RhoA GTPase and Serum Response Factor Control Selectively the Expression of MyoD without Affecting Myf5 in Mouse Myoblasts

MyoD and Myf5 belong to the family of basic helix-loop-helix transcription factors that are key operators in skeletal muscle differentiation. MyoD and Myf5 genes are selectively activated during development in a time and region-specific manner and in response to different stimuli. However, molecules that specifically regulate the expression of these two genes and the pathways involved remain to be determined. We have recently shown that the serum response factor (SRF), a transcription factor involved in activation of both mitogenic response and muscle differentiation, is required for MyoD gene expression. We have investigated here whether SRF is also involved in the control of Myf5 gene expression, and the potential role of upstream regulators of SRF activity, the Rho family G-proteins including Rho, Rac, and CDC42, in the regulation of MyoD and Myf5. We show that inactivation of SRF does not alter Myf5 gene expression, whereas it causes a rapid extinction of MyoD gene expression. Furthermore, we show that RhoA, but not Rac or CDC42, is also required for the expression of MyoD. Indeed, blocking the activity of G-proteins using the general inhibitor lovastatin, or more specific antagonists of Rho proteins such as C3-transferase or dominant negative RhoA protein, resulted in a dramatic decrease of MyoD protein levels and promoter activity without any effects on Myf5 expression. We further show that RhoA-dependent transcriptional activation required functional SRF in C2 muscle cells. These data illustrate that MyoD and Myf5 are regulated by different upstream activation pathways in which MyoD expression is specifically modulated by a RhoA/SRF signaling cascade. In addition, our results establish the first link between RhoA protein activity and the expression of a key muscle regulator.

Characterization of muscle-regulatory gene, MyoD, from flounder (Paralichthys olivaceus) and analysis of its expression patterns during embryogenesis

Specification and differentiation of skeletal muscle cells are driven by the activity of genes encoding members of the myogenic regulatory factors (MRFs). In vertebrates, the MRF family includes MyoD, Myf5, myogenin, and MRF4. The MRFs are capable of converting a variety of nonmuscle cells into myoblasts and myotubes. To better understand their roles in fish muscle development, we isolated the MyoD gene from flounder (Paralichthys olivaceus) and analyzed its structure and patterns of expression. Sequence analysis showed that flounder MyoD shared a structure similar to that of vertebrate MRFs with three exons and two introns, and its protein contained a highly conserved basic helix-loop-helix domain (bHLH). Comparison of sequences revealed that flounder MyoD was highly conserved with other fish MyoD genes. Sequence alignment and phylogenetic analysis indicated that flounder MyoD, seabream (Sparus aurata) MyoD1, takifugu (Takifugu rubripes) MyoD, and tilapia (Oreochromis aureus) MyoD were more likely to be homologous genes. Flounder MyoD expression was first detected as two rows of presomitic cells in the segmental plate. From somitogenesis, MyoD transcripts were present in the adaxial cells that give rise to slow muscles and the lateral somitic cells that give rise to fast muscles. After 30 somites formed, MyoD expression decreased in the somites except the caudal somites, coincident with somite maturation. In the hatching stage, MyoD was expressed in other muscle cells and caudal somites. It was detected only in muscle in the growing fish.

牙鲆肌肉发育调节基因的克隆、表达及功能分析

本文克隆了牙鲆的成肌因子MyoD和Myf5，以及牙鲆的Forkhead基因FoxD1、 FoxD3和FoxD5，并对其在牙鲆肌肉发育中的功能进行了分析。 牙鲆MyoD和Myf5基因都具有三个外显子，两个内含子。其编码的氨基酸序列都含有保守的bHLH；牙鲆FoxD1，FoxD3，FoxD5基因都只有一个外显子，编码的氨基酸序列都含有保守的翼状螺旋DNA结合结构域。 在胚胎发育早期，Myf5在近轴中胚层中表达，体节发生过程中，Myf5在体节中表达，MyoD基因最早在分节板的体节前细胞中表达，随后在近轴细胞、体节中表达；随着胚胎的发育，Myf5在成熟体节中表达量降低，在新生体节中表达较强；MyoD自30个体节时期后只在新生的尾部体节中表达，在成熟的体节中表达量降低；在孵化期，MyoD和 Myf5在头部及鳍的肌肉、尾部的体节中表达；生长期的牙鲆中，Myf5在骨骼肌和肠中表达，成体牙鲆中，Myf5只在肌肉中表达；生长期的牙鲆及成体牙鲆中，MyoD只在肌肉组织中表达。 牙鲆MyoD和Myf5的启动子可以驱动绿色荧光蛋白在斑马鱼肌肉纤维中表达，其包含了这两个基因正常表达所需的核心区域，并可以跨物种行使功能。 在胚胎发育早期，FoxD3在未迁移神经嵴前体细胞、体节、耳后的基板、头部和躯干的神经嵴细胞、松果体中表达。牙鲆FoxD1主要在脑，体节，肾脏及肠中表达。牙鲆FoxD5主要在体节、尾芽、前脑、耳泡中表达。 在斑马鱼中过量表达牙鲆FoxD3，并与斑马鱼的同源基因进行比较，结果表明注射牙鲆和斑马鱼FoxD3的斑马鱼胚胎表型一致，它们在中轴两侧的发育出现了不同步现象，MyoD和Myf5在近轴中胚层中的表达受到不同程度抑制。因此，FoxD3在不同物种之间保守，并且FoxD3在肌肉发育的调控通路中可能通过与MyoD和Myf5相互作用而行使功能。 在斑马鱼中过量表达FoxD1后，MyoD在一侧体节中的表达受到了严重抑制，而在近轴细胞中的表达未受影响，Myf5在体节前中胚层，近轴细胞，及体节中的表达都受到了抑制。FoxD1在胚胎发育的早期可能通过调控MyoD和Myf5的表达而参与肌肉发育的调控。 将牙鲆FoxD5在斑马鱼中过量表达，MyoD在一侧体节中的表达量有所升高，而在近轴细胞中的表达未受影响；Myf5在一侧体节和体节前中胚层中的表达也有所升高，表明牙鲆FoxD5可以调控肌肉调节因子MyoD和Myf5的表达而参与早期肌肉发育的调控。

牙鲆成肌因子myogenin和MRF4的研究

本文从牙鲆中克隆到其肌肉调节因子myogenin和MRF4的基因，并对它们的表达、启动子活性及功能进行分析。 研究结果表明牙鲆myogenin和MRF4都由三个外显子和两个内含子组成，其cDNA编码的氨基酸序列分别与几种鱼类的同源基因亲缘关系较近。 原位杂交显示最早在胚胎5-6个体节时体节中部的细胞检测到myogenin的表达，随着发育的进行，它的表达向体节两侧和体后延伸，最后在整个体节表达。当发育到30个体节的时候，在躯干的表达迅速下降，只在胚胎的尾部能检测到信号。 RT-PCR的结果显示，MyoD和Myf5的表达要早于myogenin，而MRF4的表达晚于myogenin，并且myogenin、MRF4只在成鱼的肌肉中表达，这进一步证明其在肌肉发育过程中的作用。 myogenin和MRF4的启动子中含有保守的肌肉特异调控序列，瞬时表达分析的结果证实两段启动子序列足以驱动GFP在斑马鱼胚胎肌肉中的特异表达。首次对鱼类MRF4启动子进行了调控序列的分析，发现在启动子的-181到-506的序列中含有调节MRF4表达的必需位点。 首次应用原核表达系统对MRF4蛋白进行重组表达，并利用纯化的重组蛋白制备多克隆抗体，通过western杂交分析显示所得到的MRF4多克隆抗体能够识别牙鲆内源性的MRF4蛋白。

斑马鱼肌肉发育相关基因克隆、表达与功能分析

本文主要研究在斑马鱼胚胎发育过程中参与肌肉发育的相关基因，克隆了四个在体节和肌肉中表达的基因全长，分析了基因的时空表达特征，并对其中两个基因进行了过表达，分析其在体节形成和肌肉发生过程中的功能。 从斑马鱼中克隆到甲状腺激素受体相关蛋白基因TRAP150。原位杂交分析TRAP150表达在近轴细胞的慢肌和体节的快肌，表达模式与MyoD的模式相近；并表现出肌肉特异性表达，TRAP150在体节形成和肌肉发生早期高水平说明TRAP150在肌肉分化过程发挥着重要的作用。此外，在胚胎的心脏中也检测到TRAP150的表达。在斑马鱼胚胎过量表达TRAP150造成MyoD在近轴中胚层的过量表达，而对MyoD在近轴细胞的表达影响不明显；由于MyoD在近轴中胚层的表达将诱导快肌的形成，因此过量表达TRAP150将可能导致快肌的增多；从过量表达的结果分析，TRAP150在MyoD的上游正向调控MyoD的表达，是诱导快肌的分化的重要基因。 从斑马鱼中克隆到双特性酪氨酸调控激酶新基因DYRK2。RT-PCR结果表明DYRK2具有母源性表达的特点，并且在36小时前各个时期都有表达。DYRK2在体节形成后表达在体节的近轴细胞中，大约在15个体节时检测到在快肌部位表达，18小时后在胚胎肌肉组织、脑部以及眼睛表达。DYRK2在斑马鱼胚胎发育过程中与体节和肌肉发生相关的基因之一，并可能参与脑和眼睛的发生。在斑马鱼胚胎中过量表达DYRK2导致肌肉标记基因MyoD的表达出现了很大的变化，注射侧MyoD在近轴细胞和近轴中胚层过量表达，尤其是在未形成体节的体节前体中胚层，注射侧的MyoD有大范围的高表达，而在正常一侧没有表达。MyoD的过量表达说明斑马鱼胚胎早期DYRK2通过调控MyoD的表达影响慢肌的分化。 克隆得到斑马鱼的血细胞生成的PBX1互作蛋白基因HPIP1。RT-PCR结果表明HPIP1在斑马鱼胚胎表达具有母源性，但是原位杂交的检测一直到10个体节时才检测到，说明HPIP1一直到肌肉分化后才大量表达，可能在肌肉的成熟阶段起作用。当胚胎发育到18小时，HPIP1表达在所有体节中，前端的较早形成的体节中表达量比晚形成的体节表达量高，也符合HPIP1参与肌肉成熟过程的判断。HPIP1还表达在胚胎的眼部周围，说明HPIP1可能参与到眼部肌肉的形成。 在斑马鱼中克隆的Chp-1相似蛋白基因CHORDC1。在3个体节时，CHORDC1表达在脊索两侧的近轴细胞中，而在5个体节时CHORDC1在表达在体节中，这个特点与MyoD的表达很相似，与MyoD在体节中表达不同的是，CHORDC1也在体节前体中胚层中表达，这与Myf5的表达特点相似，CHORDC1紧随着MyoD，Myf5的高表达说明CHORDC1在肌肉细胞的分化的早期即参与肌肉的发育，而其高表达量也说明CHORDC1在这个过程中可能起到非常重要的作用。CHORDC1在近轴中胚层的表达与MyoD，Myf5有不同的特点，这种不同表现在其在近轴中胚层的表达不仅仅限于快肌和慢肌，而且由后到前的逐渐扩展。而且，CHORDC1在心肌中也表达说明其不仅在骨骼肌中发挥作用。综合CHORDC1的表达特点可以认为其对肌肉的作用不限于特定肌肉类型，广泛参与到各种肌肉的发育过程

The origin, molecular regulation and therapeutic potential of myogenic stem cell populations

Satellite cells, originating in the embryonic dermamyotome, reside beneath the myofibre of mature adult skeletal muscle and constitute the tissue-specific stem cell population. Recent advances following the identification of markers for these cells (including Pax7, Myf5, c-Met and CD34) (CD, cluster of differentiation; c-Met, mesenchymal epithelial transition factor) have led to a greater understanding of the role played by satellite cells in the regeneration of new skeletal muscle during growth and following injury. In response to muscle damage, satellite cells harbour the ability both to form myogenic precursors and to self-renew to repopulate the stem cell niche following myofibre damage. More recently, other stem cell populations including bone marrow stem cells, skeletal muscle side population cells and mesoangioblasts have also been shown to have myogenic potential in culture, and to be able to form skeletal muscle myofibres in vivo and engraft into the satellite cell niche. These cell types, along with satellite cells, have shown potential when used as a therapy for skeletal muscle wasting disorders where the intrinsic stem cell population is genetically unable to repair non-functioning muscle tissue. Accurate understanding of the mechanisms controlling satellite cell lineage progression and self-renewal as well as the recruitment of other stem cell types towards the myogenic lineage is crucial if we are to exploit the power of these cells in combating myopathic conditions. Here we highlight the origin, molecular regulation and therapeutic potential of all the major cell types capable of undergoing myogenic differentiation and discuss their potential therapeutic application.

Candidate SNPs for carcass and meat traits in Nelore animals and in their crosses with Bos taurus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mecanismos celulares e moleculares que controlam o desenvolvimento e o crescimento muscular

O músculo estriado esquelético é formado pela associação de fibras musculares com a matriz extracelular. Esse tecido possui alta plasticidade e o conhecimento das características morfológicas, da miogênese, e da dinâmica do crescimento é importante para o entendimento da morfofisiologia bem como para a seleção de animais visando a melhoria na produção de carne. A maioria dos músculos estriados originam-se de células precursoras do mesoderma a partir dos somitos do embrião e o controle da diferenciação ocorre pela ação de fatores indutores ou inibidores. Um grupo de fatores transcricionais, pertencentes à família MyoD tem um papel central na diferenciação muscular. Coletivamente chamados de Fatores de Regulação Miogênica (MRFs), são conhecidos quatro tipos: MyoD, myf-5, miogenina e MRF4. Esses fatores ligam-se à seqüências de DNA conhecidas como Ebox (CANNTG) na região promotora de vários genes músculo-específicos, levando à expressão dos mesmos. As células embrionárias com potencial para diferenciação em células musculares (células precursoras miogênicas) expressam MyoD e Myf-5 e são denominadas de mioblastos. Essas células proliferam, saem do ciclo celular, expressam miogenina e MRF4, que regulam a fusão e a diferenciação da fibra muscular. Uma população de mioblastos que se diferencia mais tardiamente, as células miossatélites, são responsáveis pelo crescimento muscular no período pós natal, que pode ocorrer por hiperplasia e hipertrofia das fibras. As células satélites quiescentes não expressam os MRFs, porém, sob a ação de estímulos como fatores de crescimento ou citocinas, ocorre a ativação desse tipo celular que prolifera e expressa os MRFs de maneira similar ao que ocorre com as células precursoras miogênicas durante a miogênese. Os mecanismos de crescimento muscular são regulados pela expressão temporal dos (MRFs), que controlam a expressão dos genes relacionados com o crescimento muscular.

Differential Expression of Myogenic Regulatory Factor Genes in the Skeletal Muscles of Tambaqui Colossoma macropomum (Cuvier 1818) from Amazonian Black and Clear Water

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expressão dos fatores de regulação miogênica em cultura de mioblastos submetidos ao TNF-α/IFN-γ

A insuficiência cardíaca (IC) está associada a miopatia dos músculos esqueléticos dos membros, com perda da massa muscular, diminuição na proporção das fibras do tipo I (contração lenta) e aumento na proporção das fibras do tipo II (contração rápida). É provável que alterações na expressão de fatores de transcrição pertencentes à família “basic helix-loop-helix” (bHLH), da qual fazem parte a MyoD, Miogenina, Myf5 e o MRF-4, conhecidos como fatores de regulação miogênica (MRFs), sejam responsáveis pelas mudanças nos tipos de fibras. Enquanto que a Miogenina é expressa em níveis superiores aos da MyoD em músculos lentos, o oposto é verdadeiro para músculos rápidos. Similarmente, a MyoD está associada com a expressão das isoformas de miosina de cadeia pesada rápidas dos tipos IIX e IIB. Estudos in vitro, demonstraram que o TNF-α inibe a expressão de MyoD e miogenina diminuindo a atividade de genes músculo específicos. A ação do TNF-α diminuindo a expressão da MyoD mostra-se mais acentuada quando em associação com o IFN-γ, no entanto, há poucas informações na literatura a respeito do papel desta associação na expressão dos fatores de regulação miogênica, in vitro. Avaliar a expressão dos fatores de regulação miogênica, MyoD, miogenina, Myf5, e MRF-4 em cultura de mioblastos C2C12 submetidos ao TNF-α/IFN-γ. Nossos resultados mostraram um aumentou na expressão dos gene MyoD, Myf5 e miogenina sob tratamento com IFN-γ quando comparado aos grupos controle e TNF-α/IFN-γ. A expressão gênica do MRF-4 na cultura de células não foi detectada em nenhum dos grupos analisados. O GAPDH foi utilizado para normalizar os valores de expressão dos outros genes analisados. O presente estudo demonstrou que o IFN-γ exógeno administrado à culturas de mioblastos... (Resumo completo, clicar acesso eletrônico abaixo)

Differential Expression of Genes Involved in the Degeneration and Regeneration Pathways in Mouse Models for Muscular Dystrophies

The genetically determined muscular dystrophies are caused by mutations in genes coding for muscle proteins. Differences in the phenotypes are mainly the age of onset and velocity of progression. Muscle weakness is the consequence of myofiber degeneration due to an imbalance between successive cycles of degeneration/regeneration. While muscle fibers are lost, a replacement of the degraded muscle fibers by adipose and connective tissues occurs. Major investigation points are to elicit the involved pathophysiological mechanisms to elucidate how each mutation can lead to a specific degenerative process and how the regeneration is stimulated in each case. To answer these questions, we used four mouse models with different mutations causing muscular dystrophies, Dmd (mdx) , SJL/J, Large (myd) and Lama2 (dy2J) /J, and compared the histological changes of regeneration and fibrosis to the expression of genes involved in those processes. For regeneration, the MyoD, Myf5 and myogenin genes related to the proliferation and differentiation of satellite cells were studied, while for degeneration, the TGF-beta 1 and Pro-collagen 1 alpha 2 genes, involved in the fibrotic cascade, were analyzed. The result suggests that TGF-beta 1 gene is activated in the dystrophic process in all the stages of degeneration, while the activation of the expression of the pro-collagen gene possibly occurs in mildest stages of this process. We also observed that each pathophysiological mechanism acted differently in the activation of regeneration, with distinctions in the induction of proliferation of satellite cells, but with no alterations in stimulation to differentiation. Dysfunction of satellite cells can, therefore, be an important additional mechanism of pathogenesis in the dystrophic muscle.

Mice with a targeted disruption of the Fgfrl1 gene die at birth due to alterations in the diaphragm

FGFRL1 is a recently discovered member of the fibroblast growth factor receptor family that is lacking the intracellular tyrosine kinase domain. To elucidate the function of the novel receptor, we created mice with a targeted disruption of the Fgfrl1 gene. These mice develop normally until term, but die within a few minutes after birth due to respiratory failure. The respiratory problems are explained by a significant reduction in the size of the diaphragm muscle, which is not sufficient to inflate the lungs after birth. The remaining portion of the diaphragm muscle appears to be well developed and innervated. It consists of differentiated myofibers with nuclei at the periphery. Fast and slow muscle fibers occur in normal proportions. The myogenic regulatory factors MyoD, Myf5, myogenin and Mrf4 and the myocyte enhancer factors Mef2A, Mef2B, Mef2C and Mef2D are expressed at normal levels. Experiments with a cell culture model involving C2C12 myoblasts show that Fgfrl1 is expressed during the late stages of myotube formation. Other skeletal muscles do not appear to be affected in the Fgfrl1 deficient mice. Thus, Fgfrl1 plays a critical role in the development of the diaphragm.

ELUCIDATING FUNCTIONAL ROLES FOR MYOGENIN IN ADULT SKELETAL MUSCLE METABOLISM, EXERCISE CAPACITY, AND REGENERATION

The four basic helix-loop-helix myogenic transcription factors, myogenin, Myf5, MRF4, and MyoD are critical for embryonic skeletal muscle development. Myogenin is necessary for the terminal differentiation of myoblasts into myofibers during embryogenesis, but little is known about the roles played by myogenin in adult skeletal muscle function and metabolism. Furthermore, while metabolism is a well-studied physiological process, how it is regulated at the transcriptional level remains poorly understood. In this study, my aim was to determine the function of myogenin in adult skeletal muscle metabolism, exercise capacity, and regeneration. To investigate this, I utilized a mouse strain harboring the Myogflox allele and a Cre recombinase transgene, enabling the efficient deletion of myogenin in the adult mouse. Myogflox/flox mice were stressed physically through involuntary treadmill running and by breeding them with a strain harboring the Duchenne’s muscular dystrophy (DMDmdx) allele. Surprisingly, Myog-deleted animals exhibited an enhanced capacity for exercise, running farther and faster than their wild-type counterparts. Increased lactate production and utilization of glucose as a fuel source indicated that Myog-deleted animals exhibited an increased glycolytic flux. Hypoglycemic Myog-deleted mice no longer possessed the ability to outrun their wild-type counterparts, implying the ability of these animals to further deplete their glucose reserves confers their enhanced exercise capacity. Moreover, Myog-deleted mice exhibited an enhanced response to long-term exercise training. The mice developed a greater proportion of type 1 oxidative muscle fibers, and displayed increased levels of succinate dehydrogenase activity, indicative of increased oxidative metabolism. Mdx:Myog-deleted mice exhibited a similar phenotype, outperforming their mdx counterparts, although lagging behind wild-type animals. The morphology of muscle tissue from mdx:Myog-deleted mice appears to mimic that of mdx animals, indicating that myogenin is dispensable for adult skeletal muscle regeneration. Through global gene expression profiling and quantitative (q)RT-PCR, I identified a unique set of putative myogenin-dependent genes involved in regulating metabolic processes. These data suggest myogenin’s functions during adulthood are distinctly different than those during embryogenesis, and myogenin acts as a high-level transcription factor regulating metabolic activity in adult skeletal muscle.

A study of the {\it in vivo\/} role of myogenin

Myogenin is a member of the MyoD family of skeletal muscle specific bHLH transcription factors. All of the members of this family have been shown to initiate the muscle differentiation cascade in a variety of nonmuscle cell lines. Many of the properties of the MyoD family have been studied in vitro, but their in vivo roles had not yet been examined. In this thesis, I study the in vivo role of myogenin by creating mice that carry a mutation at the myogenin locus.^ Mice lacking the myogenin protein are born alive, but immobile. Histological examination showed that these mice are severely deficient in skeletal muscle; they show a reduction in the number and density of myofibers. In addition to the reduction in fiber number, these mice express lower levels of a variety of muscle-specific markers. The undifferentiated cells in the muscle forming regions of these mice do express some muscle-specific markers, indicating that these cells are determined but undifferentiated myoblasts. Additional studies show that the major muscle defect arises late in embryogenesis, at a time coincident with secondary myogenesis. Moreover, studies regarding the nature of the remaining myofibers indicate that they are representative of a normal population of myofibers, merely reduced in numbers. In addition, I studied the effects of combining the myogenin mutation with mutations in two other members of the MyoD family, MyoD and myf5. Mice mutant in myogenin + MyoD and myogenin + myf5 show no increase in the severity of the myogenin single mutation, as indicated by histological or molecular examination. These results reveal the unique and essential role of myogenin in mammalian skeletal myogenesis. ^

Regulation of XMyoDa transcription by the binding of XMEF2A to the TATA box

MEF2 is a $\underline{\rm m}$yocyte-specific $\underline{\rm e}$nhancer-binding $\underline{\rm f}$actor that binds a conserved DNA sequence, CTA(A/T)$\sb4$TAG. A MEF2 binding site in the XMyoDa promoter overlaps with the TATA box and is required for muscle specific expression. To examine the potential role of MEF2 in the regulation of MyoD transcription during early development, the appearance of MEF2 binding activity in developing Xenopus embryos was analyzed with the electrophoretic mobility shift assay. Two genes were isolated from a X. Laevis stage 24 cDNA library that encode factors that bind the XMyoDa TFIID/MEF2 site. Both genes are highly homologous to each other, belong to the MADS ($\underline{\rm M}$CM1-$\underline{\rm A}$rg80-agamous-$\underline{\rm d}$eficiens-$\underline{\rm S}$RF) protein family, and most highly related to the mammalian MEF2A gene, hence they are designated as XMEF2A1 and XMEF2A2. Proteins encoded by both cDNAs form specific complexes with the MEF2 binding site and show the same binding specificity as the endogenous MEF2 binding activity. XMEF2A transcripts accumulate preferentially in developing somites after the appearance of XMyoD transcripts. XMEF2 protein begins to accumulate in somites at tailbud stages. Transcriptional activation of XMyoD promoter by XMEF2A required only the MADS box and MEF2-specific domain when XMEF2A is bound at the TATA box. However, a different downstream transactivation domain was required when XMEF2A activates transcription through binding to multiple upstream sites. These results suggest that different activation mechanisms are involved, depending on where the factor is bound. Mutations in several basic amino acid clusters in the MADS box inhibit DNA binding suggesting these amino acids are essential for DNA binding. Mutation of Thr-20 and Ser-36 to the negatively charged amino acid residue, aspartic acid, abolish DNA binding. XMEF2A activity may be regulated by phosphorylation of these amino acids. A dominant negative mutant was made by mutating one of the basic amino acid clusters and deleting the downstream transactivation domain. In vivo roles of MEF2 in the regulation of MyoD transcription were investigated by overexpression of wild type MEF2 and dominant negative mutant of XMEF2A in animal caps and assaying for the effects on the level of expression of MyoD genes. Overexpression of MEF2 activates the transcription of endogenous MyoD gene family while expression of a dominant negative mutant reduces the level of transcription of XMRF4 and myogenin genes. These results suggest that MEF2 is downstream of MyoD and Myf5 and that MEF2 is involved in maintaining and amplifying expression of MyoD and Myf5. MEF2 is upstream of MRF4 and myogenin and plays a role in activating their expression. ^