Myosin Heavy Chain Promoter Activity Research Articles

DNA Methylation of a GC Repressor Element in the Smooth Muscle Myosin Heavy Chain Promoter Facilitates Binding of the Notch-Associated Transcription Factor, RBPJ/CSL1

The goal of the present study was to identify novel mechanisms that regulate smooth muscle cell (SMC) differentiation marker gene expression. We demonstrate that the CArG-containing regions of many SMC-specific promoters are imbedded within CpG islands. A previously identified GC repressor element in the SM myosin heavy chain (MHC) promoter was highly methylated in cultured aortic SMC but not in the aorta, and this difference was inversely correlated with SM MHC expression. Using an affinity chromatography/mass spectroscopy-based approach, we identified the multifunctional Notch transcription factor, recombination signal binding protein for immunoglobulin κ J region (RBPJ), as a methylated GC repressor-binding protein. RBPJ protein levels and binding to the endogenous SM MHC GC repressor were enhanced by platelet-derived growth factor-BB treatment. A methylation mimetic mutation to the GC repressor that facilitated RBPJ binding inhibited SM MHC promoter activity as did overexpression of RBPJ. Consistent with this, knockdown of RBPJ in phenotypically modulated human aortic SMC enhanced endogenous SMC marker gene expression, an effect likely mediated by increased recruitment of serum response factor and Pol II to the SMC-specific promoters. In contrast, the depletion of RBPJ in differentiated transforming growth factor-β-treated SMC inhibited SMC-specific gene activation, supporting the idea that the effects of RBPJ/Notch signaling are context dependent. Our results indicate that methylation-dependent binding of RBPJ to a GC repressor element can negatively regulate SM MHC promoter activity and that RBPJ can inhibit SMC marker gene expression in phenotypically modulated SMC. These results will have important implications on the regulation of SMC phenotype and on Notch-dependent transcription.

IGF-I activates the mouse type IIb myosin heavy chain gene

IGF-I increases skeletal muscle mass, but whether IGF-I increases type IIb myosin heavy chain (MyHC) transcriptional activity is not known. C2C12 myotubes were cultured with or without IGF-I to determine whether IGF-I increases type IIb MyHC promoter activity, and if so, what region of the promoter might IGF-I signaling regulate. At differentiation days 3 and 4, IGF-I increased type IIb MyHC mRNA and mouse 3.0-kb type IIb MyHC promoter activity. Deletion construct studies identified a potential IGF-I-responsive region between 1.25 and 1.2 kb of the type IIb MyHC promoter, which contained an exact 6-bp T-cell factor/lymphoid enhancer factor (Tcf/Lef) binding site at position -1206 to -1201. Site-specific mutation of the putative Tcf/Lef binding site reduced IGF-I-induced 1.3-kb type IIb MyHC promoter activity. To identify potential IGF-I signaling molecules, the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY-294002 were both found to markedly attenuate IGF-I activation of the 1.3-kb type IIb MyHC promoter. Downstream signaling of IGF-I can phosphorylate and inactivate GSK-3beta, thereby enhancing beta-catenin protein. The GSK-3beta inhibitor, LiCl, dramatically enhanced IGF-I induction of the 1.3-kb type IIb MyHC promoter, and constitutively active GSK-3beta attenuated IGF-I-induced 1.3-kb type IIb MyHC promoter activity. Finally, IGF-I increased nuclear beta-catenin protein, and small interfering RNA knockdown of beta-catenin attenuated IGF-I-induced 1.3-kb type IIb MyHC promoter activity and type IIb MyHC mRNA. In summary, IGF-I stimulation of C2C12 myotubes increases mouse type IIb MyHC promoter activity, likely through signaling of PI3K, GSK-3beta, beta-catenin, and a Tcf/Lef binding site at -1,206 to -1,201 bp in the promoter.

IIx myosin heavy chain promoter regulation cannot be characterized in vivo by direct gene transfer

In skeletal muscle of the adult mammal IIx is a pivotal myosin heavy chain (MHC) isoform that can be either up- or downregulated depending on both the fiber type of the target muscle and the type of external stimulus imposed. Since little is known about promoter elements of the IIx MHC gene that are important for its transcriptional regulation in vivo,the main goal of this study was to characterize IIx MHC promoter activity and identify potential regulatory elements on the IIx MHC promoter. A direct gene transfer approach was used, and this approach involved transfection of promoter-reporter constructs into intact rat soleus and plantaris muscle under control and denervated conditions, as well as hindlimb suspension (i.e., models to upregulate IIx MHC transcription). Fast-twitch (plantaris) muscle fibers were confirmed to have significantly greater IIx MHC transcriptional products (pre-mRNA and mRNA) than slow-twitch (soleus) muscle fibers. However, promoter sequences corresponding to -2671 to +1720, -1000 to +392, and -605/+392 relative to the IIx MHC transcription start site, plus an additional construct ligated to a putative embryonic MHC enhancer, failed to produce a fiber type-specific response that is characteristic of the endogenous IIx MHC promoter. Furthermore, the activity of these promoter constructs did not demonstrate the expected response to denervation or hindlimb suspension (i.e., marked upregulation), despite normal uptake and activity of a coinjected alpha-actin reference promoter. On the basis of these findings with IIx MHC promoter-reporters we conclude that the loss of the native chromatin environment as well as other necessary cis elements may preclude use of the gene transfer approach, thereby suggesting that there are hidden layers of regulation for the IIx MHC gene.

Type IIx myosin heavy chain promoter activity during hindlimb immobilization

Myosin heavy chain (MyHC) is an important skeletal muscle contractile protein that is often regulated at the transcriptional level. During atrophy, fast IIx MyHC mRNA levels are increased 35 fold in young, predominantly slow-twitch soleus muscle. The purpose of this study was to determine if the 5′-flanking region of the rat MyHC IIx gene contains an atrophy-response element. 1007 base pairs of the 5′-flanking region (−1 kb promoter) of the rat MyHC IIx gene were cloned, and deletion mutations were made. Intramuscular injections of plasmid DNA containing the firefly luciferase gene under control of the rat MyHC IIx promoter were made into the soleus of 2-mo-old rats undergoing normal cage activity or 6 days of hindlimb immobilization (HI). Aliquots of soleus muscle homogenates were used for firefly luciferase activity and plasmid DNA isolation for normalization (using real-time PCR with primers and probe designed against the firefly luciferase gene). The −1 and −0.889 kb promoter activities were 1.46-fold and 1.3-fold higher, respectively, in HI soleus compared to control. Deletion to −0.754 and −0.617 kb decreased promoter activity 70% and 30%, respectively, in HI soleus compared to control. In summary, the −1 kb MyHC IIx promoter is responsive to HI, and deletional analyses suggest that the MyHC IIx promoter potentially contains an atrophy-response element between −0.889 and −0.754 kb. Sponsored by the National Institutes of Health: 5F32AR051640-02 (RAS).

CHF1/Hey2 suppresses SM-MHC promoter activity through an interaction with GATA-6

The bHLH transcription factor CHF1/Hey2 has been previously shown to regulate neointimal formation after vascular injury, but the mechanisms have not been fully elucidated. The zinc-finger protein GATA-6 has also been shown to regulate vascular smooth-muscle phenotype through regulation of smooth-muscle contractile protein gene expression. To address the potential mechanisms by which CHF1/Hey2 regulates vascular smooth-muscle phenotype switching, we investigated the effect of CHF1/Hey2 on GATA-6-dependent smooth-muscle myosin heavy chain promoter activity. When cotransfected into NIH3T3 cells, CHF1/Hey2 reduced GATA-6-dependent activation of the promoter by 90%. Exogenous p300 was not sufficient to overcome this repression effect, demonstrating that the inhibitor effect did not involve coactivation by p300. Coimmunoprecipitation studies demonstrated that CHF1/Hey2 interacts directly with GATA-6. Mutational analysis demonstrated that the bHLH domain is required for transcriptional repression. Our findings highlight an important transcriptional mechanism by which CHF1/Hey2 may affect smooth-muscle cell phenotype.

A G/C element mediates repression of the SM22alpha promoter within phenotypically modulated smooth muscle cells in experimental atherosclerosis.

A hallmark of smooth muscle cell (SMC) phenotypic switching in atherosclerotic lesions is suppression of SMC differentiation marker gene expression. Yet little is known regarding the molecular mechanisms that control this process. Here we show that transcription of the SMC differentiation marker gene SM22alpha is reduced in atherosclerotic lesions and identify a cis regulatory element in the SM22alpha promoter required for this process. Transgenic mice carrying the SM22alpha promoter-beta-galactosidase (beta-gal) reporter transgene were crossed to apolipoprotein E (ApoE)-/- mice. Cells of the fibrous cap, intima, and underlying media showed complete loss of beta-gal activity in advanced atherosclerotic lesions. Of major significance, mutation of a G/C-rich cis element in the SM22alpha promoter prevented the decrease in SM22alpha promoter-beta-gal reporter transgene expression, including in cells that compose the fibrous cap of the lesion and in medial cells in proximity to the lesion. To begin to assess mechanisms whereby the G/C repressor element mediates suppression of SM22alpha in atherosclerosis, we tested the hypothesis that effects may be mediated by platelet-derived growth factor (PDGF)-BB-induced increases in the G/C binding transcription factor Sp1. Consistent with this hypothesis, results of studies in cultured SMCs showed that: (1) PDGF-BB increased expression of Sp1; (2) PDGF-BB and Sp1 profoundly suppressed SM22alpha promoter activity as well as smooth muscle myosin heavy chain promoter activity through mechanisms that were at least partially dependent on the G/C cis element; and (3) a short interfering RNA to Sp1 increased basal expression and attenuated PDGF-BB induced suppression of SM22alpha. Together, these results support a model whereby a G/C repressor element within the SM22alpha promoter mediates transcriptional repression of this gene within phenotypically modulated SMCs in experimental atherosclerosis and provide indirect evidence implicating PDGF-BB and Sp1 as possible mediators of these effects.

Phosphatase 2 A represses the human alpha myosin heavy chain promoter activity

Phosphatases are an important component of the signal transduction pathway. Recently, various studies have shown that phophatase (e.g. Map Kinase Phosphatases 1 and 2 (MKP 1 and 2), calcineurin, PP1 and PP2A) levels and/or activity are up-regulated in the failing heart. These various phophatases are implicated in a variety of cellular and molecular processes. Heart failure is also characterized by changes in the expression levels of many of the components of the contractile apparatus. Of the changes that are observed in failing hearts, increases in b-MyHC, skeletal a-actin, and ANF, with coordinate decreases in a-MyHC and SRCA2a, termed fetal gene induction, are perhaps the most well-known. Changes in MyHC are likely to play an important role in the contractile dysfunction observed in the failing heart. Recent experiments have shown that in single cell experiments, cardiac myocytes expressing only 12% of their MyHC as alpha have 52% greater power output than those expressing only beta. More recently, findings that a mutation in the aMyHC gene can cause either hypertrophic or dilated cardiomyopathies in humans, emphasize the importance of the regulation of aMyHC mRNA and protein expression that is observed in the human failing heart. Recent findings from our laboratory suggest that PP2A mRNA levels are increased in the human failing heart. In order to understand the significance of this increase we sought to determine the regulation of the fetal gene program in response to PP2A. At low concentrations Okadaic Acid is a specific inhibitor of PP2A. Here we report that aMyHC promoter activity is up-regulated in Okadaic Acid-treated neonate rat ventricular myocytes (NRVMs) and over expression of a wild type PP2A construct results in repression of aMyHC promoter activity. Moreover, over expression of a dominant negative PP2A construct dramatically up-regulates aMyHC promoter activity. Our results suggest that PP2A plays an important role in mediating aMyHC expression levels, and that its up-regulation in the failing heart is likely to have a dramatic effect on the regulation of the fetal gene program. Future studies are needed to determine other proteins involved in the PP2A regulation of the fetal gene program.

Activator effect of coinjected enhancers on the muscle‐specific expression of promoters in zebrafish embryos

The transient expression of reporter gene constructs in embryos provides a powerful tool to characterise cis-acting transcriptional elements of the genes involved in development. In the present study, we have analysed the expression pattern of several muscle-specific and ubiquitous regulatory sequences in microinjected zebrafish embryos. By using a fast and reproducible coinjection strategy, the mosaic expression of lacZ reporter gene was monitored in wholemount embryos injected with sequences containing putative enhancer elements and a carp myosin heavy chain promoter/lacZ reporter construct. We have found that a 0.9-kb myosin heavy chain (MyHC) proximal promoter containing several putative myogenic regulatory factors (MRF) binding sites is sufficient to restrict lacZ expression to the skeletal muscle fibres of prim-6 stage zebrafish embryos. Expression of a rat-derived foetal myosin light chain enhancer (MyLC) and different fragments of a carp β-actin regulatory region together with the MyHC promoter were compared by accumulating the type, number and spatial distribution of β-galactosidase-expressing cells on an expression map. β-galactosidase activity increased similarly whether the MyLC enhancer was ligated to the promoter/reporter construct directly or when coinjected as a separate fragment whilst skeletal muscle specificity was retained. The coinjection of two different forms of the β-actin regulatory elements also showed a marked effect on the MyHC promoter activity. The coinjection of putative enhancers with minimal promoter constructs and subsequent analysis of the transient expression pattern in the developing embryos provides a rapid and simple technique to identify cis acting activator elements of genes expressed in the vertebrate embryo. Mol. Reprod. Dev. 47:404–412, 1997. © 1997 Wiley-Liss, Inc.

Activator effect of coinjected enhancers on the muscle-specific expression of promoters in zebrafish embryos.

The transient expression of reporter gene constructs in embryos provides a powerful tool to characterise cis-acting transcriptional elements of the genes involved in development. In the present study, we have analysed the expression pattern of several muscle-specific and ubiquitous regulatory sequences in microinjected zebrafish embryos. By using a fast and reproducible coinjection strategy, the mosaic expression of lacZ reporter gene was monitored in wholemount embryos injected with sequences containing putative enhancer elements and a carp myosin heavy chain promoter/lacZ reporter construct. We have found that a 0.9-kb myosin heavy chain (MyHC) proximal promoter containing several putative myogenic regulatory factors (MRF) binding sites is sufficient to restrict lacZ expression to the skeletal muscle fibres of prim-6 stage zebrafish embryos. Expression of a rat-derived foetal myosin light chain enhancer (MyLC) and different fragments of a carp beta-actin regulatory region together with the MyHC promoter were compared by accumulating the type, number and spatial distribution of beta-galactosidase-expressing cells on an expression map. beta-galactosidase activity increased similarly whether the MyLC enhancer was ligated to the promoter/ reporter construct directly or when coinjected as a separate fragment whilst skeletal muscle specificity was retained. The coinjection of two different forms of the beta-actin regulatory elements also showed a marked effect on the MyHC promoter activity. The coinjection of putative enhancers with minimal promoter constructs and subsequent analysis of the transient expression pattern in the developing embryos provides a rapid and simple technique to identify cis acting activator elements of genes expressed in the vertebrate embryo.