other. Perhaps additional regulatory mechanisms, such as dif-
fering stabilities of miR-1 and miR-133 as well as different
regulation of their target mRNAs and signaling inputs differen-
tially modulate the actions of these miRNAs in the muscle
differentiation pathway downstream of MEF2.
miR-1-1/133a-2 enhancer. Fractions of F0transgenic embryos showing cardiac and skeletal muscle (SKM) expression at E12.5 are indicated in the right column.
(B) Representative transgenic embryo showing lacZ expression from construct 1 and transverse section revealing lacZ expression in the outflow tract and four
chambers of the heart as well as in somites. ra, right atrium; la, left atrium; rv, right ventricle; lv, left ventricle; m, somite myotomes.
Delineation of an intragenic miR-1-1/133a-2 muscle-specific enhancer. (A) Summary of transgenic constructs used to delineate the intragenic
www.pnas.org?cgi?doi?10.1073?pnas.0710558105Liu et al.
In addition to its central role in the control of muscle
development, MEF2 modulates the growth, gene expression
patterns, and functions of cardiac and skeletal muscle in re-
sponse to activity and extracellular signaling (1, 18). MEF2
promotes cardiac hypertrophy in response to increased workload
or excessive neurohumoral signaling, and miR-133 has been
implicated in this process (18, 19). In skeletal muscle, MEF2
drives the slow myofiber gene program in response to motor
innervation and exercise (20). In each of these settings, the
actions of MEF2 are governed by a variety of signaling systems
and the signal-dependent association of MEF2 with class II
HDACs. Thus, manipulating the signaling pathways that control
MEF2 activity provides possibilities for modulating the expres-
sion of miR-1 and miR-133 and the regulatory programs they
govern in striated muscle.
Materials and Methods
RNA Analyses. A 5?- and 3?-RACE was performed by using mouse embryo
(10–12 days) FirstChoice RACE-ready cDNA kit (Ambion). RNA was isolated by
using TRIzol (Invitrogen) and treated with Turbo RNase-free DNase (Ambion)
performed as described (20). Methods and primer sequences are described in
SI Materials and Methods.
Generation and Analysis of Transgenic Mice and Knockout Mice. Transgenes
were generated by cloning DNA fragments into the hsp68 basal promoter
upstream of a LacZ reporter gene (21). Transgenic mice were generated, and
lacZ staining was analyzed as described (21). See SI Materials and Methods for
Mef2c?/?, Mef2c, and Mef2d conditional knockout mice were described
(14, 22, 23). Cardiac-specific deletion of Mef2c and Mef2d was achieved by
using Nkx2.5-Cre transgenic mice expressing Cre recombinase under the
Nkx2.5 basal promoter and cardiac enhancer (24).
Electrophoretic Mobility Shift Assays. Details of electrophoretic mobility shift
assays can be found in SI Materials and Methods.
Site-Directed Mutagenesis. Mutagenesis of the MEF2 site and E-box was
achieved by using the overlap extension method as described (25). Same
mutations were introduced within each site as those used in electrophoretic
mobility shift assays (SI Materials and Methods). Mutant fragments were
cloned into the transgenic expression vector.
ACKNOWLEDGMENTS. We thank Dr. E. van Rooij for insightful comments on
the manuscript, A. Tizenor for graphics, J. Brown for editorial assistance, and
Drs. W. Klein and J. Martin for comments on the manuscript. N.L. was sup-
ported by a grant from the American Heart Association. Work in the labora-
tory of E.N.O. was supported by grants from the National Institutes of Health,
the Donald W. Reynolds Cardiovascular Clinical Research Center, and the
Robert A. Welch Foundation.
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