MEF2 Site Research Articles

A non-coding SCN5A variant that reduces sodium channel function in hiPSC-derived cardiomyocytes is significantly enriched in Brugada syndrome patients from Thailand

Abstract Background/Introduction Brugada syndrome (BrS) is an inherited arrhythmia condition that can cause sudden cardiac death. Rare coding and common non-coding genetic variation in the Nav1.5 cardiac sodium channel-encoding SCN5A gene is robustly associated with BrS, but the role of rare and low frequency non-coding variants remains unexplored. BrS is several times more prevalent in Southeast Asia compared to other populations, indicating ancestry-specific genetic risk factors remain to be discovered. Purpose To identify and characterise novel genetic risk factors in BrS patients from Southeast Asia. Methods We performed genome sequencing in 231 BrS probands from Thailand and 500 population-matched controls to identify novel disease associated variants across the entire SCN5A locus. The candidate variant was engineered into human induced pluripotent stem cells (hiPSCs) by CRISPR-Cas9 and its effect was evaluated by single cell electrophysiology analysis on hiPSC-derived cardiomyocytes. Results We identified a rare (absent in gnomAD), non-coding variant in a regulatory element of the SCN5A gene (GRCh38:3-38580380-A-C) that was significantly enriched in cases (3.9%) vs controls (0.2%) (OR=20.2[2.5-160.6], p=2e-04). Transcription factor motif scanning analysis suggested the variant is likely to disrupt a Mef2 site that is conserved across species (Figure 1) – the MEF2 family of transcription factors play a critical role in cardiac transcriptional regulation. In hiPSC-derived cardiomyocytes, a significant reduction of peak Nav1.5-mediated sodium-current (INa) density (30% decrease at -20mV, p=8e-03), without changes in gating properties, was observed in cardiomyocytes carrying the variant in heterozygosity compared to isogenic controls (Figure 2). The variant also significantly reduced luciferase activity of the candidate regulatory element in HEK cells in the presence of cardiac transcription factors (Tbx5, Gata4, Mef2A, Mef2D). Conclusion A novel, non-coding variant in an SCN5A enhancer region is associated with BrS and was functionally validated using single cell electrophysiology in hiPSC-derived cardiomyocytes. This variant is found in ∼1 in every 25 BrS patients from Thailand and therefore may at least partially explain the increased prevalence of BrS in this region. Our study highlights the importance of research in understudied populations to understand the genetic aetiology of disease in diverse ancestries and to identify novel disease risk factors.

Cooperative activation of cardiac transcription through myocardin bridging of paired MEF2 sites.

Enhancers frequently contain multiple binding sites for the same transcription factor. These homotypic binding sites often exhibit synergy, whereby the transcriptional output from two or more binding sites is greater than the sum of the contributions of the individual binding sites alone. Although this phenomenon is frequently observed, the mechanistic basis for homotypic binding site synergy is poorly understood. Here, we identify a bona fide cardiac-specific Prkaa2 enhancer that is synergistically activated by homotypic MEF2 binding sites. We show that two MEF2 sites in the enhancer function cooperatively due to bridging of the MEF2C-bound sites by the SAP domain-containing co-activator protein myocardin, and we show that paired sites buffer the enhancer from integration site-dependent effects on transcription in vivo Paired MEF2 sites are prevalent in cardiac enhancers, suggesting that this might be a common mechanism underlying synergy in the control of cardiac gene expression in vivo.

HDAC Inhibitor Sodium Butyrate Augments the MEF2C Enhancement of Nampt Expression under Hypoxia

Nicotinamide phosphoribosyl transferase (Nampt) is the rate-limiting enzyme for the salvage biosynthesis of nicotinamide adenine dinucleotide (NAD). Although elevated level of Nampt expression has been observed in various cancers, the involvement of Nampt promoter regulation was not well understood. We have identified a cluster of MEF2 recognition sites upstream of the functional hypoxia response elements (HREs) within the human Nampt promoter, and demonstrated that the two MEF2 sites at -1272 and -1200 were functional to upregulate the promoter activity by luciferase reporter assays. The Nampt promoter was able to be activated cooperatively following hypoxic stimulation by CoCl₂ treatment with associated MEF2C overexpression. During the investigation on MEF2C regulation of endogenous Nampt expression in HeLa cells, the most significant enhancement of Nampt expression observed was by overexpression of MEF2C in combination with sodium butyrate exposure. By chromatin immunoprecipitation with a MEF2C anti-body, we found that MEF2C indeed interacted with endogenous Nampt promoter. The requirement of HDAC inhibition for the MEF2C enhancement of Nampt transcription was verified by RNAi of HDAC. Our results were in support of reports indicating that MEF2 family transcription factors interacted with HDACs and regulated downstream gene expression at the epigenetic levels. Our study provided important evidence to demonstrate the sophisticated mechanism of endogenous Nampt promoter regulation, and therefore, will help to better understand the Nampt overexpression in cancer progression, especially in the context of MEF2C upregulation which frequently occurred in cancer development and drug resistance.

Contribution of Myocyte Enhancer Factor 2 Family Transcription Factors to BZLF1 Expression in Epstein-Barr Virus Reactivation from Latency

Reactivation of Epstein-Barr virus (EBV) from latency is dependent on expression of the viral transactivator BZLF1 protein, whose promoter (Zp) normally exhibits only low basal activity but is activated in response to chemical or biological inducers. Using a reporter assay system, we screened for factors that can activate Zp and isolated genes, including those encoding MEF2B, KLF4, and some cellular b-Zip family transcription factors. After confirming their importance and functional binding sites in reporter assays, we prepared recombinant EBV-BAC, in which the binding sites were mutated. Interestingly, the MEF2 mutant virus produced very low levels of BRLF1, another transactivator of EBV, in addition to BZLF1 in HEK293 cells. The virus failed to induce a subset of early genes, such as that encoding BALF5, upon lytic induction, and accordingly, could not replicate to produce progeny viruses in HEK293 cells, but this restriction could be completely lifted by exogenous supply of BRLF1, together with BZLF1. In B cells, induction of BZLF1 by chemical inducers was inhibited by point mutations in the ZII or the three SP1/KLF binding sites of EBV-BAC Zp, while leaky BZLF1 expression was less affected. Mutation of MEF2 sites severely impaired both spontaneous and induced expression of not only BZLF1, but also BRLF1 in comparison to wild-type or revertant virus cases. We also observed that MEF2 mutant EBV featured relatively high repressive histone methylation, such as H3K27me3, but CpG DNA methylation levels were comparable around Zp and the BRLF1 promoter (Rp). These findings shed light on BZLF1 expression and EBV reactivation from latency.

Differentiation and fiber type-specific activity of a muscle creatine kinase intronic enhancer

BackgroundHundreds of genes, including muscle creatine kinase (MCK), are differentially expressed in fast- and slow-twitch muscle fibers, but the fiber type-specific regulatory mechanisms are not well understood.ResultsModulatory region 1 (MR1) is a 1-kb regulatory region within MCK intron 1 that is highly active in terminally differentiating skeletal myocytes in vitro. A MCK small intronic enhancer (MCK-SIE) containing a paired E-box/myocyte enhancer factor 2 (MEF2) regulatory motif resides within MR1. The SIE's transcriptional activity equals that of the extensively characterized 206-bp MCK 5'-enhancer, but the MCK-SIE is flanked by regions that can repress its activity via the individual and combined effects of about 15 different but highly conserved 9- to 24-bp sequences. ChIP and ChIP-Seq analyses indicate that the SIE and the MCK 5'-enhancer are occupied by MyoD, myogenin and MEF2. Many other E-boxes located within or immediately adjacent to intron 1 are not occupied by MyoD or myogenin. Transgenic analysis of a 6.5-kb MCK genomic fragment containing the 5'-enhancer and proximal promoter plus the 3.2-kb intron 1, with and without MR1, indicates that MR1 is critical for MCK expression in slow- and intermediate-twitch muscle fibers (types I and IIa, respectively), but is not required for expression in fast-twitch muscle fibers (types IIb and IId).ConclusionsIn this study, we discovered that MR1 is critical for MCK expression in slow- and intermediate-twitch muscle fibers and that MR1's positive transcriptional activity depends on a paired E-box MEF2 site motif within a SIE. This is the first study to delineate the DNA controls for MCK expression in different skeletal muscle fiber types.

The MADS box transcription factor MEF2C regulates melanocyte development and is a direct transcriptional target and partner of SOX10.

Waardenburg syndromes are characterized by pigmentation and autosensory hearing defects, and mutations in genes encoding transcription factors that control neural crest specification and differentiation are often associated with Waardenburg and related disorders. For example, mutations in SOX10 result in a severe form of Waardenburg syndrome, Type IV, also known as Waardenburg-Hirschsprung disease, characterized by pigmentation and other neural crest defects, including defective innervation of the gut. SOX10 controls neural crest development through interactions with other transcription factors. The MADS box transcription factor MEF2C is an important regulator of brain, skeleton, lymphocyte and cardiovascular development and is required in the neural crest for craniofacial development. Here, we establish a novel role for MEF2C in melanocyte development. Inactivation of Mef2c in the neural crest of mice results in reduced expression of melanocyte genes during development and a significant loss of pigmentation at birth due to defective differentiation and reduced abundance of melanocytes. We identify a transcriptional enhancer of Mef2c that directs expression to the neural crest and its derivatives, including melanocytes, in transgenic mouse embryos. This novel Mef2c neural crest enhancer contains three functional SOX binding sites and a single essential MEF2 site. We demonstrate that Mef2c is a direct transcriptional target of SOX10 and MEF2 via this evolutionarily conserved enhancer. Furthermore, we show that SOX10 and MEF2C physically interact and function cooperatively to activate the Mef2c gene in a feed-forward transcriptional circuit, suggesting that MEF2C might serve as a potentiator of the transcriptional pathways affected in Waardenburg syndromes.

Conserved proximal promoter elements control repulsive guidance molecule c/hemojuvelin (Hfe2) gene transcription in skeletal muscle

Repulsive guidance molecule c (RGMc; gene symbol: Hfe2) plays a critical role in iron metabolism. Inactivating mutations cause juvenile hemochromatosis, a severe iron overload disorder. Understanding mechanisms controlling RGMc biosynthesis has been hampered by minimal information about the RGMc gene. Here we define the structure, examine the evolution, and establish mechanisms of regulation of the mouse RGMc gene. RGMc is a 4-exon gene that undergoes alternative RNA splicing to yield 3 mRNAs with 5′ different untranslated regions. Gene transcription is induced during myoblast differentiation, producing all 3 mRNAs. We identify 3 critical promoter elements responsible for transcriptional activation in skeletal muscle, comprising paired E-boxes, a putative Stat and/or Ets element, and a MEF2 site, and muscle transcription factors myogenin and MEF2C stimulate RGMc promoter function in non-muscle cells. As these elements are conserved in RGMc genes from multiple species, our results suggest that RGMc has been a muscle-enriched gene throughout its evolutionary history.

Interplay between DNA Methylation and Transcription Factor Availability: Implications for Developmental Activation of the Mouse Myogenin Gene

During development, gene activation is stringently regulated to restrict expression only to the correct cell type and correct developmental stage. Here, we present mechanistic evidence that suggests DNA methylation contributes to this regulation by suppressing premature gene activation. Using the mouse Myogenin promoter as an example of the weak CpG island class of promoters, we find that it is initially methylated but becomes demethylated as development proceeds. Full hypersensitive site formation of the Myogenin promoter requires both the MEF2 and SIX binding sites, but binding to only one site can trigger the partial chromatin opening of the nonmethylated promoter. DNA methylation markedly decreases hypersensitive site formation that now occurs at a detectable level only when binding to both MEF2 and SIX binding sites is possible. This suggests that the probability of activating the methylated promoter is low until two of the factors are coexpressed within the same cell. Consistent with this, the single-cell analysis of developing somites shows that the coexpression of MEF2A and SIX1, which bind the MEF2 and SIX sites, correlates with the fraction of cells that demethylate the Myogenin promoter. Taken together, these studies imply that DNA methylation helps to prevent inappropriate gene activation until sufficient activating factors are coexpressed.

TRIM72, a novel negative feedback regulator of myogenesis, is transcriptionally activated by the synergism of MyoD (or myogenin) and MEF2

TRIM72 is known to be involved in the negative feedback regulation of myogenesis by targeting insulin receptor substrate-1. Here, we found that TRIM72 was more highly expressed in oxidative muscle with the higher activity of MEF2, compared to glycolytic muscle. Indeed, TRIM72 promoter contained an evolutionarily conserved MEF2 site juxtaposed to E-box. TRIM72 promoter activity was decreased by the site-directed mutagenesis of either E-boxes or a MEF2 site and synergistically enhanced by MyoD (or myogenin) and MEF2, which were associated with proximal E-box, and MEF2 site of the TRIM72 promoter, respectively. Taken together all these data, we concluded that the synergism of MyoD (or myogenin) and MEF2 is necessary for TRIM72 expression during C2C12 differentiation.

Transcriptional induction of MMP-10 by TGF-β, mediated by activation of MEF2A and downregulation of class IIa HDACs

Transforming growth factor (TGF)-beta regulates the expression of matrix metalloproteinases (MMPs) and components of the extracellular matrix, thereby profoundly affecting the microenvironment of cells including cancerous ones. We studied MMP-10 induction by TGF-beta in mammary epithelial cells and found that the induction was dependent on the myocyte enhancer factor (MEF)-2 transcription factor. TGF-beta upregulated the gene promoter through the MEF2 site, and knockdown of the MEF2A transcription factor negatively affected MMP-10 induction, whereas its overexpression had a positive effect on the induction. In response to TGF-beta, acetylation and concomitant binding of MEF2A to the promoter region increased, thus suggesting a critical role of MEF2A in transactivation of MMP-10 by TGF-beta. Consistent with the fact that class IIa histone deacetylases (HDACs) interact with MEF2 and suppress transcription, knockdown of HDACs increased and their overexpression inhibited MMP-10 expression. Intriguingly, TGF-beta promoted proteasome-dependent degradation of HDACs. Consistent with this, acetylation of core histones was increased around the MEF2 site of the MMP-10 promoter by TGF-beta and alleviated by overexpression of HDACs. Collectively, it is possible that TGF-beta transcriptionally upregulated MMP-10 through activation of MEF2A, concomitant with acetylation of core histones increasing around the promoter, as a consequence of degradation of the class IIa HDACs.

Secondary enhancers synergise with primary enhancers to guarantee fine-tuned muscle gene expression

Although tight quantitative control of gene expression is required to ensure that organs and tissues function correctly, the transcriptional mechanisms underlying this process still remain poorly understood. Here, we describe novel and evolutionary conserved secondary enhancers that are needed for the regulation of the expression of Troponin I genes. Secondary enhancers are silent when tested individually in electroporated muscles but interact with the primary enhancers and are required to precisely control the appropriate timing, the tissue and fibre specificity, and the quantitative expression of these genes during muscle differentiation. Synergism is completely dependent of the fully conserved MEF2 site present on the primary enhancers core of skeletal muscle Troponin I genes. Thus, while each of these paired enhancers has a different function, the concerted action of both is crucial to recapitulate endogenous gene expression. Through comparative genomics, we predict that this mechanism has also arisen in other mammalian muscle genes. Our results reveal the existence of a novel mechanism, conserved from flies to mammals, to fine-tune gene expression in each muscle and probably other tissues.

CaMK activation during exercise is required for histone hyperacetylation and MEF2A binding at the MEF2 site on theGlut4gene

The role of CaMK II in regulating GLUT4 expression in response to intermittent exercise was investigated. Wistar rats completed 5 x 17-min bouts of swimming after receiving 5 mg/kg KN93 (a CaMK II inhibitor), KN92 (an analog of KN93 that does not inhibit CaMK II), or an equivalent volume of vehicle. Triceps muscles that were harvested at 0, 6, or 18 h postexercise were assayed for 1) CaMK II phosphorylation by Western blot, 2) acetylation of histone H3 at the Glut4 MEF2 site by chromatin immunoprecipitation (ChIP) assay, 3) bound MEF2A at the Glut4 MEF2 cis-element by ChIP, and 4) GLUT4 expression by RT-PCR and Western blot. Compared with controls, exercise caused a twofold increase in CaMK II phosphorylation. Immunohistochemical stains indicated increased CaMK II phosphorylation in nuclear and perinuclear regions of the muscle fiber. Acetylation of histone H3 in the region surrounding the MEF2 binding site on the Glut4 gene and the amount of MEF2A that bind to the site increased approximately twofold postexercise. GLUT4 mRNA and protein increased approximately 2.2- and 1.8-fold, respectively, after exercise. The exercise-induced increases in CaMK II phosphorylation, histone H3 acetylation, MEF2A binding, and GLUT4 expression were attenuated or abolished when KN93 was administered to rats prior to exercise. KN92 did not affect the increases in pCaMK II and GLUT4. These data support the hypothesis that CaMK II activation by exercise increases GLUT4 expression via increased accessibility of MEF2A to its cis-element on the gene.

CaMK activation during exercise is required for histone hyper‐acetylation and MEF2A binding at the MEF2 site on the Glu4 gene.

Regular exercise protects against type II diabetes in part by increasing GLUT4 content. Previous work from our laboratory has established that GLUT4 expression is mediated by CaMK‐dependent binding of MEF2A to its cis element on the GLUT4 promoter. To further investigate how MEF2A binding to the GLUT4 gene is regulated, we injected rats with KN93 to inhibit CaMK II activation and measured; 1) MEF2A binding to, and histone H3 acetylation of, the MEF2 site on the GLUT4 gene by ChIP assay, 2) nuclear HDAC5 and GLUT4 contents by Western blot and 3) GLUT4 mRNA by RT‐PCR after exercise. Exercise increased CaMK II activity ∼50%, H3 acetylation and MEF2A binding to DNA ∼2‐fold and GLUT4 expression significantly. All the above increases by exercise were attenuated by KN93. Our data show that CaMK II activity modulates chromatin structure to increase MEF2A binding to the GLUT4 gene during exercise and suggests that drugs that mimic these effects might be worthy targets for controlling diabetes.

Mef2c is an essential regulatory element required for unique expression of the cardiac‐specific CARK gene

The cardiac ankyrin repeat kinase (CARK) gene, also named TNNI3K for its interaction with cardiac troponin I, is both a unique expression and heart-enriched gene. To understand the mechanisms of CARK gene expression and regulation, we first cloned the full-length mRNA sequence and mapped the transcription start site of the mouse CARK gene and characterized its promoter regions. Two transcriptional isoforms of the CARK gene were identified in mouse heart tissue. Truncation analysis of the CARK promoter identified a minimal 151 bp region that has strong basal transcription activity. Mutational analysis revealed five conserved cis-acting elements in this 151-bp long minimal promoter. Mutational and loss-of-functional analysis and co-transfection studies indicated that MEF2 binding region is the most critical cis-acting element in the CARK promoter, and CARK transcription level can be down-regulated by MEF2C antisense. Binding to the MEF2 sites by Mef2c protein was confirmed by electrophoretic mobility shift assay and competition and supershift electrophoretic mobility shift assays.

Mutant p53 Protects Cells from 12-O-Tetradecanoylphorbol-13-Acetate–Induced Death by Attenuating Activating Transcription Factor 3 Induction

Mutations in p53 are ubiquitous in human tumors. Some p53 mutations not only result in loss of wild-type (WT) activity but also grant additional functions, termed "gain of function." In this study, we explore how the status of p53 affects the immediate response gene activating transcription factor 3 (ATF3) in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-protein kinase C (PKC) pathway. We show that high doses of TPA induce ATF3 in a WT p53-independent manner correlating with PKCs depletion and cell death. We show that cells harboring mutant p53 have attenuated ATF3 induction and are less sensitive to TPA-induced death compared with their p53-null counterparts. Mutagenesis analysis of the ATF3 promoter identified the regulatory motifs cyclic AMP-responsive element binding protein/ATF and MEF2 as being responsible for the TPA-induced activation of ATF3. Moreover, we show that mutant p53 attenuates ATF3 expression by two complementary mechanisms. It interacts with the ATF3 promoter and influences its activity via the MEF2 site, and additionally, it attenuates transcriptional expression of the ATF3 activator MEF2D. These data provide important insights into the molecular mechanisms that underlie mutant p53 gain of function.

Divergent transcriptional enhancer factor-1 regulates the cardiac troponin T promoter

MCAT elements are essential for cardiac gene expression during development. Avian transcriptional enhancer factor-1 (TEF-1) proteins are muscle-enriched and contribute to MCAT binding activities. However, direct activation of MCAT-driven promoters by TEF-1-related proteins has not been uniformly achieved. Divergent TEF (DTEF)-1 is a unique member of the TEF-1 multigene family with abundant transcripts in the heart but not in skeletal muscle. Herein we show that DTEF-1 proteins are highly expressed in the heart. Protein expression is activated at very early stages of chick embryogenesis (Hamburger-Hamilton stage 4, 16-18 h), after which DTEF-1 becomes abundant in the sinus venosus and is expressed in the trabeculated ventricular myocardium and ventricular outflow tracts. By chromatin immunoprecipitation, DTEF-1 interacts with the cardiac troponin T (cTnT) promoter in vivo. DTEF-1 also interacts with MEF- 2 by coimmunoprecipitation and independently or cooperatively (with MEF-2) trans-activates the cTnT promoter. DTEF-1 isoforms do not activate the cTnT promoter in fibroblasts or skeletal muscle. DTEF-1 expression occurs very early in chick embryogenesis (16-18 h), preceding sarcomeric protein expression, and it activates cardiac promoters. As such, DTEF-1 may be an early marker of the myocardial phenotype. DTEF-1 trans-activates the cTnT promoter in a tissue-specific fashion independent of AT-rich, MEF-2, or GATA sites. The observed spatial pattern suggests decreasing levels of expression from the cardiac inlet to the ventricular outflow tracts, which may mark a cardiogenic or differentiation pathway that parallels the direction of flow through the developing chick heart.

Functional interaction between peroxisome proliferator-activated receptors-alpha and Mef-2C on human carnitine palmitoyltransferase 1beta (CPT1beta) gene activation.

Muscle-type carnitine palmitoyltransferase 1 (CPT1beta) is considered to be the gene that controls fatty acid mitochondrial beta-oxidation. A functional peroxisome proliferator-activated receptor (PPAR) responsive element (PPRE) and a myocite-specific (MEF2) site that binds MEF2A and MEF2C in the promoter of this gene had been previously identified. We investigated the roles of the PPRE and the MEF2 binding sites and the potential interaction between PPARalpha and MEF2C regulating the CPT1beta gene promoter. Mutation analysis indicated that the MEF2 site contributed to the activation of the CPT1beta promoter by PPAR in C2C12 cells. The reporter construct containing the PPRE and the MEF2C site was synergistically activated by co-expression of PPAR, retinoid X receptor (RXR) and MEF2C in non-muscle cells. Moreover, protein-binding assays demonstrated that MEF2C and PPAR specifically bound to one another in vitro. Also for the synergistic activation of the CPT1beta gene promoter by MEF2C and PPARalpha-RXRalpha, a precise arrangement of its binding sites was essential.

The MEF2 site is necessary for induction of the myosin light chain 2 slow promoter in overloaded regenerating plantaris muscle

Functional overload (OV) of the rat plantaris muscle results in a fast to slow change in muscle phenotype with induction of the slow contractile protein genes including myosin light chain 2 slow (MLC2s). To identify potential cis-acting DNA sites regulating MLC2s following ablation, plasmid constructs were transfected in vivo into regenerating overloaded plantaris muscles. Activity of the 270bp promoter (−270MLC2s) was increased in OV muscles at 28 days. Mutation of the MEF2 site (−270MEF2) knocked out the overload-induced activity of the promoter. Mutation of the Ebox (−270Ebox) resulted in an earlier induction with OV and mutation of the CACC site (–270CACC) resulted in increased activity in the CON PLN with OV induction detected by 21 days. These results demonstrate that the –270MLC2s promoter contains the elements necessary for expression of MLC2s in regenerating OV PLN. More importantly, mutation analysis of −270MLC2s promoter demonstrates that mechanical loading induced expression shares some common molecular mechanisms with slow nerve dependent model regulation. In these two models of physiological induction of MLC2s, the CACC site acts as a repressor region (on/off switch) and the MEF2 site acts to modulate quantitative expression.

Muscle-specific expression of myogenin in zebrafish embryos is controlled by multiple regulatory elements in the promoter

Myogenin is a member of the basic Helix–Loop–Helix transcription factor family that play key roles in myoblast specification and differentiation. Myogenin is specifically expressed in developing somite and skeletal muscles in zebrafish embryos. To determine the regulation of myogenin expression, we reported here the characterization of zebrafish myogenin gene and analysis of its promoter activity in zebrafish embryos. Our data showed that a 0.8-kb myogenin promoter was sufficient to direct correct temporal and spatial muscle-specific green fluorescence protein expression in zebrafish embryos. Sequence analysis identified two putative E box sites in the myogenin gene promoter. In addition, a MEF2 recognition site and a MEF3 binding site were also found in the promoter. Mutation of the E boxes, MEF2 or MEF3 binding site individually had little effect on the muscle-specificity and activity of the myogenin promoter. However, mutating these sites in various combinations, e.g. E boxes and MEF2 binding site, or MEF2 and MEF3 sites significantly reduced the activity of the promoter. Moreover, mutating the E boxes, MEF2 and MEF3 sites together almost abolished the activity of the promoter. These data indicate that muscle-specific expression of myogenin in zebrafish embryos is controlled by multiple regulatory elements in the promoter. In addition, because these regulatory elements control myogenin expression in mouse and human embryos, these data suggest that the regulatory mechanism controlling myogenin expression might be conserved during evolution.

ATF1 Phosphorylation by the ERK MAPK Pathway Is Required for Epidermal Growth Factor-induced c-jun Expression

Epidermal growth factor induction of c-jun expression requires ATF1 and MEF2 sites in the c-jun promoter. We find that activation of the c-jun promoter through the ATF1 site requires phosphorylation of ATF1 at serine 63. A serine 63 to alanine mutation of ATF1 acts to block epidermal growth factor (EGF) induction of a transfected c-jun gene. ATF1 can be phosphorylated by mitogen- and stress-activated protein kinase 1 (MSK1), which is activated by EGF and ERK1/2. Kinase-dead MSK1 mutants blocked EGF induction of a transfected c-jun gene suggesting that MSK1 or a similar family member is required for induced c-jun expression. Use of the MEK1 inhibitor U0126 and dominant negative MEK1 further showed that MSK1 activation and c-jun induction require the ERK pathway. In contrast, a JNK inhibitor blocked EGF induction of c-jun expression but not ATF1 phosphorylation. These results show that the two MAPK pathways, ERK and JNK, are required for EGF-induced c-jun expression and that the ERK pathway acts through downstream phosphorylation of ATF1.