Functional analysis of protein interactions using coupled bi-fluorescence complementation/GFP nanobody techniques.

Transcription factors (TFs) form homo- or hetero-dimeric DNA binding complexes along with associated co-regulators that can have transcriptional repressor or activator functions. Defining the specific composition of the complexes is therefore key to understanding their biological role. Here, we utilized bimolecular fluorescence complementation (BiFC) to visualize the formation of defined TF dimers and associated co-regulators derived from the activator protein-1 (AP-1) and myocyte enhancer factor 2 (MEF2) families. Firstly, BiFC signals were observed in cells co-expressing TFs tagged with complimentary combinations of the split fluorescent protein, demonstrating the engineered formation of defined dimer complexes. Next, we applied this approach and determined that defined AP-1 dimers localized at discrete sub-nuclear locations. Subsequently, a combination of BiFC coupled with GFP binding peptide (GBP)-nanotrap allowed observation of protein-protein interactions between a co-regulator, HDAC4, and defined BiFC-MEF2 engineered dimers. To determine transactivation properties of defined TF dimers in a cellular system, the Gal4-DNA binding domain fused to GBP was utilized to assess the transcriptional properties of the BiFC-TF dimers using a generically applicable Gal4/UAS luciferase reporter gene assay system. Here, we report efficacy of a BiFC/GBP-nanobody approach that allows engineering, visualization, and functional analysis of defined TF dimers.

Re-organization of nucleolar architecture in myogenic differentiation.

Myogenesis, the process of muscle differentiation, requires an extensive remodeling of the cellular transcriptome and proteome. Whereas the transcriptional program underpinning myogenesis is well characterized, the required adaptation in protein synthesis is incompletely understood. Enhanced protein synthesis necessitates ribosome biogenesis at the nucleolus. Nucleolar size and activity are inextricably linked with altered gene expression. Here, we report changes in nucleolar morphology and function during myogenic differentiation. Immunofluorescence analysis revealed alterations in nucleolar morphology that were dependent on the cellular state - proliferative or quiescent myogenic progenitors (myoblasts or reserve cells) contained multiple small nucleoli with a characteristic spherical shape, whereas multinucleated myotubes typically contained one large, often irregularly shaped nucleolus. These morphological alterations are consistent with changes to nucleolar phase separation properties. Re-organization of the nucleolar structure was correlated with enhanced rRNA production and protein translation. Inhibition of mTOR signaling with rapamycin perturbed nucleolar re-organization. Conversely, hyperactivated mTOR enhanced alterations in nucleolar morphology. These findings support the idea that there is an mTOR dependent re-organization of nucleolar structure during myogenesis, enhancing our understanding of myogenesis and possibly facilitating new approaches to therapeutic interventions in muscle pathologies.

TAZ exhibits phase separation properties and interacts with Smad7 and ß-catenin to repress skeletal myogenesis.

Hippo signaling in Drosophila and mammals is prominent in regulating cell proliferation, death and differentiation. Hippo signaling effectors (YAP and TAZ; also known as YAP1 and WWTR1, respectively) exhibit crosstalk with transforming growth factor-ß (TGF-ß)-Smad and Wnt/ß-catenin pathways. Previously, we implicated Smad7 and ß-catenin in mammalian myogenesis. Therefore, we assessed a potential role of TAZ on the Smad7-ß-catenin complex in muscle cells. Here, we document functional interactions between Smad7, TAZ and ß-catenin in mouse myogenic cells. Ectopic TAZ expression resulted in repression of the muscle-specific creatine kinase muscle (Ckm) gene promoter and its corresponding protein level. Depletion of endogenous TAZ enhanced Ckm promoter activation. Ectopic TAZ, while potently active on a TEAD reporter (HIP-HOP), repressed myogenin (Myog) and Myod1 enhancer regions and myogenin protein level. Additionally, a Wnt/ß-catenin readout (TOP flash) demonstrated TAZ-mediated inhibition of ß-catenin activity. In myoblasts, TAZ was predominantly localized in nuclear speckles, while in differentiation conditions TAZ was hyperphosphorylated at Ser89, leading to enhanced cytoplasmic sequestration. Finally, live-cell imaging indicated that TAZ exhibits properties of liquid-liquid phase separation (LLPS). These observations indicate that TAZ, as an effector of Hippo signaling, suppresses the myogenic differentiation machinery.

The importance of biological sex in cardiac cachexia.

Cardiac cachexia is a catabolic muscle-wasting syndrome observed in approximately 1 in 10 patients with heart failure. Increased skeletal muscle atrophy leads to frailty and limits mobility, which impacts quality of life, exacerbates clinical care, and is associated with higher rates of mortality. Heart failure is known to exhibit a wide range of prevalence and severity when examined across individuals of different ages and with comorbidities related to diabetes, renal failure, and pulmonary dysfunction. It is also recognized that men and women exhibit striking differences in the pathophysiology of heart failure, as well as skeletal muscle homeostasis. Given that both skeletal muscle and heart failure physiology are in part sex-dependent, the diagnosis and treatment of cachexia in patients with heart failure may depend on a comprehensive examination of how these organs interact. In this review, we explore the potential for sex-specific differences in cardiac cachexia. We summarize advantages and disadvantages of clinical methods used to measure muscle mass and function and provide alternative measurements that should be considered in preclinical studies. In addition, we summarize sex-dependent effects on muscle wasting in preclinical models of heart failure, disuse, and cancer. Lastly, we discuss the endocrine function of the heart and outline unanswered questions that could directly impact patient care.

TNAP limits TGF-ß-dependent cardiac and skeletal muscle fibrosis by inactivating the SMAD2/3 transcription factors.

Fibrosis is associated with almost all forms of chronic cardiac and skeletal muscle diseases. The accumulation of extracellular matrix impairs the contractility of muscle cells contributing to organ failure. Transforming growth factor ß (TGF-ß) plays a pivotal role in fibrosis, activating pro-fibrotic gene programmes via phosphorylation of SMAD2/3 transcription factors. However, the mechanisms that control de-phosphorylation of SMAD2 and SMAD3 (SMAD2/3) have remained poorly characterized. Here, we show that tissue non-specific alkaline phosphatase (TNAP, also known as ALPL) is highly upregulated in hypertrophic hearts and in dystrophic skeletal muscles, and that the abrogation of TGF-ß signalling in TNAP-positive cells reduces vascular and interstitial fibrosis. We show that TNAP colocalizes and interacts with SMAD2. The TNAP inhibitor MLS-0038949 increases SMAD2/3 phosphorylation, while TNAP overexpression reduces SMAD2/3 phosphorylation and the expression of downstream fibrotic genes. Overall our data demonstrate that TNAP negatively regulates TGF-ß signalling and likely represents a mechanism to limit fibrosis.

Identification of the Most Important Subset of Doppler, Laboratory, and Clinical Parameters for Serial TIPS Evaluation.

OBJECTIVE. The purpose of the present study was to identify the subset of a wide range of serial Doppler, laboratory, and clinical parameters most predictive (both individually and in combination) of TIPS dysfunction in a large patient sample. MATERIALS AND METHODS. The medical records of 189 patients who had undergone TIPS procedures were analyzed. The patients (mean age, 52 years; 62% of whom were men) had undergone 1139 Doppler studies and 323 portovenograms. Laboratory parameters included model for end-stage liver disease (MELD) scores, serum albumin levels, presence of ascites, and time since last intervention. Doppler parameters included intrashunt velocities, temporal change in intrashunt velocities, main portal vein velocity, direction of flow in the left portal hepatic vein, and venous pulsatility index. Statistical analysis used ROC, univariate, and multivariate regression models to assess the parameters both individually and in combination. Shunt dysfunction was defined by a portosystemic gradient of more than 12 mm Hg. RESULTS. The laboratory and clinical parameters of greatest predictive value included the MELD score and the time since the last intervention. The Doppler parameters that were of greatest predictive value included the change in velocity at the hepatic venous end and the left portal vein flow direction. Multivariate models produced an AUC of 0.74. Differences between functional and dysfunctional shunts were also statistically significant for absolute velocity at the hepatic venous end, the change in velocity within the stent, and the temporal change in the mid shunt velocity. CONCLUSION. The subset of serial parameters most predictive of TIPS dysfunction are the temporal change in the velocity at the hepatic venous end, the absolute velocity at the hepatic venous end, the direction of flow in the left portal venous branch, and changes in the MELD score.

FMRP recruitment of ß-catenin to the translation pre-initiation complex represses translation.

Canonical Wnt/ß-catenin signaling is an essential regulator of various cellular functions throughout development and adulthood. Aberrant Wnt/ß-catenin signaling also contributes to various pathologies including cancer, necessitating an understanding of cell context-dependent mechanisms regulating this pathway. Since protein-protein interactions underpin ß-catenin function and localization, we sought to identify novel ß-catenin interacting partners by affinity purification coupled with tandem mass spectrometry in vascular smooth muscle cells (VSMCs), where ß-catenin is involved in both physiological and pathological control of cell proliferation. Here, we report novel components of the VSMC ß-catenin interactome. Bioinformatic analysis of the protein networks implies potentially novel functions for ß-catenin, particularly in mRNA translation, and we confirm a direct interaction between ß-catenin and the fragile X mental retardation protein (FMRP). Biochemical studies reveal a basal recruitment of ß-catenin to the messenger ribonucleoprotein and translational pre-initiation complex, fulfilling a translational repressor function. Wnt stimulation antagonizes this function, in part, by sequestering ß-catenin away from the pre-initiation complex. In conclusion, we present evidence that ß-catenin fulfills a previously unrecognized function in translational repression.

Maintenance of the Undifferentiated State in Myogenic Progenitor Cells by TGFß Signaling is Smad Independent and Requires MEK Activation.

Transforming growth factor ß (TGFß) is a pluripotent cytokine and regulates a myriad of biological processes. It has been established that TGFß potently inhibits skeletal muscle differentiation; however, the molecular mechanism is not clearly defined. Previously, we reported that inhibition of the TGFß canonical pathway by an inhibitory Smad, Smad7, does not reverse this effect on differentiation, suggesting that activation of receptor Smads (R-Smads) by TGFß is not responsible for repression of myogenesis. In addition, pharmacological blockade of Smad3 activation by TGFß did not reverse TGFß's inhibitory effect on myogenesis. In considering other pathways, we observed that TGFß potently activates MEK/ERK, and a pharmacological inhibitor of MEK reversed TGFß's inhibitory effect on myogenesis, as indicated by a myogenin promoter-reporter gene, sarcomeric myosin heavy chain accumulation, and phenotypic myotube formation. Furthermore, we found that c-Jun, a known potent repressor of myogenesis, which is coincidently also a down-stream target of MEK/ERK signaling, was phosphorylated and accumulates in the nucleus in response to TGFß activation. Taken together, these observations support a model in which TGFß activates a MEK/ERK/c-Jun pathway to repress skeletal myogenesis, maintaining the pluripotent undifferentiated state in myogenic progenitors.

Regulation of Hspb7 by MEF2 and AP-1: implications for Hspb7 in muscle atrophy.

Mycocyte enhancer factor 2 (MEF2) and activator protein 1 (AP-1) transcription complexes have been individually implicated in myogenesis, but their genetic interaction has not previously been addressed. Using MEF2A, c-Jun and Fra-1 chromatin immunoprecipitation sequencing (ChIP-seq) data and predicted AP-1 consensus motifs, we identified putative common MEF2 and AP-1 target genes, several of which are implicated in regulating the actin cytoskeleton. Because muscle atrophy results in remodelling or degradation of the actin cytoskeleton, we characterized the expression of putative MEF2 and AP-1 target genes (Dstn, Flnc, Hspb7, Lmod3 and Plekhh2) under atrophic conditions using dexamethasone (Dex) treatment in skeletal myoblasts. Heat shock protein b7 (Hspb7) was induced by Dex treatment and further analyses revealed that loss of MEF2A using siRNA prevented Dex-regulated induction of Hspb7. Conversely, ectopic Fra-2 or c-Jun expression reduced Dex-mediated upregulation of Hspb7 whereas AP-1 depletion enhanced Hspb7 expression. In vivo, expression of Hspb7 and other autophagy-related genes was upregulated in response to atrophic conditions in mice. Manipulation of Hspb7 levels in mice also impacted gross muscle mass. Collectively, these data indicate that MEF2 and AP-1 confer antagonistic regulation of Hspb7 gene expression in skeletal muscle, with implications for autophagy and muscle atrophy.

Global MEF2 target gene analysis in cardiac and skeletal muscle reveals novel regulation of DUSP6 by p38MAPK-MEF2 signaling.

MEF2 plays a profound role in the regulation of transcription in cardiac and skeletal muscle lineages. To define the overlapping and unique MEF2A genomic targets, we utilized ChIP-exo analysis of cardiomyocytes and skeletal myoblasts. Of the 2783 and 1648 MEF2A binding peaks in skeletal myoblasts and cardiomyocytes, respectively, 294 common binding sites were identified. Genomic targets were compared to differentially expressed genes in RNA-seq analysis of MEF2A depleted myogenic cells, revealing two prominent genetic networks. Genes largely associated with muscle development were down-regulated by loss of MEF2A while up-regulated genes reveal a previously unrecognized function of MEF2A in suppressing growth/proliferative genes. Several up-regulated (Tprg, Mctp2, Kitl, Prrx1, Dusp6) and down-regulated (Atp1a2, Hspb7, Tmem182, Sorbs2, Lmod3) MEF2A target genes were chosen for further investigation. Interestingly, siRNA targeting of the MEF2A/D heterodimer revealed a somewhat divergent role in the regulation of Dusp6, a MAPK phosphatase, in cardiac and skeletal myogenic lineages. Furthermore, MEF2D functions as a p38MAPK-dependent repressor of Dusp6 in myoblasts. These data illustrate that MEF2 orchestrates both common and non-overlapping programs of signal-dependent gene expression in skeletal and cardiac muscle lineages.

Adiponectin is required for cardiac MEF2 activation during pressure overload induced hypertrophy.

Cardiomyocyte (CM) hypertrophy and increased heart mass in response to pressure overload are associated with hyper-activation of the myocyte enhancer factor-2 (MEF2) family of transcriptional regulators, and concomitant initiation of the fetal gene program. Adiponectin, an adipokine that is reduced in individuals with obesity and diabetes, has been characterized both as a negative regulator or permissive factor in cardiac hypertrophy. We therefore sought to analyze temporal regulation of MEF2 activity in response to pressure overload (PO) and changes in adiponectin status. To address this we crossed a well characterized transgenic MEF2 "sensor" mouse (MEF2-lacZ) with adiponectin null mice (Ad-KO) to create compound MEF2 lacZ/Ad-KO mice. Initially, we established that transverse aortic banding induced PO in wild-type (WT) mice increased heart mass and CM hypertrophy from 1 to 4weeks following surgery, indicated by increased CM diameter and heart weight/tibia length ratio. This was associated with cardiac dysfunction determined by echocardiography. Hypertrophic changes and dysfunction were observed in Ad-KO mice 4weeks following surgery. MEF2 lacZ activity and endogenous ANF mRNA levels, used as indicators of hypertrophic gene activation, were both robustly increased in WT mice after MTAB but attenuated in the Ad-KO background. Furthermore, activation of the pro-hypertrophic molecule p38 was increased following MTAB surgery in WT mice, but not in Ad-KO animals, and treatment of primary isolated CM with recombinant adiponectin induced p38 phosphorylation in a time dependent manner. Adiponectin also increased MEF2 activation in primary cardiomyocytes, an effect attenuated by p38 MAPK inhibition. In conclusion, our data indicate that robust hypertrophic MEF2 activation in the heart in vivo requires a background of adiponectin signaling and that adiponectin signaling in primary isolated CM directly enhances MEF2 activity through activation of p38 MAPK. We conclude that adiponectin is required for full induction of cardiomyocyte MEF2 activation, thus contributing to the myocardial hypertrophic gene expression program in response to PO.

FoxP1 Represses MEF2A in Striated Muscle.

Myocyte enhancer factor 2 (MEF2) proteins are involved in multiple developmental, physiological, and pathological processes in vertebrates. Protein-protein interactions underlie the plethora of biological processes impacted by MEF2A, necessitating a detailed characterization of the MEF2A interactome. A nanobody based affinity-purification/mass spectrometry strategy was employed to achieve this goal. Specifically, the MEF2A protein complexes were captured from myogenic lysates using a GFP-tagged MEF2A protein immobilized with a GBP-nanobody followed by LC-MS/MS proteomic analysis to identify MEF2A interactors. After bioinformatic analysis, we further characterized the interaction of MEF2A with a transcriptional repressor, FOXP1. FOXP1 coprecipitated with MEF2A in proliferating myogenic cells which diminished upon differentiation (myotube formation). Ectopic expression of FOXP1 inhibited MEF2A driven myogenic reporter genes (derived from the creatine kinase muscle and myogenin genes) and delayed induction of endogenous myogenin during differentiation. Conversely, FOXP1 depletion enhanced MEF2A transactivation properties and myogenin expression. The FoxP1:MEF2A interaction is also preserved in cardiomyocytes and FoxP1 depletion enhanced cardiomyocyte hypertrophy. FOXP1 prevented MEF2A phosphorylation and activation by the p38MAPK pathway. Overall, these data implicate FOXP1 in restricting MEF2A function in order to avoid premature differentiation in myogenic progenitors and also to possibly prevent re-activation of embryonic gene expression in cardiomyocyte hypertrophy.

Suppression of a MEF2-KLF6 survival pathway by PKA signaling promotes apoptosis in embryonic hippocampal neurons.

In the mammalian nervous system, regulation of transcription factor activity is a crucial determinant of neuronal cell survival, differentiation, and death. The myocyte enhancer factor 2 (MEF2) transcription factors have been implicated in cellular processes underlying neuronal survival and differentiation. A core component of the MEF2 complex is the MEF2D subunit. Recently, we reported that cAMP-dependent protein kinase (cAMP/PKA) signaling negatively regulates MEF2D function in myogenic cells. Here, we assessed whether cAMP signaling converges on the prosurvival role of MEF2D in Sprague Dawley rat embryonic (E18) hippocampal neurons. Initially, we observed that experimental induction of cAMP/PKA signaling promotes apoptosis in primary hippocampal neurons as indicated by TUNEL and FACS analysis. Luciferase reporter gene assays revealed that PKA potently represses MEF2D trans-activation properties in neurons. This effect was largely reversed by engineered neutralizing mutations of PKA phospho-acceptor sites on MEF2D (S121/190A). Krüppel-like factor 6 (KLF6) was identified as a key transcriptional target of MEF2 in hippocampal neurons, and siRNA-mediated knockdown of KLF6 expression promotes neuronal cell death and also antagonizes the prosurvival role of MEF2D. These observations have important implications for understanding the pathways controlling cell survival and death in the mammalian nervous system.

A novel RhoA/ROCK-CPI-17-MEF2C signaling pathway regulates vascular smooth muscle cell gene expression.

Differentiation of vascular smooth muscle cells (VSMC) is a fundamental aspect of normal development and vascular disease. During contraction, VSMCs modulate calcium sensitivity through RhoA/ROCK-mediated inhibition of the myosin light chain phosphatase complex (MLCP). Previous studies have demonstrated that this signaling pathway functions in parallel to increase the expression of smooth muscle genes through the myocardin-family of co-activators. MEF2C fulfills a critical role in VSMC differentiation and regulates myocardin expression, leading us to investigate whether the RhoA/ROCK signaling cascade might regulate MEF2 activity. Depolarization-induced calcium signaling increased the expression of myocardin, which was sensitive to ROCK and p38 MAPK inhibition. We previously identified protein phosphatase 1α (PP1α), a known catalytic subunit of the MLCP in VSMCs, as a potent repressor of MEF2 activity. PP1α inhibition resulted in increased expression of myocardin, while ectopic expression of PP1α inhibited the induction of myocardin by MEF2C. Consistent with these data, shRNA-mediated suppression of a PP1α inhibitor, CPI-17, reduced myocardin expression and inhibited VSMC differentiation, suggesting a pivotal role for CPI-17 in regulating MEF2 activity. These data constitute evidence of a novel signaling cascade that links RhoA-mediated calcium sensitivity to MEF2-dependent myocardin expression in VSMCs through a mechanism involving p38 MAPK, PP1α, and CPI-17.

Identification of differentially regulated secretome components during skeletal myogenesis.

Myogenesis is a well-characterized program of cellular differentiation that is exquisitely sensitive to the extracellular milieu. Systematic characterization of the myogenic secretome (i.e. the ensemble of secreted proteins) is, therefore, warranted for the identification of novel secretome components that regulate both the pluripotency of these progenitor mesenchymal cells, and also their commitment and passage through the differentiation program. Previously, we have successfully identified 26 secreted proteins in the mouse skeletal muscle cell line C2C12 (1). In an effort to attain a more comprehensive picture of the regulation of myogenesis by its extracellular milieu, quantitative profiling employing stable isotope labeling by amino acids in cell culture was implemented in conjunction with two parallel high throughput online reverse phase liquid chromatography-tandem mass spectrometry systems. In summary, 34 secreted proteins were quantified, 30 of which were shown to be differentially expressed during muscle development. Intriguingly, our analysis has revealed several novel up- and down-regulated secretome components that may have critical biological relevance for both the maintenance of pluripotency and the passage of cells through the differentiation program. In particular, the altered regulation of secretome components, including follistatin-like protein-1, osteoglycin, spondin-2, and cytokine-induced apoptosis inhibitor-1, along with constitutively expressed factors, such as fibulin-2, illustrate dynamic changes in the secretome that take place when differentiation to a specific lineage occurs.

MEF2 in cardiac hypertrophy in response to hypertension.

Hypertension is a globally prevalent pathological condition and an underlying risk factor for the development of cardiac hypertrophy leading to heart failure. Myocyte enhancer factor 2 (Mef2) has been identified as one of the primary effectors of morphological changes in the hypertensive heart, as part of a complex network of molecular signaling controlling cardiac gene expression. Experimental chronic pressure-overload models that mimic hypertension in the mammalian heart lead to the activation of various pathological mechanisms that result in structural changes leading to debilitating cardiac hypertrophy and ultimately heart failure. The purpose here is to survey the literature implicating Mef2 in hypertension induced cardiac hypertrophy, towards illuminating points of interest for understanding and potentially treating heart failure.

The MEF2A transcription factor interactome in cardiomyocytes.

Transcriptional regulators encoded by the Myocyte Enhancer Factor 2 (MEF2) gene family play a fundamental role in cardiac development, homeostasis and pathology. Previous studies indicate that MEF2A protein-protein interactions serve as a network hub in several cardiomyocyte cellular processes. Based on the idea that interactions with regulatory protein partners underly the diverse roles of MEF2A in cardiomyocyte gene expression, we undertook a systematic unbiased screen of the MEF2A protein interactome in primary cardiomyocytes using an affinity purification-based quantitative mass spectrometry approach. Bioinformatic processing of the MEF2A interactome revealed protein networks involved in the regulation of programmed cell death, inflammatory responses, actin dynamics and stress signaling in primary cardiomyocytes. Further biochemical and functional confirmation of specific protein-protein interactions documented a dynamic interaction between MEF2A and STAT3 proteins. Integration of transcriptome level data from MEF2A and STAT3-depleted cardiomyocytes reveals that the balance between MEF2A and STAT3 activity exerts a level of executive control over the inflammatory response and cardiomyocyte cell survival and experimentally ameliorates Phenylephrine induced cardiomyocyte hypertrophy. Lastly, we identified several MEF2A/STAT3 co-regulated genes, including the MMP9 gene. Herein, we document the cardiomyocyte MEF2A interactome, which furthers our understanding of protein networks involved in the hierarchical control of normal and pathophysiological cardiomyocyte gene expression in the mammalian heart.

Nucleolar localization of c-Jun.

Nucleoli are well defined for their function in ribosome biogenesis, but only a small fraction of the nucleolar proteome has been characterized. Here, we report that the proto-oncogene, c-Jun, is targeted to the nucleolus. Using live cell imaging in myogenic cells, we document that the c-Jun basic domain contains a unique, evolutionarily conserved motif that determines nucleolar targeting. Fos family Jun dimer partners, such as Fra2, while nuclear, do not co-localize with c-Jun in the nucleolus. A point mutation in c-Jun that mimics Fra2 (M260E) in its Nucleolar Localization sequence (NoLS) results in loss of c-Jun nucleolar targeting while still preserving nuclear localization. Fra2 can sequester c-Jun in the nucleoplasm, indicating that the stoichiometric ratio of heterodimeric partners regulates c-Jun nucleolar targeting. Finally, nucleolar localization of c-Jun modulates nucleolar architecture and ribosomal RNA accumulation. These studies highlight a novel role for Jun family proteins in the nucleolus, having potential implications for a diverse array of AP-1-regulated cellular processes.

Menin expression modulates mesenchymal cell commitment to the myogenic and osteogenic lineages.

Menin plays an established role in the differentiation of mesenchymal cells to the osteogenic lineage. Conversely, whether Menin influences the commitment of mesenschymal cells to the myogenic lineage, despite expression in the developing somite was previously unclear. We observed that Menin is down-regulated in C2C12 and C3H10T1/2 mesenchymal cells when muscle differentiation is induced. Moreover, maintenance of Menin expression by constitutive ectopic expression inhibited muscle cell differentiation. Reduction of Menin expression by siRNA technology results in precocious muscle differentiation and concomitantly attenuates BMP-2 induced osteogenesis. Reduced Menin expression antagonizes BMP-2 and TGF-beta1 mediated inhibition of myogenesis. Furthermore, Menin was found to directly interact with and potentiate the transactivation properties of Smad3 in response to TGF-beta1. Finally in concert with these observations, tissue-specific inactivation of Men1 in Pax3-expressing somite precursor cells leads to a patterning defect of rib formation and increased muscle mass in the intercostal region. These data invoke a pivotal role for Menin in the competence of mesenchymal cells to respond to TGF-beta1 and BMP-2 signals. Thus, by modulating cytokine responsiveness Menin functions to alter the balance of multipotent mesenchymal cell commitment to the osteogenic or myogenic lineages.