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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:8.)
© 2008 American Heart Association, Inc.

Brief Reviews

MCAT Elements and the TEF-1 Family of Transcription Factors in Muscle Development and Disease

Tadashi Yoshida

From the Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville.

Correspondence to Tadashi Yoshida, MD, PhD, Department of Molecular Physiology and Biological Physics, University of Virginia, MR5 Room 1226, 415 Lane Road, Charlottesville, Virginia 22908. E-mail ty2c{at}virginia.edu

	Abstract

Top Abstract Introduction TEF-1 Family Members Regulate... The MCAT Element and... Multiple Mechanisms Modulate the... Conclusion and Perspectives References

 
MCAT elements are located in the promoter-enhancer regions of cardiac, smooth, and skeletal muscle-specific genes including cardiac troponin T, β-myosin heavy chain, smooth muscle -actin, and skeletal -actin, and play a key role in the regulation of these genes during muscle development and disease. The binding factors of MCAT elements are members of the transcriptional enhancer factor-1 (TEF-1) family. However, it has not been fully understood how these transcription factors confer cell-specific expression in muscle, because their expression patterns are relatively broad. Results of recent studies revealed multiple mechanisms whereby TEF-1 family members control MCAT element-dependent muscle-specific gene expression, including posttranslational modifications of TEF-1 family members, the presence of muscle-selective TEF-1 cofactors, and cell-selective control of TEF-1 accessibility to MCAT elements. In addition, of particular interest, recent studies regarding MCAT element-dependent transcription of the myocardin gene and the smooth muscle -actin gene in muscle provide evidence for the transcriptional diversity among distinct cell types and subtypes. This article summarizes the role of MCAT elements and the TEF-1 family of transcription factors in muscle development and disease, and reviews recent progress in our understanding of the transcriptional regulatory mechanisms involved in MCAT element-dependent muscle-specific gene expression.

This article summarizes the role of MCAT elements and the transcriptional enhancer factor-1 family of transcription factors in cardiac, smooth, and skeletal muscle development and disease, and reviews recent progress in our understanding of the transcriptional regulatory mechanisms involved in MCAT element-dependent muscle-specific gene expression.

Key Words: transcriptional enhancer factor-1 • MCAT element • cardiac muscle • smooth muscle • skeletal muscle • myofibroblasts

	Introduction

Top Abstract Introduction TEF-1 Family Members Regulate... The MCAT Element and... Multiple Mechanisms Modulate the... Conclusion and Perspectives References

 
Cardiovascular disease is the leading cause of mortality in the world according to the World Health Report from the World Health Organization. For the advancement of therapeutic and preventive approaches, it is important to understand the mechanisms whereby the genes specifically expressed in the heart and the vasculature are controlled under physiological and pathological conditions. Identification of signaling pathways and molecules that activate these genes will provide insights regarding the pathophysiology of cardiovascular disease, and elucidation of cell type-specific DNA cis-elements in the cardiovascular system may shed light on therapeutic gene delivery into these tissues.

Cardiomyocytes and vascular smooth muscle cells (SMCs) are the major components of the cardiovascular system. Transcription of cell-specific genes in these cell types has been shown to be regulated by multiple cis-elements and their cognate transcription factors, including CArG elements and serum response factor (SRF)/myocardin, E-boxes and the basic Helix-Loop-Helix proteins, AT-rich elements and the myocyte enhancer factor-2 (MEF2) family members, GATA factors, homeodomain proteins, and Krüppel-like factors.^1,2 In addition, MCAT elements and their trans-binding factors, the transcriptional enhancer factor-1 (TEF-1) family of transcription factors, have also been recognized as critical regulators of multiple cardiac and smooth muscle-specific genes during cardiovascular development and disease, as well as regulators of skeletal muscle-specific genes in skeletal muscle. However, it has not been fully understood how these factors confer muscle-specific gene transcription, because expression of TEF-1 family members is not restricted to muscles. This review article will summarize the contribution of MCAT elements and TEF-1 family members to cardiovascular development and disease with particular focus on in vivo studies, and consider recent advances in our understanding of the molecular mechanisms whereby the TEF-1 family of transcription factors controls expression of muscle-specific genes in cardiac, smooth, and skeletal muscle. For a more comprehensive review of the transcriptional regulation of cardiac, smooth, or skeletal muscle-specific genes, the reader should refer to several excellent review articles.^1–4

	TEF-1 Family Members Regulate Transcription of Muscle-Specific Genes Through the Binding to MCAT Elements

Top Abstract Introduction TEF-1 Family Members Regulate... The MCAT Element and... Multiple Mechanisms Modulate the... Conclusion and Perspectives References

 
The MCAT element was originally identified as a muscle-specific cytidine-adenosine-thymidine sequence, 5'-CATTCCT-3', in the chicken cardiac troponin T promoter.⁵ To date, the MCAT element has been found in a number of cardiac, smooth, and skeletal muscle-specific genes, including cardiac troponin T⁶, β-myosin heavy chain (β-MHC),⁷ smooth muscle -actin (SM -actin),⁸ and skeletal -actin⁹ (Table 1). It has been shown to play a key role in the transcriptional regulation of these genes, although it is also present in the promoter regions of nonmuscle genes, including the simian virus 40 enhancer,^10,11 the human papillomavirus type 16 E6 and E7 oncogenes,¹² the chorionic somatomammotropin gene,¹³ and the Foxa2 gene.¹⁴ The consensus sequence of MCAT elements has been reported as 5'-CATTCC-3', based on the binding site selection assays for TEF-1.¹⁵ As shown in Table 1, some muscle-specific genes contain two MCAT elements in their promoter-enhancer regions, whereas the others only contain a single MCAT element. Mutational analysis of the cardiac troponin T promoter that contains two MCAT elements has shown that both MCAT elements are required for the full transcriptional activity in cultured muscle cells.⁶ In addition, the spatial relationship between these two MCAT elements has been shown to be a critical determinant for gene transcriptional activity.¹⁶ These results suggest that the transcriptional activity of a subset of muscle-specific genes is cooperatively regulated by multiple MCAT elements and their binding factors.

View this table: [in this window] [in a new window]   Table 1. MCAT Elements in the Muscle-Specific Genes

The proteins that bind to the MCAT element belong to the TEF-1 family of transcription factors.^11,17 This family shares a highly conserved DNA binding domain called the TEA domain and consists of four members including TEF-1 (NTEF-1/Tead1),¹¹ RTEF-1 (TEF-3/ETFR-2/FR-19/Tead4),^18–22 ETF (TEF-4/Tead2),^21,23 and DTEF-1 (TEF-5/ETFR-1/Tead3)^13,20,24 (Table 2). Each TEF-1 family member has multiple names as shown in parentheses above and in Table 2, and this review article uses the nomenclature proposed by Stewart and his colleagues.²⁵ The TEA domain is also referred to as the ATTS domain, because it appears in yeast, vertebrate, plant, and fly transcription factors AbaA, TEC1, TEF-1, and Scalloped.^26,27 AbaA regulates development of the asexual spores in Aspergillus nidulans and terminates vegetative growth, TEC1 is involved in the activation of the Ty1 retrotransposon in yeast Saccharomyces cerevisiae, and the Drosophila gene Scalloped plays an important role in sensory neuron and wing development. Conservation of the TEA domain in multiple organisms indicates its critical role in regulation of gene transcription. TEF-1 family members have several interesting features. First, TEF-1, RTEF-1, and DTEF-1, respectively, initiate translation at an isoleucine (AUU), leucine (UUG), and isoleucine (AUA) codon that lies upstream of the first methionine codon.^11,13,18 ETF, however, uses the methionine (AUG) codon for the initiation of translation.²³ Second, TEF-1 family members bind to the double-stranded form of the MCAT element, but not to the single-stranded MCAT element.²⁸ The tissue distribution of TEF-1 family members has been examined by a number of studies.^{11,13,18–20,23,24,29} Indeed, TEF-1, DTEF-1, and RTEF-1 are widely expressed in multiple tissues including the skeletal muscle, pancreas, placenta, lung, and heart. In contrast to these three factors, ETF is selectively expressed in a subset of embryonic tissues including the cerebellum, testis, and distal portion of the forelimb and hindlimb buds as well as the tail bud, but it is essentially absent from the adult tissues.²³ ETF has also been shown to be expressed from the 2-cell stage during development.³⁰

View this table: [in this window] [in a new window]   Table 2. The TEF-1 Family Members

In summary, MCAT elements are located within the promoter-enhancer regions of a number of cardiac, smooth, and skeletal muscle-specific genes, and the relatively widely expressed TEF-1 family members are the binding factor of MCAT elements. In the next section, the roles of MCAT elements and TEF-1 family members in muscle development and disease will be described.

	The MCAT Element and TEF-1 Family Members Play Important Roles in Muscle Development and Disease

Top Abstract Introduction TEF-1 Family Members Regulate... The MCAT Element and... Multiple Mechanisms Modulate the... Conclusion and Perspectives References

 
Cardiac Development
There is evidence showing that TEF-1 is involved in cardiac development. TEF-1 knockout mice were generated by a retroviral gene trap approach.³¹ TEF-1 knockout mice exhibited an enlarged pericardial cavity, bradycardia, a dilated fourth ventricle in the brain, and died by embryonic day (E)12.5. Histological examination revealed that the ventricular wall in the heart of TEF-1 knockout mouse embryos was abnormally thin with a reduced number of trabeculae. These results indicate that TEF-1 is required for cardiac morphogenesis and that other TEF-1 family members cannot compensate for TEF-1 function during embryogenesis. However, the studies did not detect any changes in the expression of MCAT element-containing muscle-specific genes including cardiac troponin T and MHC. This might be attributable to the experimental procedure in which expression levels of muscle-specific genes were examined in extracts from the whole embryos. In situ hybridization and immunohistochemistry are needed to further determine the expression of MCAT element-containing genes in the heart of TEF-1 knockout mouse embryos.

Cardiac Hypertrophy
Cardiac hypertrophy is an adaptive response of the heart to increased workload and injury. It occurs in a number of pathophysiological conditions such as hypertension, valvular disease, myocardial infarction, and cardiomyopathy. At the cellular level, it is characterized by an increase in cell size and protein synthesis and by reactivation of the fetal cardiac genes including β-MHC and skeletal -actin.³² Stimulation of ₁-adrenergic signaling has been shown to induce cardiac hypertrophy and activate transcription of the β-MHC gene and the skeletal -actin gene in cultured neonatal rat cardiomyocytes. Interestingly, ₁-adrenergic receptor-mediated induction of these genes is abolished by mutation of the MCAT element within the promoters.^9,33 In addition, RTEF-1, but not TEF-1, potentiates the ₁-adrenergic response of the β-MHC and skeletal -actin promoters.²⁵ However, it remains to be determined whether this mechanism is applicable to the in vivo condition of cardiac hypertrophy. Two groups independently examined whether MCAT elements contribute to the induction of β-MHC in aortic constriction-induced left ventricular hypertrophy in rats.^34,35 They showed that, although simultaneous mutation of two MCAT elements in the β-MHC promoter dramatically decreased the basal transcriptional activity, the same MCAT element-mutated promoter still responded to aortic constriction in vivo. However, each of the experiments was performed at limited time points relatively late after the onset of hemodynamic overload. Therefore, it remains possible that MCAT elements are involved in the induction of β-MHC at earlier time points. In addition, MCAT elements might contribute to the induction of fetal cardiac genes in other in vivo models of cardiac hypertrophy.

Cardiac Arrhythmia
Cardiac muscle-specific overexpression of RTEF-1 has been shown to induce arrhythmias in mice in vivo.³⁶ Transgenic mice expressing RTEF-1 under the control of the -MHC promoter exhibited prolongation of the PR, QRS, and AH intervals, which indicate a conduction delay in the atrial myocardium, in the ventricular myocardium, and above the bundle of His, respectively. Accompanying the conduction defects, spontaneous atrial tachycardia and progressive atrial dilatation were detected in RTEF-1 transgenic mice. To clarify the mechanism of arrhythmias in RTEF-1 transgenic mice, the authors examined the phosphorylation status of connexins. They found that expression of protein phosphatase 1β was upregulated in the heart of RTEF-1 transgenic mice, which resulted in the dephosphorylation of cardiac connexin40 and connexin43. Because chronic dephosphorylation of connexins impairs the gap-junction conductance, this is likely to be the cause of arrhythmias in RTEF-1 transgenic mice. In future studies, it will be interesting to determine whether RTEF-1 directly regulates expression of protein phosphatase 1β in cardiomyocytes.

Smooth Muscle Development and Myofibroblast Formation
Results of recent studies provide novel evidence that the MCAT element plays a critical role in smooth muscle development.^37,38 Myocardin is exclusively expressed in SMCs and cardiomyocytes, and it extraordinarily induces a number of smooth and cardiac muscle-specific genes by forming a ternary complex with SRF and the CArG element.^4,39 Transcriptional regulation of the myocardin gene was recently examined in transgenic mice harboring the promoter-enhancer region of the mouse myocardin gene linked to the LacZ reporter gene.³⁷ A DNA fragment located between –32.5 kb and –22.6 kb upstream of the first exon was identified as a sequence required for directing LacZ expression in the heart and vessels, and this region contained an MCAT element as well as a MEF2 binding site and multiple Foxo binding sites. Of interest, mutation of the MCAT element selectively abolished LacZ expression in SMCs, but not in the heart, whereas mutation of either the MEF2 binding site or Foxo binding sites dramatically reduced LacZ expression in both SMCs and cardiomyocytes. These results suggest that the MCAT element behaves as a SMC-specific upstream signaling pathway for the induction of the myocardin gene. However, it remains undetermined which TEF-1 family members bind to this MCAT element, and how the MCAT element functions selectively in SMCs. In addition, because the studies were performed in early developmental stages of mouse embryos, further studies are needed to determine whether the MCAT element also regulates myocardin expression in adult differentiated SMCs.

The MCAT element has also been shown to play a key role in the regulation of the SM -actin gene in SMCs during embryogenesis.³⁸ SM -actin is one of the most frequently used cell-selective markers for SMCs, although it is also transiently expressed in cardiac and skeletal muscle during embryogenesis and in activated myofibroblasts. Recently, we examined the role of MCAT elements in SM -actin expression in vivo using transgenic mice that harbor an SM -actin promoter-enhancer-LacZ reporter construct containing MCAT element mutations.³⁸ In wild-type SM -actin promoter-enhancer-LacZ transgenic mice, LacZ expression was detectable in smooth muscle-containing vessels, heart, and skeletal muscle from E10.5 (Figure 1A), and in the adult it was seen exclusively in smooth muscle-containing tissues such as the aorta, stomach, intestine, colon, and bladder (Figure 1B). In contrast, of major significance, LacZ expression was not detected in any smooth muscle-containing tissues in MCAT element-mutated SM -actin promoter-enhancer-LacZ transgenic mice at E10.5 and E12.5, although it was detectable in smooth muscle-containing tissues after E13.5 through adulthood (Figure 1A and 1B). Mutation of MCAT elements also completely abolished LacZ expression in cardiac and skeletal muscle throughout embryonic development. Results indicate that MCAT elements are required for the initial induction of SM -actin expression in SMCs, as well as in skeletal and cardiac myoblasts, whereas they are dispensable in differentiated SMCs. Moreover, results also suggest that the initial induction of SMC differentiation marker genes including SM -actin is dependent on MCAT elements and their binding factors but not myocardin, because induction of myocardin expression in the descending aorta is delayed until E12.5 as determined by in situ hybridization assays.⁴⁰

View larger version (76K): [in this window] [in a new window]   Figure 1. MCAT elements are required for SM -actin expression in embryonic SMCs (A), skeletal/cardiac myoblasts (A) and myofibroblasts (C), but not in adult SMCs (B). LacZ staining in mice harboring a MCAT element-mutated SM -actin promoter-enhancer-LacZ construct and wild-type construct is shown. Scale bar=20 µm. Adapted from Gan et al.38

Myofibroblasts are produced de novo in multiple pathological states such as the granulation tissue of contracting wounds, vein graft remodeling, and fibrocontractive diseases, and play a major role in the inflammatory response.⁴¹ Requirement of MCAT elements for the induction of the SM -actin gene in myofibroblasts was tested in MCAT element-mutated SM -actin promoter-enhancer-LacZ transgenic mice.³⁸ Results showed that LacZ was expressed in myofibroblasts within granulation tissues of skin wounds in wild-type SM -actin promoter-enhancer-LacZ mice, whereas it was not expressed in MCAT element-mutated SM -actin promoter-enhancer-LacZ mice (Figure 1C). As such, results provide evidence that MCAT elements are required for SM -actin expression in myofibroblasts.

Studies analyzing the roles of MCAT elements in the transcription of the myocardin gene and the SM -actin gene both indicate the diversity in the transcriptional control mechanisms among different cell types. That is, although the genes are similarly expressed in multiple cell types or subtypes, these cell types/subtypes can be distinguished at the molecular level. The transcriptional diversity is not unique to the MCAT element-containing genes, but is also seen in other muscle-specific genes including the smooth muscle-MHC (SM-MHC) gene and the SM22 gene.^42–45 Results of studies using transgenic mice harboring an SM-MHC promoter-enhancer-LacZ construct provide evidence that transcription of the SM-MHC gene employs distinct transcriptional control mechanisms among SMC subtypes, in that mutation of intronic CArG element in the SM-MHC gene abolished LacZ reporter expression selectively in the large elastic arteries such as the abdominal aorta, but not in small arteries.⁴² Likewise, a 400 bp–length 5'-flanking region of the SM22 gene has been shown to be active in arterial SMCs and skeletal and cardiac muscle in embryos, but inactive in venous and visceral SMCs, even though the endogenous SM22 gene is expressed.^43–45 Although precise mechanisms of the transcriptional diversity remain unknown, both inherent factors (eg, distinct embryological origins) and plastic factors (eg, different microenvironmental cues) appear to contribute to the diversity, as extensively discussed in our previous review article.⁴ However, based on the findings that embryonic SMCs transitioned from MCAT element-dependent SM -actin transcription to MCAT element-independent one within one day from E12.5 to E13.5 (Figure 1A), the transcriptional diversity is likely to be plastic rather than to be an inherent property to a particular cell type.

Skeletal Muscle Hypertrophy and Regeneration
The MCAT element has been shown to be involved in the regulation of muscle-specific genes in skeletal muscle. For example, stretch overload-induced muscle hypertrophy is accompanied by the increased activity of the skeletal -actin gene in chicken anterior latissimus dorsi,⁴⁶ and this induction is attenuated by mutation of the MCAT element in the skeletal -actin promoter.⁴⁷ Electrophoretic mobility shift assays (EMSA) have shown that binding of nuclear proteins to the MCAT element is dramatically induced by the stretch overload.⁴⁷ Likewise, the 600-bp upstream region of the β-MHC gene, which contains two MCAT elements, is sufficient to exhibit muscle-specific gene expression in mice.⁷ Mutational analyses have revealed that functional overload-induced activity of the β-MHC gene is mediated through the proximal MCAT element in rat plantaris muscle.⁴⁸ Moreover, denervation-induced decrease in β-MHC expression is also mediated by the proximal MCAT element in rat soleus.⁴⁹ Taken together, these results clearly indicate the contribution of the MCAT element to the regulation of muscle-specific genes in skeletal muscle. However, a series of studies by Tsika and colleagues showed that induction of the β-MHC gene in overloaded skeletal muscle was not mediated by the MCAT elements, but, by the association of TEF-1 proteins with the A/T-rich element located between two MCAT elements.^50–52 Additional work is required to reconcile the discrepancy.

Zhao et al⁵³ showed that the MCAT element and ETF were implicated in a process of skeletal muscle regeneration. They showed that cardiotoxin-induced degeneration/regeneration of skeletal muscle occurred abnormally in fibroblast growth factor receptor 4 (FGFR4) knockout mice as compared with wild-type mice. By 14 days after cardiotoxin injection, much of the skeletal muscle exhibited impaired regeneration and was replaced by fat and calcifications in FGFR4 knockout mice. They found that ETF expression was induced in skeletal muscle during regeneration in vivo, and that ETF induced the promoter activity of the FGFR4 gene via a MCAT element in C2C12 myoblasts. These results suggest the importance of the MCAT element and ETF in muscle regeneration, although evidence is indirect. Further studies are needed to determine whether knockout of ETF also exhibits abnormal skeletal muscle regeneration in vivo.

Neural Crest Development
Pax3 is a transcription factor that functions in the embryonic central nervous system, neural crest, and somatic mesoderm. Milewski et al⁵⁴ identified an enhancer region within the Pax3 promoter-enhancer that was sufficient to induce expression in neural crest precursors, but not in somatic mesoderm. They found a MCAT-like element (5'-CATTCAT-3') in this enhancer region, and identified ETF as a binding factor of this enhancer by a yeast one-hybrid screen. They showed that expression of Pax3 was dramatically reduced by either mutation of the ETF binding site or dominant-negative ETF in neural crest. These results suggest that ETF regulates Pax3 expression via a MCAT-like element selectively in neural crest, but not in somatic mesoderm. However, recent studies showed that Pax3 expression was normal in ETF knockout mouse embryos at E11.5, although mice exhibited a defect in neural tube closure.⁵⁵ It is possible that ETF-mediated expression of Pax3 was compensated by other factors and mechanisms in ETF knockout mice.

	Multiple Mechanisms Modulate the Transcriptional Activity of MCAT Element-Dependent Muscle-Specific Genes

Top Abstract Introduction TEF-1 Family Members Regulate... The MCAT Element and... Multiple Mechanisms Modulate the... Conclusion and Perspectives References

 
As described above, MCAT elements and the TEF-1 family of transcription factors have been shown to play important roles in regulation of muscle-specific genes during muscle development and disease. One of the most interesting questions in the field is how the widely expressed TEF-1 family members confer muscle-specific expression of MCAT element-containing genes. The potential mechanisms will be reviewed in this section.

Phosphorylation of TEF-1 Family Members
₁-Adrenergic agonists have been shown to activate expression of MCAT element-dependent genes including β-MHC and skeletal -actin in neonatal rat cardiomyocytes.³² This induction is potentiated by RTEF-1, but not TEF-1, although both TEF-1 and RTEF-1 are expressed in cardiomyocytes.²⁵ Results of EMSA have shown that TEF-1 accounts for 85% of the MCAT element-binding activity on the skeletal -actin promoter, whereas other TEF-1 family members account for the rest,⁵⁶ suggesting that the simple binding of TEF-1 family members to MCAT elements does not correlate with transcriptional activity. Ueyama et al⁵⁶ examined the phosphorylation status of TEF-1 family members in cardiomyocytes and found that the unique phosphorylation site in RTEF-1 at Serine 322 is required for ₁-adrenergic response. Subsequently, ₁-adrenergic stimulation-induced activation of MCAT element-dependent genes is also shown to be mediated in part by phosphorylation of DTEF-1.⁵⁷

TEF-1 also undergoes phosphorylation. Activation of protein kinase A induces phosphorylation of TEF-1 at Serine 102 and activates the transcription of the -MHC gene in cardiomyocytes.⁵⁸ Phosphorylated TEF-1 exhibits a reduced binding activity to the MCAT element within the -MHC gene, suggesting that TEF-1 binding to the MCAT element functions as a repressive mechanism. As such, the phosphorylation status of TEF-1 family members is likely to be a mechanism modulating muscle-specific transcription of MCAT element-containing genes.

Cofactors of TEF-1 Family Members
Ectopic expression of TEF-1 is entirely inactive in cell lines in which the endogenous TEF-1 protein is absent.¹¹ Overexpression of TEF-1 in cells that express TEF-1 family members results in the transcriptional repression of MCAT element-containing genes.¹¹ Because these observations are consistent with a squelching phenomenon of coactivator activity, the presence of cofactors for TEF-1 family members has long been predicted. Recently, multiple cofactors for TEF-1 family members have been identified. They include the p160 family of nuclear receptor coactivators (SRC1, TIF2, and RAC3),⁵⁹ a Src/Yes-associated protein YAP65,⁶⁰ TAZ,⁶¹ Vgl-2,⁶² and Vgl-4.⁶³ Of these, Vgl-2 is expressed in a tissue-specific manner and contributes to the cell-specific transcription of MCAT element-containing genes.

Vgl-2, (Vestigial-like 2, also called as VITO-1) was identified as a human homolog of a Drosophila protein, Vestigial, a transcriptional coactivator of Scalloped that contains the TEA domain and is required for wing formation.⁶⁴ Vgl-2 is expressed in the differentiating somites and branchial arches during embryogenesis and is exclusively expressed in skeletal muscle in the adult.⁶² During differentiation of C2C12 skeletal muscle cells, expression of Vgl-2 is increased and Vgl-2 protein is translocated from the cytoplasm to the nucleus. Vgl-2 interacts with TEF-1 and RTEF-1,⁶² and regulates the binding activity of TEF-1 family members to MCAT elements.^65,66 Cotransfection assays have shown that RTEF-1 and Vgl-2 cooperatively increase the promoter activity of the skeletal -actin gene in cultured cells.⁶² Of importance, suppression of Vgl-2 by antisense morpholino decreases MHC expression in C2C12 myocytes and chicken limb muscles in vivo.⁶⁵ As such, Vgl-2 is a key cofactor of TEF-1 family members regulating muscle-specific gene transcription in skeletal muscle.

In contrast, a recently cloned Vgl family member, Vgl-4, does not exhibit cell type–specific expression patterns and functions differently from Vgl-2.⁶³ Vgl-4 is relatively widely expressed in multiple tissues including the heart, brain, kidney, small intestine, lung, and placenta. Vgl-4 physically interacts with TEF-1 and MEF2. Overexpression of Vgl-4 interferes with the basal and ₁-adrenergic agonist-induced activity of the skeletal -actin promoter in neonatal cardiomyocytes. In addition, ₁-adrenergic signaling elicits nuclear export of Vgl-4 in cardiomyocytes. These results suggest that Vgl-4 acts as a repressor of TEF-1 family member-dependent gene transcription under normal conditions, and that it translocates to the cytoplasm and modifies the transcriptional activity of these genes after stimulation.

Physical Interaction With SRF, MEF2, and Max
Multiple transcription factors including SRF, myocardin and myocardin-related transcription factors (MKL1/MRTF-A and MKL2/MRTF-B), MEF2, homeodomain proteins, the GATA family, Krüppel-like factors, basic Helix-Loop-Helix proteins, and TEF-1 family members have been shown to be involved in the control of muscle-specific gene transcription, although most of these transcription factors are not specific for a particular muscle cell type.⁴ Therefore, it has been proposed that expression of muscle-specific genes is controlled by unique combinations of transcription factors that are expressed ubiquitously or cell-selectively. In this regard, it is worth noting that TEF-1 family members interact with several transcription factors including SRF, MEF2, and a basic Helix-Loop-Helix zipper protein, Max, to control muscle-specific genes.^29,67–69

Gupta et al⁶⁷ showed that SRF, a trans-binding factor for CArG elements, physically interacted with TEF-1 to regulate the skeletal -actin gene transcription. They showed that the MADS domain of SRF and the TEA domain of TEF-1 were responsible for direct interaction as determined by coimmunoprecipitation assays and GST pull-down assays. They showed the functional interaction of these factors in cultured cells, in that SRF and TEF-1 synergistically induced the promoter activity of the skeletal -actin gene and mutation of either the CArG element or the MCAT element within the skeletal -actin promoter abolished the synergistic effect. Max has also been shown to interact with TEF-1 to positively regulate -MHC expression in primary culture of cardiomyocytes.⁶⁸ Although overexpression of TEF-1 alone or Max alone does not increase the activity of -MHC transcription in cardiomyocytes, coexpression of TEF-1 and Max increases the activity. Moreover, TEF-1 and DTEF-1 have been reported to interact with MEF2.^29,69 DTEF-1 and MEF-2 cooperatively increase the activity of the cardiac troponin T promoter in cardiomyocytes,²⁹ although the functional consequence of interaction between TEF-1 and MEF2 is unclear.⁶⁹

Combinatorial interactions of TEF-1 family members with other transcription factors that colocalize with TEF-1 family members in muscle are likely to confer cell-specific expression of MCAT element-dependent genes. However, the interactive model cannot fully explain the muscle-specific expression of these genes, because colocalization patterns do not exactly match the expression patterns of MCAT element-dependent genes. Further studies are needed to better understand the complex interactive network of multiple transcription factors that control MCAT element-containing genes.

Flanking Sequence of MCAT Elements and the Single-Stranded DNA Binding Factors, Pur, Purβ, and MSY1
Flanking sequence of MCAT elements and its binding factors are capable of modulating the transcriptional activity of MCAT element-containing genes. Strauch, Kelm, and their colleagues have extensively studied the role of flanking sequence of MCAT elements in regulation of the SM -actin gene transcription in myofibroblasts.^28,70–74 They showed that, although mutation of MCAT1 element at –182/–176 bp within the mouse SM -actin promoter decreased transcriptional activity of the SM -actin gene, mutation of the flanking regions increased the activity in AKR-2B fibroblasts.⁷⁰ They found that the flanking sequence surrounding the MCAT1 element exhibited a high degree of polypurine/polypyrimidine (Pu/Py) asymmetry,⁷¹ and it was the binding site for three single-stranded DNA-binding proteins, Pur, Purβ, and MSY1.^28,72,73 In addition, they showed that small interfering RNA (siRNA)-induced knockdown of Purβ, but not Pur, increased the transcriptional activity of the SM -actin gene, whereas overexpression of Purβ, but not Pur, decreased the activity in AKR-2B cells.⁷⁴ Taken together, these results suggest that the flanking sequence of MCAT elements and its binding factors, including Pur, Purβ, and MSY1, contribute to the regulation of MCAT element-dependent genes. However, results of recent studies showed that Pur and Purβ also regulated expression of -MHC and β-MHC in cardiomyocytes and skeletal muscle, respectively, and in these gene promoters, the DNA binding sites for Pur and Purβ were located far from the MCAT elements.^75,76 Further studies are needed to analyze the spatial relationship between the single-stranded DNA binding sites and MCAT elements in more detail and to determine if Pur and Purβ directly alter the binding of TEF-1 family members to MCAT elements on the promoter.

Accessibility of TEF-1 Family Members to MCAT Elements
Results of our recent studies provide evidence that expression of the SM -actin gene uses distinct transcriptional control mechanisms among different cell types.³⁸ We explored the underlying mechanisms by which MCAT elements play different roles in SM -actin gene transcription between embryonic SMCs and myofibroblasts versus adult differentiated SMCs.³⁸ Although TEF-1, RTEF-1, and DTEF-1 were expressed in a similar manner in these cell types, siRNA-induced knockdown of each of TEF-1 family members showed that only RTEF-1 regulated SM -actin transcription in myofibroblasts and embryonic SMCs, but not in differentiated SMCs (Figure 2). Moreover, quantitative chromatin immunoprecipitation assays revealed that RTEF-1 was a major binding factor for MCAT elements within the SM -actin promoter in myofibroblasts, whereas TEF-1 was associated with the same region in differentiated SMCs. In addition, no TEF-1 family members were associated with MCAT elements within the SM -actin promoter in cells in which SM -actin was not expressed. At present, binding factors of MCAT elements within the SM -actin promoter in skeletal and cardiac myoblasts are unknown. These results indicate that distinct TEF-1 family members associate with MCAT elements in different cell types, and this is likely to be a critical determinant for MCAT element-dependent SM -actin transcription.

View larger version (20K): [in this window] [in a new window]   Figure 2. Distinct TEF-1 family members are associated with MCAT elements within the SM -actin promoter in embryonic SMCs and myofibroblasts (A) versus differentiated SMCs (B), as determined by siRNA-induced knockdown experiments and quantitative chromatin immunoprecipitation assays.38 No TEF-1 family members are associated with MCAT elements in non-SM -actin expressing cells (C).

Alternative Splicing
The TEF-1 family of transcription factors has been reported to consist of multiple alternative splicing isoforms.^77–79 Some TEF-1 isoforms are localized in the cytoplasm, whereas most isoforms are located in the nucleus.⁷⁸ Several TEF-1 isoforms exhibit tissue-restricted expression patterns and a distinct DNA binding affinity as compared with the prototype.⁷⁹ The presence of these isoforms may contribute to muscle-specific expression of MCAT element-dependent genes, although this mechanism alone is unlikely to be sufficient to confer cell-specific expression. For example, we did not detect any difference in size or in subcellular localization of TEF-1, RTEF-1, and DTEF-1 between cell types in which SM -actin transcription is MCAT element-dependent versus -independent.³⁸ However, future studies need to take consideration of this possibility.

	Conclusion and Perspectives

Top Abstract Introduction TEF-1 Family Members Regulate... The MCAT Element and... Multiple Mechanisms Modulate the... Conclusion and Perspectives References

 
Multiple lines of evidence indicate that MCAT elements and the TEF-1 family of transcription factors play important roles in the regulation of muscle-specific genes during embryonic development and various disease states in cardiac, smooth, and skeletal muscle. In addition to the genes described in this review, other muscle-specific genes, such as the rat atrial natriuretic factor gene (5'-CATTCTT-3' at –207/–213 bp) and the mouse SM22 gene (5'-CATTCTC-3' at –195/–201 bp), also contain a MCAT-like sequence in the promoter-enhancer regions. Expression of these muscle-specific genes is not controlled by the simple presence or absence of a single transcription factor, rather, it is controlled by multiple mechanisms including combinatorial interactions of multiple cis-elements, cognate transcription factors and cofactors, posttranslational modifications of transcription factors, and accessibility of transcription factors to DNA cis-elements. Because each of these mechanisms alone cannot explain the cell specificity of MCAT element-containing genes, they are likely to be interrelated and cooperatively regulate muscle-specific gene transcription. For example, it is possible that changes in the phosphorylation status of TEF-1 family members alter the binding affinity of TEF-1 family members with Vgl cofactors or other transcription factors such as SRF. Alternatively, the phosphorylation state of TEF-1 family members or the existence of Vgl cofactors might regulate accessibility of TEF-1 family members to MCAT elements. Moreover, different splicing isoforms of TEF-1 family members may exhibit distinct association patterns with Vgl cofactors and other transcription factors. These mechanisms are not mutually exclusive (Figure 3), and further studies are needed to elucidate their precise interactions.

View larger version (22K): [in this window] [in a new window]   Figure 3. TEF-1 family member-dependent transcription of MCAT element-containing muscle-specific genes is modulated by: (A) phosphorylation of TEF-1 family members; (B) interaction with cofactors; (C) interaction with SRF/MEF2/Max; (D) flanking sequence and its binding factors; (E) accessibility of TEF-1 family members to MCAT elements; and (F) alternative splicing of TEF-1 family members.

Recently, analyses of ETF knockout mice⁵⁵ and RTEF-1 knockout mice⁸⁰ have been reported, in addition to TEF-1 knockout mice.³¹ It is interesting to determine the effects of knockout of TEF-1 family members on cardiac, smooth, and skeletal muscle development and disease in detail, because the initial studies did not address these issues. Analyses of cell type–specific conditional knockout of each of these factors may also be required in future studies, because the phenotype in conventional knockout animals might be confounded by the phenotype that appears in other organs and tissues. Moreover, because of the possible functional overlap between TEF-1 family members, analyses of combinatorial knockout of these factors are also warranted.

A number of epigenetic studies provide evidence that changes in chromatin structure affect gene transcription. It is highly possible that alterations in epigenetic modifications such as histone modification patterns and DNA methylation patterns confer cell-specific expression of MCAT element-dependent genes. In this regard, it is interesting to note that TEF-1 and RTEF-1 have been shown to interact with poly(ADP-ribose) polymerase, a chromatin modifying enzyme.⁸¹ Further studies are needed to determine whether TEF-1 family members have an ability to change the chromatin structure to regulate muscle-specific gene transcription.

Finally, it is hoped that a further clarification of the molecular mechanisms for muscle-specific gene expression will contribute to the advancement of therapeutic strategies in multiple disease states of cardiac, smooth, and skeletal muscle.

	Acknowledgments

 
Sources of Funding

This work was supported by American Heart Association National Scientist Development Grant (0635253N).

Disclosures

None.

	Footnotes

 
Original received September 10, 2007; final version accepted October 16, 2007.

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