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(Circulation Research. 1996;79:4-13.)
© 1996 American Heart Association, Inc.

Articles

Factors Involved in Cardiogenesis and the Regulation of Cardiac-Specific Gene Expression

John D. Mably, Choong-Chin Liew

the Laboratory for Molecular Cardiology, The Centre for Cardiovascular Research, The Toronto Hospital, and the Departments of Clinical Biochemistry and Medicine, University of Toronto (Canada).

Correspondence to Dr C.-C. Liew, The Cardiac Gene Unit, 100 College St, Banting Institute, Rm 418, University of Toronto, Toronto, Ontario, Canada M5G 1L5. E-mail liewcc@gpu.utcc.utoronto.ca.

	Abstract

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The delineation of the mechanisms that regulate cardiac gene expression is central to our understanding of cardiac growth and development. Much progress has been made toward the identification of factors involved in tissue-restricted gene expression, especially in skeletal muscle cells. However, the mechanisms regulating the expression of cardiac-specific genes remain less well understood. Certain homeodomain proteins have been implicated in commitment to the cardiac phenotype. Among the best characterized are the murine proteins Csx, Nkx-2.5, and Nkx-2.6, related to the protein tinman, which is essential for heart formation in Drosophila. The expression of these genes precedes that of cardiac-specific genes and is therefore believed to play a critical role in the development of the heart. The GATA proteins are a family of zinc finger proteins that are also expressed early in cardiac development and may act separately from, or in concert with, the homeodomain proteins as crucial regulators of heart development. The myosin heavy and light chain genes, the actin genes, the troponin genes, and the atrial natriuretic factor and muscle creatine kinase genes have served as excellent paradigms for the study of cardiac gene expression. Although differences in cis-acting elements and their behavior in binding assays have been observed between different genes, there exist similarities that are noteworthy. In this review, we will discuss the factors involved in the regulation of cardiac-specific gene expression in an attempt to provide a better understanding of the process of cardiogenesis.

Key Words: cardiogenesis • cardiac-specific gene regulation

	Introduction

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The determination of factors involved in cardiogenesis and cardiac-specific gene regulation is fundamental to our understanding of heart growth and development. For several years, investigators have examined the biochemistry of nuclear proteins in an attempt to define their roles in DNA replication, transcription, and posttranscriptional events. Their role in myocardial development and the genesis of cardiomyopathy, as well as in the regulation of muscle gene regulation, has also been examined (for review, see Reference 1). However, although the complex network of myogenic regulatory proteins in skeletal muscle is well established, less is known of the mechanisms involved in cardiac tissue development. Although some of the factors involved in cardiogenesis have been determined, the entire regulatory cascade has yet to be fully delineated (see Fig 1). Studies in Drosophila have contributed greatly to our understanding of this process through the description of several homeodomain proteins involved in early stages of cardiac development and have provided a basis for analyses in mammalian systems (for review, see Reference 2). We begin the present review with a description of some of the factors implicated in the development of cardiac tissue, followed by a description of the factors involved in cardiac-specific gene expression.

View larger version (14K): [in this window] [in a new window]   Figure 1. Interplay between the factors involved in skeletal muscle and cardiac development. Either MyoD or Myf-5 is required early in the developmental process for determination in myoblasts. Myogenin is essential for the differentiation of myoblasts into multinucleated myotubes, but the expression pattern of MRF4 suggests that it acts late in the developmental program. Interaction with the HLH protein Id inhibits the binding activity of the MyoD proteins. A complicated interplay also exists between the MyoD family of myogenic regulators and the MEF-2 proteins. During the development of cardiac muscle, no MyoD family proteins are expressed. However, other factors involved in cardiogenesis have been identified and shown to be expressed during the earliest stages of heart development. dHAND/eHAND may interact with Id, providing an explanation for Id expression in cardiac tissue.

	Factors Involved in Cardiogenesis

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Homeodomain Proteins
Recent studies in Drosophila have implicated several members of the NK class of homeodomain proteins in the development of myogenic lineages during embryogenesis. For example, the homeobox genes tinman (also called NK-4 or msh-2) and bagpipe are responsible for the determination of cell fates in the dorsal mesoderm,³ and the tinman gene is also essential for subdividing the mesoderm into somatic, visceral, and cardiac primordia.² Loss of tinman expression results in the loss of appearance of heart or visceral muscles, whereas restoration of tinman expression results in partial restoration of heart cell formation. During the first phase of tinman regulation, the gene twist is believed to be essential for its activation and expression in all mesodermal cells until after gastrulation.² Another gene, dpp, is then expressed in dorsal ectodermal cells only. Dpp is believed to regulate tinman expression and play a role in heart development, since dorsal tinman expression is lost in dpp mutant embryos and results in a failure to develop cardiac mesoderm.⁴ The segment polarity gene wingless has also been implicated in the development of cardiac tissue by contributing to the specification of the primordial cells of the Drosophila heart.⁵ These studies have formed much of the basis for our understanding of the early events in heart development.

The murine homeobox genes Nkx-2.5 and Csx were isolated by polymerase chain reaction amplification using degenerate primers to amplify the third helix of the homeodomain region of the Drosophila NK family of genes (NK-4/msh-2 and NK-3, respectively).^{6 7} This helix is virtually invariant among homeodomain proteins and is responsible for high-affinity, sequence-specific DNA binding.⁸ Nkx-2.5 and Csx are expressed predominantly in adult heart muscle, with no significant expression in adult skeletal muscle or other tissues.^{6 7} Both genes are expressed very early in development (by day E8.5, 8.5 days postcoitum), preceding that of other cardiac-specific genes. Although Nkx-2.5 expression is also detected in lingual myoblasts, in the visceral mesoderm capping the distal end of the stomach, and within mesenteric tissue next to the stomach,⁷ its expression in embryonic myocardial progenitors and adult hearts suggests a role in the differentiation of the myocardial lineage and maintenance of the cardiac muscle phenotype.

Analysis of several Nkx-2.5 deletion mutants revealed a COOH-terminal inhibitory domain containing clusters of alanines and prolines that appeared to mask a potent activation domain within the amino terminus of the protein.⁸ It is possible that certain conditions alter the conformation of this protein sufficiently to prevent this interaction and allow Nkx-2.5 to serve as an activator of transcription. The targeted disruption of Nkx-2.5 results in abnormal heart morphogenesis, growth retardation, and embryonic lethality at day E9-10.⁹ Although heart tube formation proceeds normally, looping morphogenesis, which is critical for heart formation, is not initiated at the linear tube stage (day E8.25-8.5). The expression of most myofilament genes and myofibrillogenesis was normal; MLC-2v was not expressed in either the mutant hearts or the mutant embryonic stem cell-derived cardiocytes, although regional expression of ß-MyHC and cyclinD2 was normal.⁹ MLC-2v is normally expressed only in ventricular cells and is one of the earliest known markers of ventricular differentiation,¹⁰ and the loss of its proper expression pattern demonstrates that Nkx-2.5 is an important regulator of at least one component pathway of the cardiac myogenic program. Recently, an additional member of the NK family, XNkx-2.3, has been isolated, and studies reveal an expression pattern largely restricted to the heart during Xenopus embryogenesis.¹¹ There is considerable overlap with the expression patterns of Csx/Nkx-2.5, suggesting some redundancy in function among members of this family, as has been demonstrated for the MyoD proteins.

GATA Proteins
The GATA factors are a family of transcriptional regulators that are expressed in a tissue-restricted manner. Three members of this family, GATA-4, -5, and -6, demonstrate patterns of expression consistent with a role in cardiogenesis. The GATA-4 mRNA is most abundant in heart, with lower levels in ovary and small intestine.^{12 13 14} The expression of GATA-4 is generally lower than either GATA-5 or -6 and is largely restricted to the endocardial cells within the heart.¹³ GATA-5 is also transcribed during the very early stages of cardiac development and is detectable in the cardiogenic region at the time progenitors are becoming committed to form heart tissue.¹³ The expression of these genes at day E10 in embryonic quail heart and day E7.0-7.5 in mouse embryos demonstrates that these genes are valuable markers of cardiogenesis.

Evidence is also accumulating to implicate the GATA factors in cardiac gene regulation. The expression of the cardiac muscle-specific troponin C and -MyHC genes is influenced by the expression of GATA-4.^{15 16} The promoters of both the atrial and brain natriuretic peptides also contain GATA motifs and are transactivated by transient expression of GATA-4 in neonatal rat cardiomyocytes.^{17 18} Studies on the function of the zinc finger motifs of GATA-1 suggest that they are important for both protein-DNA and protein-protein interactions, possibly serving to bring together or stabilize loops between proteins bound to distant regulatory elements.¹⁹ This mechanism of activation may be shared by other members of the GATA protein family, as well as other regulators of cardiac gene expression.

CT-1
A 21.5-kD protein has recently been identified that has the potential to induce cardiac myocyte hypertrophy in vitro.²⁰ This protein, named CT-1, is a member of the IL-6/IL-11/LIF/CNTF/oncostatin M family of cytokines that signal through the activation of the transmembrane protein gp130.²¹ Other members of this family also act through gp130 and are able to stimulate hypertrophy in cardiac myocytes, supporting a role for this pathway in general cardiac hypertrophy. The 1.4-kb cardiotrophin mRNA is expressed in several mouse tissues, including heart.²⁰ However, Sheng et al²² report that CT-1 is predominantly expressed in the early mouse embryonic heart tube (day E8.5-10.5) and is restricted to myocardial cells. CT-1 and LIF, both of which signal through the activation of gp130, are able to promote myocyte survival and DNA synthesis, whereas IL-6 and CNTF do not.²² Since LIF is not expressed in the embryonic, neonatal, or adult heart, CT-1 may be essential to the survival and proliferation of immature myocytes during cardiac growth and development.

Retinoid Receptors
Some new studies have begun to implicate the retinoid signaling pathway in cardiogenesis and cardiac gene regulation. A null mutation generated in the mouse RXR gene resulted in growth deficiency in heterozygote mice, whereas homozygotes died in utero between days E13.5 and E16.5 and displayed myocardial and ocular defects.^{23 24} However, the absence of RXR does not prevent the expression of cardiomyocyte-specific genes, such as the -MyHC gene.²³ Embryonic vitamin A deficiency exhibits a similar phenotype, implicating RXR as a genetic component of the vitamin A signaling pathway in cardiogenesis. Dyson et al²⁵ extend these observations by demonstrating that RXR (-/-) embryos show depressed ventricular function, ventricular septal defects, and various degrees of atrioventricular block. This phenotype is also associated with the aberrant expression of the atrial-specific isoform MLC-2a in the ventricle of the RXR (-/-) embryos.²⁵

Retinoids are also able to suppress the onset of the embryonic gene program in an in vitro model of hypertrophy.²⁶ The increase in cell size and induction of atrial natriuretic factor observed in rat neonatal cardiomyocytes after addition of the -adrenergic receptor agonist phenylephrine is suppressed by the addition of physiological concentrations of retinoic acid; retinoic acid also suppresses the endothelin-1 pathways for cardiomyocyte hypertrophy but does not prevent the increase in cell size and induction of atrial natriuretic factor associated with serum stimulation.²⁶ Further analysis using synthetic agonists of retinoic acid, which selectively bind to RXR or RAR, were used in trans-activation transient transfection assays, demonstrating that RAR/RXR heterodimers mediate suppression of -adrenergic receptor-dependent hypertrophy.²⁶ The formation of myocardial defects in RXR-deficient mice together with the implication of retinoic acid in cardiomyocyte hypertrophy suggests an important role for this pathway in heart development.

	Regulatory Elements Involved in Myogenesis

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We have chosen to examine and discuss the regulation of contractile protein gene expression at the transcriptional level. Although the mechanisms of regulation in skeletal and cardiac muscles are distinct, the overlap in the expression patterns of several genes between these two compartments has served as a basis for many insights into mechanisms of transcriptional control in cardiac tissue. We provide only a brief review of the factors regulating skeletal muscle gene expression, since detailed reviews of these protein families and their roles in myocyte differentiation and growth have been published previously (for reviews, see References 27 and 28). We will instead discuss the role that these or related factors may play in the regulation of cardiac gene expression.

The MyoD Family
The original characterization of MyoD was quickly followed by the discovery of three related factors, myogenin, Myf-5, and MRF4/herculin/Myf-6.²⁷ Each gene is expressed exclusively in skeletal muscle and can activate skeletal muscle-specific genes when introduced into nonmuscle cells. The levels of expression of these MyoD family members and the order in which they are expressed are believed to be essential to the appropriate control of the myogenic process, as illustrated in Fig 1. Heterodimerization of the MyoD family members may control certain aspects of myogenesis as well as cell lineage determinations. This regulation may involve the interplay of ubiquitous (E2A, E12, and E47) and tissue-specific (MyoD and myogenin) gene products.^{29 30} The HLH protein Id is capable of forming heterodimers with members of the bHLH family of myogenic regulators but lacks a DNA-binding domain.³¹ The interaction between other HLH proteins and Id therefore results in a suppression of their activity due to the loss of DNA-binding activity. Id expression can be inhibited by mitogen depletion and is essential for myogenic differentiation.³²

Although Id activity is also detected in cardiac tissue,³¹ MyoD family members are not expressed in cardiac muscle.³³ However, a novel bHLH protein was recently identified using the yeast two-hybrid technique to screen a mouse embryo cDNA library for novel bHLH transcription factors that heterodimerize with the bHLH E12. This protein, which was called eHAND, appears to define a new subclass of the class B bHLH proteins that includes the myogenic regulatory factors of the MyoD family.³⁴ The first site of eHAND expression in embryos was the heart, where high levels were expressed between days E8.5 and E10.5; transcript levels then declined abruptly and were eventually localized to regions of valve formation by day E13.5. eHAND is also detected in extraembryonic tissues, the autonomic nervous system, and neural crest derivatives as well as the heart, suggesting a role in the development of these tissues as well.³⁴

A second member of this subclass, named dHAND, has been cloned by low-stringency screening and exhibits an overlapping expression pattern with eHAND. dHAND expression is first detected in the lateral mesoderm of day E7.75 embryos and, by day E8.5, is expressed throughout the developing heart.³⁵ By day E13.5, dHAND expression was barely detectable in the heart, and by day E16, levels were not detectable within the embryo. Further support for the role of these proteins in cardiac development is provided by experiments in which antisense dHAND or eHAND oligomers were incubated with stage 8 chick embryos. Alone, neither oligomer had an effect on embryonic development, but when used together, they arrested development at the looping heart tube stage.³⁵ Thus, these two proteins may play redundant roles in the regulation of cardiac morphogenesis, as suggested for MyoD and Myf-5 in skeletal muscle development.

The MEF-2 Family
Members of the bHLH family alone, however, are not sufficient to regulate all muscle-specific genes. The MEF proteins, members of the MADS box family that includes SRF (p67^SRF), are also involved in the regulation of muscle gene expression (for review, see Reference 28). There is a close link between the MyoD and MEF-2 families, since the expression of both MyoD and myogenin leads to the induction of MEF-2 activity.³⁶ MEF-2A and MyoD physically interact through the MADS domain of MEF-2A and three amino acids within the bHLH motif of MyoD and together can activate skeletal muscle gene expression.³⁷ As well, the myogenin promoter contains a binding site for MEF-2, which in turn can be induced by myogenin.³⁸ The interplay between the myogenic regulators of the MyoD family and MEF-2 suggests the existence of a complex regulatory circuit to amplify expression of these genes, which is necessary to stabilize the myogenic program (see Fig 1). It will be interesting to observe if similar interactions occur between the newly identified dHAND/eHAND proteins and other members of the MADS box family, including the MEF proteins.

In fact, cardiac factors that bind MEF-2 sites have been identified. For example, a protein named BBF-1 can interact with the MEF site, but the expression of this protein is restricted to cardiac tissue.³⁹ Analysis of chicken embryos revealed that binding activity of BBF-1 is distinguishable from cardiac MEF-2 and appears before that of MEF-2.³⁹ The presence of BBF-1 binding activity before MEF-2 suggests a role for BBF-1 in determination of the cardiogenic cell lineage and a role for cardiac MEF-2 in the maintenance of the differentiated state. The MEF-2 site in the PGAM-M gene⁴⁰ and a MEF-2-like motif and its flanking sequence in the rat cardiac troponin T gene⁴¹ also appear to be necessary for cardiac-specific expression of these genes.

Regulatory Elements Involved in Cardiac Contractile Gene Regulation
The study of contractile protein gene regulation has been responsible for the determination of many of the cis-acting elements and trans-acting factors involved in cardiac-specific gene expression. Although some elements are present in only one gene, the DNA binding sites as well as the binding activities themselves demonstrate similarities between genes. Early studies have provided evidence that distinct cis-acting regulatory elements mediate cardiac versus skeletal muscle-specific expression of genes that are normally expressed in both tissue types (for review, see Reference 1). As well, the analysis of heterokaryons of cardiac myocytes and fibroblasts demonstrated that the cardiac phenotype is not expressed over the embryonic fibroblast phenotype, an observation distinct from that noted with skeletal muscle cells.⁴² Thus, different factors must exist in the two muscle types that are necessary for the development of the two tissues. We have summarized in Table 1 many of the elements and factors responsible for the regulation of several contractile protein genes, and we have presented them in the text according to the gene in which they were first identified.

View this table: [in this window] [in a new window]   Table 1. Regulatory Elements and Associated Binding Factors Detected in Several Muscle Gene Promoters

The Actin Genes
The CArG motif is a well-characterized cis-acting element that has been demonstrated in several sarcomeric protein genes. Even though p67^SRF has been shown to interact with the sarcomeric actin gene CArG boxes in vitro, this factor is not likely responsible for the observed tissue specificity associated with certain CArG elements. Among the proteins known to interact with the CArG is YY1. Overexpression of YY1 represses basal expression from both the c-fos and skeletal -actin promoters, but this inhibition is eliminated by overexpression of the SRE-binding protein p67^SRF, suggesting that the two proteins have antagonistic functions in vivo.⁴³ The properties of YY1 are similar to those observed for p62^DBF/MAPF1, another nuclear protein that has been shown to interact with the c-fos SRE and skeletal -actin CArG.⁴⁴

Another factor has been cloned that binds to the CArG motif but has properties different from those of p67^SRF and the other cloned SRE- and CArG-binding factors. This factor, denoted CBF-A, has no homology to p67^SRF and has a repressor-like activity similar to YY1.⁴⁵ It does not contain a well-defined DNA-binding motif but does possess structural similarity to single-stranded DNA-binding or RNA-binding proteins and binds effectively to single-stranded DNA. Although this single-stranded DNA-binding activity has not been demonstrated in vivo, it may have a functional significance, since such proteins have been found in skeletal muscle extracts and shown to bind regulatory elements in the skeletal -actin⁴⁶ and vascular smooth muscle -actin⁴⁷ promoters. Factors that possess a sequence-specific single-stranded DNA-binding activity of this nature could also play roles in DNA replication or repair, if not transcription. Other proteins that interact with the SRE and CArG elements have been identified, several of which are illustrated in Fig 2.

View larger version (39K): [in this window] [in a new window]   Figure 2. Structure of the c-fos SRE and the binding sites of various interacting factors. The CArG motif is shaded, and the guanine residues implicated in SRF binding are indicated. The shaded figures below the sequence overlap the various binding sites of the indicated factors.

The Troponin Genes
The M-CAT motif, like the CArG, has been detected in the regulatory regions of several sarcomeric protein genes. This motif has been shown to interact with non-muscle-specific factors, yet its deletion results in the loss of troponin T expression in muscle cells.⁴⁸ This suggests that cooperativity between the M-CAT binding factor and proteins that bind further upstream is required for transcriptional activation of the cardiac troponin T gene. Isoforms of a protein called TEF-1, isolated from chicken heart, have been shown to bind M-CAT elements with high affinity in a sequence-specific manner and demonstrate an ability to activate transcription.⁴⁸ The GT-IIC enhancer of simian virus 40, which interacts with TEF-1, is identical to the M-CAT motif except for one residue in its 7-bp core sequence and can substitute for the M-CAT motif within the cardiac troponin T promoter.⁴⁸ Therefore, the factor(s) interacting with the M-CAT and GT-IIC motifs, as well as the ßF-1 site in the ß-MyHC gene, appears to be TEF-1 or a closely related protein.

A gene-trapping approach has provided unequivocal evidence that TEF-1 is required for the maintenance of normal cardiogenesis. When the gene trap integrates into a transcriptional unit, the normal expression pattern of the tagged gene can be followed by monitoring expression of the reporter and also creates a mutation of the gene at that site. Disruption of the TEF-1 gene results in an embryonic lethal phenotype between days E11 and E12 that is associated with heart dysfunction.⁴⁹ Although cardiogenesis is initiated and the expression of several TEF-1 targets appears to be normal, the mutant embryo heart exhibits an abnormally thin ventricle wall, establishing the role of TEF-1 in cardiac myogenesis.

The MLC Genes
A composite binding element has been characterized in the MLC-2 promoter that confers cardiac-specific expression to this gene. This element, called HF-1, consists of two binding motifs: one motif (HF-1a) interacts with a ubiquitous factor, and the second, which is an AT-rich sequence (HF-1b), binds to a factor preferentially expressed in differentiated cardiac and skeletal muscle cells.⁵⁰ A series of transgenic mice harboring mutations in regulatory elements within a 250-bp MLC-2 luciferase construct containing the proximal promoter demonstrated that both components of the HF-1 site (HF-1a and HF-1b) are required for the maintenance of ventricular-specific expression.⁵⁰ Mutation of an E-box within this region results in upregulation of reporter activity in soleus, gastrocnemius, and uterus, as does mutation of another conserved element (HF-3), suggesting that these elements act as negative regulators of expression.⁵⁰

The binding activity of HF-1b and the MEF-2 motif appear to be very similar, and a zinc finger protein that interacts with this element has been cloned by expression library screening with the HF-1b sequence.⁵¹ This protein is expressed in cardiac and skeletal muscle as well as in brain, suggesting a role in the regulation of gene expression in terminally differentiated tissues. A CArG-binding zinc finger protein that interacts with AT-rich elements, such as HF-1b/MEF-2 and CArG, has also been cloned, and this interaction may be involved in tissue-specific regulation.⁵² The bHLH factor USF can interact with both the HF-1a site and a region in the MLC-2 promoter identified as MLE1 and is able to influence expression of this gene via these independent sites.⁵³ Another component of the HF-1a binding activity has been characterized as EFI_A, the rat homologue of YB-1.⁵⁴ Together with another protein isolated by immunoprecipitation, p30, and in conjunction with HF-1b/MEF-2, it appears that these factors are involved in the mediation of ventricular-specific expression of the MLC-2 gene.⁵⁴

Recent evidence suggests that elements exist within the promoter region of the MLC-3F gene that are able to mediate expression in the right versus the left ventricular chambers during development.⁵⁵ The MLC-3F promoter and 3'-enhancer were linked to the lacZ gene, and although transgenic mice demonstrated strong expression in skeletal muscle, the developing heart was the earliest site of transgene expression (day E7.5).⁵⁵ The regulatory elements responsible for the cardiac expression of this transgene are probably located in the promoter region, since the 3'-enhancer does not drive reporter gene expression in the heart of transgenic mice generated with constructs containing the MLC-1F gene promoter and enhancer.⁵⁵ MLC-3F transgenic mice lacking the 3'-enhancer also express a high level of the lacZ reporter in the heart, providing further evidence for the role of these promoter elements in the regulation of cardiac-specific gene expression.⁵⁵

The Cardiac -MyHC Gene
The transcriptional regulation of the -MyHC gene and the role of thyroid hormone in this process has been well established, and T₃ response elements have been demonstrated in the upstream sequences of both the rat and human genes (for review, see Reference 56). Transgenic mice with mutations in these sites have also been generated to examine the effect of T₃ on the regulation of -MyHC gene expression.⁵⁷ In fact, the effect on transcription by many of the identified cis-acting elements is minimal unless thyroid hormone is added to the cell cultures.⁵⁶ A murine -MyHC/CAT gene construct containing 3 kb of the -MyHC upstream region is sufficient to drive developmental stage and tissue-specific expression, similar to that described for the rat gene.⁵⁸ Transgenic mice containing constructs of the proximal 138 bp exhibit no CAT activity in either muscle or nonmuscle cells.⁵⁸ Although this small construct contains some of the putative regulatory elements believed to be responsible for muscle gene expression, other more distal elements are essential to the proper expression of this gene.

Other factors that can influence the expression of the -MyHC gene include MEF-2,^{59 60} the M-CAT binding factor,⁶¹ endothelin,⁶² and Egr-1.⁶³ Egr-1 is a serum-inducible, early-response gene that encodes a zinc finger protein expressed at high levels in adult rat cardiomyocytes. Cotransfection of Egr-1 with a rat -MyHC/CAT chimera containing 3 kb of the 5' flanking sequence stimulates CAT expression dramatically, and endogenous levels of the -MyHC gene also increase.⁶³ The location of the cis-acting element mediating this response has been localized between -1698 and -1283 bp, substantially upstream from the transcriptional start site. The presence of this response in rat cardiomyocytes but not in myotubes or myoblasts suggests that auxiliary factors are necessary for the upregulation of -MyHC expression by Egr-1.

The competition for overlapping DNA-binding motifs within composite sites is a likely mechanism used in the complex regulation of cardiac gene expression, as suggested by the proximity of MEF-2 sites to E-boxes in several genes. Within the rat -MyHC gene promoter is an E-box/M-CAT hybrid motif, the EM element, that interacts with a protein believed to be TEF-1.⁴⁸ The EM element also interacts with another factor similar to that which interacts with the HF-1a site of the MLC-2 promoter. In the MLC-2 gene, the HF-1a site is adjacent to a MEF-2-like site.⁵⁰ Within the human -MyHC gene is another AT-rich element similar to the CArG called the GArC motif.⁶⁴ This site was identified by our laboratory using DNase I footprint analysis of the human cardiac MyHC genes. Further analysis by methylation interference has revealed a second site 3' to the GArC that interacts with a distinct protein factor (J.D. Mably and C.C. Liew, unpublished data, 1996), and between the two sites is a consensus E-box motif (see Fig 3). For all three elements shown in Fig 3, there is a 5' CArG-like sequence and a second site in proximity to an E-box motif. The proximity of the sites in the promoters of these genes suggests that some interaction between the factors bound to these sites may occur that may vary depending on the tissue type. Whereas some of these factors may be tissue specific, others appear to be ubiquitous, suggesting that this interplay determines how these DNA-binding sites influence transcription.

View larger version (35K): [in this window] [in a new window]   Figure 3. Composite elements within the promoters of the MLC-2 and -MyHC genes. An E-box motif is present in the vicinity of the characterized binding sites, suggesting that interactions between binding factors in different tissues may influence the differential expression of these genes.

The Cardiac ß-MyHC Gene
The regulation of ß-MyHC gene expression has also been examined in several species (for review, see Reference 56). In vitro studies suggest that elements within 600 bp of the start site can direct muscle-specific expression, whereas transgenic analysis with the mouse ß-MyHC gene promoter reveals that 5 kb of the upstream sequence is required for high-level expression.⁶⁵ A strong enhancer in the human ß-MyHC gene, extending from -298 to -273 bp, has been described by Flink et al,⁶⁶ as well as a factor with which it interacts. This factor, designated ßF1, binds a sequence within the element similar to the M-CAT motif and requires an adjacent sequence containing an E-box and another sequence containing a potential stem-loop structure for full activation.⁶⁶ An element in the rabbit ß-MyHC gene that corresponds to the one described in the human has been identified, and it also requires an adjacent sequence (element B) for activity.⁶⁷ Analysis of the binding properties of the two rabbit elements suggests that the enhancer (element A) interacts with muscle-specific factors, whereas element B binds non-tissue-specific proteins.⁶⁷ Sequences equivalent to elements A and B have also been characterized in the rat ß-MyHC gene.⁶⁸ These elements are also conserved in the rat gene, and another element, ße1, is similar to the HF-1a element of the MLC-2 gene.⁵⁰ Table 2 summarizes the conservation observed in the sequence of several elements in the ß-MyHC gene promoter among five mammalian species.

View this table: [in this window] [in a new window]   Table 2. Four Regulatory Elements Detected in the ß-MyHC Gene Are Highly Conserved Between Species

Although the elements required for skeletal muscle-specific expression are contained within the shorter 600-bp basal promoter, at least 5600 bp of the 5' region is required for regulation of cardiac-specific gene expression.⁶⁵ The ße2, ße3, and C-rich regulatory elements together are important for regulation of the ß-MyHC gene; the simultaneous mutation of all three sites within the context of the 5600-bp ß-MyHC promoter/CAT chimeric construct significantly reduces expression of the gene in transgenic mice.⁶⁹ In the context of the 5600-bp promoter, none of these three elements alone is essential for expression of the gene, although mutations in any one of these elements significantly affect expression within the context of a 600-bp promoter fragment.⁶⁹ The apparent discrepancies between the in vitro analyses, which stress the importance of each individual element versus the transgenic data, may be a result of the shorter promoter construct used in the cell culture assays.

The human ß-MyHC gene also contains an inhibitory region upstream from the ßF1 binding site that is conserved in the rabbit and rat genes.⁷⁰ The DNase I-protected region within this negative element does not overlap the ßF1 binding site but may still prevent binding to proximal enhancers, since it is able to repress transcription when positioned 5', but not 3', to three different promoters.⁷⁰ As the distance between the negative element and the ßF1 binding site is increased, the negative element is less effective. Although this observation supports the hypothesis that the inhibitory element prevents binding of appropriate activator proteins to the ßF1 site, increasing the distance from the transcription start site could be responsible for decreasing the influence of the negative element.⁷⁰

Using a double-stranded oligomer to the sequence of the inhibitory element, a heart cDNA expression library was screened, and a cDNA encoding a potential repressor protein was identified.⁷¹ The cDNA that was isolated coded for a 19-kD protein with seven zinc finger repeats, an isoform of CNBP originally identified in liver.⁷¹ Two isoforms of CNBP were observed, CNBP and CNBPß, which result from variations in mRNA splicing. Although transfection of CNBP into cardiomyocytes repressed activity of a human ß-MyHC/CAT construct containing the repressor region in a dose-dependent manner, CNBPß did not.⁷¹ The competition for a common suppressor motif between isoforms of CNBP with different trans-activating properties may contribute to the differential regulation of the ß-MyHC gene.

	Prospectus

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Some studies have demonstrated a discordance between in vitro cell culture studies and transgenic mouse or in vivo injection models, suggesting that the secondary structure of DNA plays a role in tissue-restricted gene expression.^{69 72} For example, although MEF-2 has been shown to positively regulate transcription of the -MyHC gene in rat heart,⁵⁹ mutation of the MEF-2 site in the mouse -MyHC gene causes an increase in the level of expression of this gene in transgenic mice.⁶⁰ This observation is in contrast to the significant decrease in expression associated with mutation of this element in the context of the MLC-2 gene.⁵¹ Such variations may be explained by the presence of trans-acting factors in the hearts of the transgenic mice that are absent in the cardiomyocyte cultures used for the transfection analyses. In vitro binding assays and cell culture transfections with plasmid constructs are not truly in vivo assays, so future studies will continue to emphasize developmental models such as the mouse to assess the importance of putative cardiac regulatory factors.

The contrasting roles of DNA-binding sites such as MEF-2 in transfection assays versus transgenic analysis illustrate that the regulation of transcription requires the interaction of multiple cis-acting elements with their appropriate trans-acting factors. In reality, genomic DNA is complexed with nuclear binding proteins into a complicated chromatin structure that limits the accessibility of such protein-binding sites within the promoters of these genes. An examination of the myoD CpG island revealed that there is hypermethylation during immortalization and transformation, coinciding with changes in chromatin structure.⁷³ Although some trans-acting factors, such as GAL4, seem to have the ability to displace the nucleosome structure, not all activators have this ability, since both NF1 and heat shock factor are unable to bind their respective sites on nucleosomal DNA.⁷⁴ Proteins that are able to influence cardiac gene transcription in cultured cells may be unable to do so in vivo if they do not have access to nucleosome-free DNA. Therefore, the significance of the role of proteins, such as the many CArG-binding factors illustrated in Fig 2, in the activation of transcription in an in vivo context is uncertain.

The study of cardiac gene expression and the description of the mechanisms that maintain muscle and cardiac cells in a terminally differentiated state are essential to any future attempts to repair or replace damaged tissue. The study of contractile protein gene expression in the heart has provided a basis for our understanding of this process. Further analysis will determine more of the mechanisms and factors involved in cardiogenesis and help to define the regulatory network involved in the formation of the heart. The delineation of this process will allow us to determine ways in which the transcription of these genes can be manipulated in vivo and should lead us to a better understanding of cardiac growth and development.

	Selected Abbreviations and Acronyms

 

bHLH = basic HLH CArG = C, AT-rich, G sequence CBF-A = CArG-binding factor A CNBP = cellular nucleic acid binding protein CNTF = ciliary neurotrophic factor CT-1 = cardiotrophin-1 dpp = Decapentaplegic E (followed by number) = embryonic day EM element = E-box/M-CAT hybrid motif GArC = G, AT-rich, C sequence HLH = helix-loop-helix Id = inhibitor of DNA binding IL-6, IL-11 = interleukin-6, interleukin-11 LIF = leukemia inhibitory factor MEF = myocyte enhancer factor MLC = myosin light chain MyHC = myosin heavy chain PGAM = human phosphoglycerate mutase RAR = retinoic acid receptor RXR = retinoid X receptor SRE = serum response element SRF = serum response factor TEF-1 = transcription enhancer factor-1 USF = upstream stimulating factor

	Acknowledgments

 
This study has been supported by grants from the Medical Research Council of Canada and the Heart and Stroke Foundation of Ontario. Dr Mably is supported by a research fellowship from the Ontario Heart and Stroke Foundation. We wish to thank Dr Douglas B. Cowan and David Hwang for their helpful discussions and appraisal of this manuscript.

Received September 11, 1995; accepted March 18, 1996.

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