Let-7i-5p enhances cell proliferation, migration and invasion of ccRCC by targeting HABP4.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is one of the best-characterized and most pervasive renal cancers. The present study aimed to explore the effects and potential mechanisms of let-7i-5p in ccRCC cells. METHODS: Using bioinformatics analyses, we investigated the expression of let-7i-5p in The Cancer Genome Atlas (TCGA) database and predicted biological functions and possible target genes of let-7i-5p in ccRCC cells. Cell proliferation assay, wound healing assay and transwell invasion assay were conducted to characterize the effects of let-7i-5p in ccRCC cells. To verify the interactions between let-7i-5p and HABP4, dual-luciferase reporter assay, quantitative real-time polymerase chain reaction, and western blotting were conducted. Rescue experiments were used to investigate the relationship between let-7i-5p and HABP4. RESULTS: TCGA data analysis revealed that ccRCC tissues had significantly increased let-7i-5p expression, which was robustly associated with poor overall survival. Further verification showed that ccRCC cell proliferation, migration and invasion were inhibited by let-7i-5p inhibitor but enhanced by let-7i-5p mimics. Subsequently, HABP4 was predicted to be the target gene of let-7i-5p. TCGA data showed that ccRCC tissues had decreased expression of HABP4 and that HABP4 expression was negatively correlated with let-7i-5p. Further verification showed that downregulation of HABP4 expression promoted cell proliferation, migration and invasion. The dual-luciferase reporter gene assay suggested that the let-7i-5p/HABP4 axis was responsible for the development of ccRCC. CONCLUSION: Our results provide evidence that let-7i-5p functions as a tumor promoter in ccRCC and facilitates cell proliferation, migration and invasion by targeting HABP4. These results clarify the pathogenesis of ccRCC and offer a potential target for its treatment.

Regulation of nuclear factor of activated T cells (NFAT) and downstream myogenic proteins during dehydration in the African clawed frog.

Xenopus laevis, otherwise known as the African clawed frog, undergoes natural dehydration of up to 30% of its total body water during the dry season in sub-Saharan Africa. To survive under these conditions, a variety of physiological and biochemical changes take place in X. laevis. We were interested in understanding the role that the calcineurin-NFAT pathway plays during dehydration stress response in the skeletal muscles of X. laevis. Immunoblotting was performed to characterize the protein levels of NFATc1-4, calcium signalling proteins, in addition to myogenic proteins (MyoD, MyoG, myomaker). In addition, DNA-protein interaction ELISAs were used to assess the binding of NFATs to their consensus binding sequence, and to identify the effect of urea on NFAT-binding. Our results showed that NFATc1 and c4 protein levels decreased during dehydration, and there were no changes in NFATc2, c3, and calcium signalling proteins. However, MyoG and myomaker both showed increases in protein levels during dehydration, thus indicating that the late myogenic program involving myoblast differentiation, but not satellite cell activation and myoblast proliferation, could be involved in preserving the skeletal muscle of X. laevis during dehydration. In addition, we observed that urea seems to reduce NFATc3-binding to DNA during control, but not during dehydration, possibly indicating that NFATc3 is protected from the denaturing effects of urea as it accumulates during dehydration. These findings expand upon our knowledge of adaptive responses to dehydration, and they identify specific protein targets that could be used to protect the skeletal muscle from damage during stress.

Macrophage depletion impairs skeletal muscle regeneration: The roles of regulatory factors for muscle regeneration.

Though macrophages are essential for skeletal muscle regeneration, which is a complex process, the roles and mechanisms of the macrophages in the process of muscle regeneration are still not fully understood. The objective of this study is to explore the roles of macrophages and the mechanisms involved in the regeneration of injured skeletal muscle. One hundred and twelve C57BL/6 mice were randomly divided into muscle contusion and macrophages depleted groups. Their gastrocnemius muscles were harvested at the time points of 12 h, 1, 3, 5, 7, 14 d post-injury. The changes in skeletal muscle morphology were assessed by hematoxylin and eosin (HE) stain. The gene expression was analyzed by real-time polymerase chain reaction. The data showed that CL-liposomes treatment did affect the expression of myogenic regulatory factors (MyoD, myogenin) after injury. In addition, CL-liposomes treatment decreased the expression of regulatory factors of muscle regeneration (HGF, uPA, COX-2, IGF-1, MGF, FGF6) and increased the expression of inflammatory cytokines (TGF-ß1, TNF-α, IL-1ß, RANTES) in the late stage of regeneration. Moreover, there were significant correlations between macrophages and some regulatory factors (such as HGF, uPA) for muscle regeneration. These results suggested that macrophages depletion impairs skeletal muscle regeneration and that the regulatory factors for muscle regeneration may play important roles in this process.

"Triplet" polycistronic vectors encoding Gata4, Mef2c, and Tbx5 enhances postinfarct ventricular functional improvement compared with singlet vectors.

OBJECTIVE: The in situ reprogramming of cardiac fibroblasts into induced cardiomyocytes by the administration of gene transfer vectors encoding Gata4 (G), Mef2c (M), and Tbx5 (T) has been shown to improve ventricular function in myocardial infarction models. The efficacy of this strategy could, however, be limited by the need for fibroblast targets to be infected 3 times--once by each of the 3 transgene vectors. We hypothesized that a polycistronic "triplet" vector encoding all 3 transgenes would enhance postinfarct ventricular function compared with use of "singlet" vectors. METHODS: After validation of the polycistronic vector expression in vitro, adult male Fischer 344 rats (n=6) underwent coronary ligation with or without intramyocardial administration of an adenovirus encoding all 3 major vascular endothelial growth factor (VEGF) isoforms (AdVEGF-All6A positive), followed 3 weeks later by the administration to AdVEGF-All6A-positive treated rats of singlet lentivirus encoding G, M, or T (1×10(5) transducing units each) or the same total dose of a GMT "triplet" lentivirus vector. RESULTS: Western blots demonstrated that triplet and singlet vectors yielded equivalent GMT transgene expression, and fluorescence activated cell sorting demonstrated that triplet vectors were nearly twice as potent as singlet vectors in generating induced cardiomyocytes from cardiac fibroblasts. Echocardiography demonstrated that GMT triplet vectors were more effective than the 3 combined singlet vectors in enhancing ventricular function from postinfarct baselines (triplet, 37%±10%; singlet, 13%±7%; negative control, 9%±5%; P<.05). CONCLUSIONS: These data have confirmed that the in situ administration of G, M, and T induces postinfarct ventricular functional improvement and that GMT polycistronic vectors enhance the efficacy of this strategy.

Commentary on the special issue on the impact of myogenic tone in health and disease.

Autoregulation is a vital homeostatic mechanism that helps maintain constant delivery of oxygen to organs despite fluctuations in arteriolar pressure. Autoregulation of blood flow to elevations in pressure is largely mediated by the myogenic response of small arteries and arterioles which constrict in response to elevations in distending pressure. There is now general agreement that the myogenic response is an intrinsic property of vascular smooth muscle cells in the vessel wall that involves depolarization and calcium influx through L-type voltage-gated calcium channels (VGCC), calcium/ calmodulin-dependent phosphorylation of myosin light chain kinase and actin myosin-based contraction. Despite intensive investigation, however, the mechanotransduction events that initiate the myogenic response and the signaling pathways involved remain uncertain. This special issue on the Impact of Myogenic Tone in Health and Disease includes 9 papers that address current thought regarding the molecular mechanisms underlying myogenic control of vascular tone in the renal, cerebral and coronary circulations and the evidence that impairments in the myogenic response contribute to the development of vascular and end organ damage associated with hypertension, diabetes and aging.

The transcription factor Mef2 links the Drosophila core clock to Fas2, neuronal morphology, and circadian behavior.

The transcription factor Mef2 regulates activity-dependent neuronal plasticity and morphology in mammals, and clock neurons are reported to experience activity-dependent circadian remodeling in Drosophila. We show here that Mef2 is required for this daily fasciculation-defasciculation cycle. Moreover, the master circadian transcription complex CLK/CYC directly regulates Mef2 transcription. ChIP-Chip analysis identified numerous Mef2 target genes implicated in neuronal plasticity, including the cell-adhesion gene Fas2. Genetic epistasis experiments support this transcriptional regulatory hierarchy, CLK/CYC- > Mef2- > Fas2, indicate that it influences the circadian fasciculation cycle within pacemaker neurons, and suggest that this cycle also contributes to circadian behavior. Mef2 therefore transmits clock information to machinery involved in neuronal remodeling, which contributes to locomotor activity rhythms.

Caprine sex affects skeletal muscle profile and MRFs expression during postnatal development.

The important roles of myogenic regulatory factors (MRFs) have been well addressed in the process of mammalian skeletal myogenesis, while limited research has been performed in small ruminants. Furthermore, the effects of gender on the development of skeletal muscle and MRFs expression remain unknown. In this study, we identified the caprine Myf5, Myf6, MyoD and myogenin genes and quantified their expressions at six different postnatal time points by real-time RT-PCR. The sex has a marked effect on the expression differences of Myf5, MyoD and myogenin in the five investigated skeletal muscles, while minor influence on the expression difference of Myf6 except for Semitendinosus and Quadriceps femoris tissues (P < 0.001). The histological sections of muscles revealed a constant increase of muscle fiber diameter with aging but non-significant differences between genders. We provide novel evidence for MRFs expression in age- and gender-dependent manners, which will contribute to prioritizing these genes as potential candidate genes for trait-associated study and provide a foundation for understanding the molecular control of skeletal muscle growth in goat species.

Apelin-APJ signaling is a critical regulator of endothelial MEF2 activation in cardiovascular development.

RATIONALE: The peptide ligand apelin and its receptor APJ constitute a signaling pathway with numerous effects on the cardiovascular system, including cardiovascular development in model organisms such as xenopus and zebrafish. OBJECTIVE: This study aimed to characterize the embryonic lethal phenotype of the Apj-/- mice and to define the involved downstream signaling targets. METHODS AND RESULTS: We report the first characterization of the embryonic lethality of the Apj-/- mice. More than half of the expected Apj-/- embryos died in utero because of cardiovascular developmental defects. Those succumbing to early embryonic death had markedly deformed vasculature of the yolk sac and the embryo, as well as poorly looped hearts with aberrantly formed right ventricles and defective atrioventricular cushion formation. Apj-/- embryos surviving to later stages demonstrated incomplete vascular maturation because of a deficiency of vascular smooth muscle cells and impaired myocardial trabeculation and ventricular wall development. The molecular mechanism implicates a novel, noncanonical signaling pathway downstream of apelin-APJ involving Gα13, which induces histone deacetylase (HDAC) 4 and HDAC5 phosphorylation and cytoplasmic translocation, resulting in activation of myocyte enhancer factor 2. Apj-/- mice have greater endocardial Hdac4 and Hdac5 nuclear localization and reduced expression of the myocyte enhancer factor 2 (MEF2) transcriptional target Krüppel-like factor 2. We identify a number of commonly shared transcriptional targets among apelin-APJ, Gα13, and MEF2 in endothelial cells, which are significantly decreased in the Apj-/- embryos and endothelial cells. CONCLUSIONS: Our results demonstrate a novel role for apelin-APJ signaling as a potent regulator of endothelial MEF2 function in the developing cardiovascular system.

Functional studies of the Ciona intestinalis myogenic regulatory factor reveal conserved features of chordate myogenesis.

Ci-MRF is the sole myogenic regulatory factor (MRF) of the ascidian Ciona intestinalis, an invertebrate chordate. In order to investigate its properties we developed a simple in vivo assay based on misexpressing Ci-MRF in the notochord of Ciona embryos. We used this assay to examine the roles of three structural motifs that are conserved among MRFs: an alanine-threonine (Ala-Thr) dipeptide of the basic domain that is known in vertebrates as the myogenic code, a cysteine/histidine-rich (C/H) domain found just N-terminal to the basic domain, and a carboxy-terminal amphipathic α-helix referred to as Helix III. We show that the Ala-Thr dipeptide is necessary for normal Ci-MRF function, and that while eliminating the C/H domain or Helix III individually has no demonstrable effect on Ci-MRF, simultaneous loss of both motifs significantly reduces its activity. Our studies also indicate that direct interaction between CiMRF and an essential E-box of Ciona Troponin I is required for the expression of this muscle-specific gene and that multiple classes of MRF-regulated genes exist in Ciona. These findings are consistent with substantial conservation of MRF-directed myogenesis in chordates and demonstrate for the first time that the Ala/Thr dipeptide of the basic domain of an invertebrate MRF behaves as a myogenic code.

Regulation of MEF2 transcriptional activity by calcineurin/mAKAP complexes.

The calcium/calmodulin-dependent protein phosphatase calcineurin is required for the induction of transcriptional events that initiate and promote myogenic differentiation. An important effector for calcineurin in striated muscle is the transcription factor myocyte enhancer factor 2 (MEF2). The targeting of the enzyme and substrate to specific intracellular compartments by scaffold proteins often confers specificity in phosphatase activity. We now show that the scaffolding protein mAKAP organizes a calcineurin/MEF2 signaling complex in myocytes, regulating gene transcription. A calcineurin/mAKAP/MEF2 complex can be isolated from C2C12 cells and cardiac myocytes, and the calcineurin/MEF2 association is dependent on mAKAP expression. We have identified a peptide comprising the calcineurin binding domain in mAKAP that can disrupt the binding of the phosphatase to the scaffold in vivo. Dominant interference of calcineurin/mAKAP binding blunts the increase in MEF2 transcriptional activity seen during myoblast differentiation, as well as the expression of endogenous MEF2-target genes. Furthermore, disruption of calcineurin binding to mAKAP in cardiac myocytes inhibits adrenergic-induced cellular hypertrophy. Together these data illustrate the importance of calcineurin anchoring by the mAKAP scaffold for MEF2 regulation.

MEF2A regulates the Gtl2-Dio3 microRNA mega-cluster to modulate WNT signaling in skeletal muscle regeneration.

Understanding the molecular mechanisms of skeletal muscle regeneration is crucial to exploiting this pathway for use in tissue repair. Our data demonstrate that the MEF2A transcription factor plays an essential role in skeletal muscle regeneration in adult mice. Injured Mef2a knockout mice display widespread necrosis and impaired myofiber formation. MEF2A controls this process through its direct regulation of the largest known mammalian microRNA (miRNA) cluster, the Gtl2-Dio3 locus. A subset of the Gtl2-Dio3 miRNAs represses secreted Frizzled-related proteins (sFRPs), inhibitors of WNT signaling. Consistent with these data, Gtl2-Dio3-encoded miRNAs are downregulated in regenerating Mef2a knockout muscle, resulting in upregulated sFRP expression and attenuated WNT activity. Furthermore, myogenic differentiation in Mef2a-deficient myoblasts is rescued by overexpression of miR-410 and miR-433, two miRNAs in the Gtl2-Dio3 locus that repress sFRP2, or by treatment with recombinant WNT3A and WNT5A. Thus, miRNA-mediated modulation of WNT signaling by MEF2A is a requisite step for proper muscle regeneration, and represents an attractive pathway for enhancing regeneration of diseased muscle.

CKIP-1 inhibits cardiac hypertrophy by regulating class II histone deacetylase phosphorylation through recruiting PP2A.

BACKGROUND: Sustained cardiac pressure overload-induced hypertrophy and pathological remodeling frequently leads to heart failure. Casein kinase-2 interacting protein-1 (CKIP-1) has been identified to be an important regulator of cell proliferation, differentiation, and apoptosis. However, the physiological role of CKIP-1 in the heart is unknown. METHODS AND RESULTS: The results of echocardiography and histology demonstrate that CKIP-1-deficient mice exhibit spontaneous cardiac hypertrophy with aging and hypersensitivity to pressure overload-induced pathological cardiac hypertrophy, as well. Transgenic mice with cardiac-specific overexpression of CKIP-1 showed resistance to cardiac hypertrophy in response to pressure overload. The results of GST pull-down and coimmunoprecipitation assays showed the interaction between CKIP-1 and histone deacetylase 4 (HDAC4), through which they synergistically inhibited transcriptional activity of myocyte-specific enhancer factor 2C. By directly interacting with the catalytic subunit of phosphatase 2A, CKIP-1 overexpression enhanced the binding of catalytic subunit of phosphatase-2A to HDAC4 and promoted HDAC4 dephosphorylation. CONCLUSIONS: CKIP-1 was found to be an inhibitor of cardiac hypertrophy by upregulating the dephosphorylation of HDAC4 through the recruitment of protein phosphatase 2A. These results demonstrated a unique function of CKIP-1, by which it suppresses cardiac hypertrophy through its capacity to regulate HDAC4 dephosphorylation and fetal cardiac genes expression.

[Regulation of myostatin promoter activity by myocyte enhancer factor 2].

Myostatin (Mstn) is a member of the transforming growth factor-beta superfamily that functions as a negative regulator of skeletal muscle growth and differentiation in mammals. The transcriptional regulation of Mstn is controlled by multiple genes including MEF2, which raise the importance of identifying the binding sites of MEF2 on myostatin promoter region and mechanisms underlying. In this study, we investigated the transcriptional regulation of MEF2 on porcine Mstn promoter activity in C2C12 cells. Sequence analysis of the 1 969 bp porcine Mstn promoter region revealed that it contained three potential MEF2 motifs. Using a serial deletion strategy, we tested the activity of several promoter fragments by luciferase assay. Overexpression of MEF2C, but not MEF2A increased Mstn promoter activity in all the promoter fragments with MEF2 motifs by two to six folds, in both C2C12 myoblasts and myotubes. When we transfected exogenous MEF2C, Mstn mRNA level was also upregulated in C2C12 cells, but the protein level was only significantly increased in myotubes. Thus, we propose that MEF2C could modulate and restrain myogenesis by Mstn activation and Mstn-dependent gene processing in porcine. Our research also provided potential targets and an effective molecule to regulate Mstn expression and gave a new way to explore the functional performance of Mstn.

Induction of cardiomyocyte-like cells in infarct hearts by gene transfer of Gata4, Mef2c, and Tbx5.

RATIONALE: After myocardial infarction (MI), massive cell death in the myocardium initiates fibrosis and scar formation, leading to heart failure. We recently found that a combination of 3 cardiac transcription factors, Gata4, Mef2c, and Tbx5 (GMT), reprograms fibroblasts directly into functional cardiomyocytes in vitro. OBJECTIVE: To investigate whether viral gene transfer of GMT into infarcted hearts induces cardiomyocyte generation. METHODS AND RESULTS: Coronary artery ligation was used to generate MI in the mouse. In vitro transduction of GMT retrovirus converted cardiac fibroblasts from the infarct region into cardiomyocyte-like cells with cardiac-specific gene expression and sarcomeric structures. Injection of the green fluorescent protein (GFP) retrovirus into mouse hearts, immediately after MI, infected only proliferating noncardiomyocytes, mainly fibroblasts, in the infarct region. The GFP expression diminished after 2 weeks in immunocompetent mice but remained stable for 3 months in immunosuppressed mice, in which cardiac induction did not occur. In contrast, injection of GMT retrovirus into α-myosin heavy chain (αMHC)-GFP transgenic mouse hearts induced the expression of αMHC-GFP, a marker of cardiomyocytes, in 3% of virus-infected cells after 1 week. A pooled GMT injection into the immunosuppressed mouse hearts induced cardiac marker expression in retrovirus-infected cells within 2 weeks, although few cells showed striated muscle structures. To transduce GMT efficiently in vivo, we generated a polycistronic retrovirus expressing GMT separated by 2A "self-cleaving" peptides (3F2A). The 3F2A-induced cardiomyocyte-like cells in fibrotic tissue expressed sarcomeric α-actinin and cardiac troponin T and had clear cross striations. Quantitative RT-PCR also demonstrated that FACS-sorted 3F2A-transduced cells expressed cardiac-specific genes. CONCLUSIONS: GMT gene transfer induced cardiomyocyte-like cells in infarcted hearts.

MEF2 negatively regulates learning-induced structural plasticity and memory formation.

Memory formation is thought to be mediated by dendritic-spine growth and restructuring. Myocyte enhancer factor 2 (MEF2) restricts spine growth in vitro, suggesting that this transcription factor negatively regulates the spine remodeling necessary for memory formation. Here we show that memory formation in adult mice was associated with changes in endogenous MEF2 levels and function. Locally and acutely increasing MEF2 function in the dentate gyrus blocked both learning-induced increases in spine density and spatial-memory formation. Increasing MEF2 function in amygdala disrupted fear-memory formation. We rescued MEF2-induced memory disruption by interfering with AMPA receptor endocytosis, suggesting that AMPA receptor trafficking is a key mechanism underlying the effects of MEF2. In contrast, decreasing MEF2 function in dentate gyrus and amygdala facilitated the formation of spatial and fear memory, respectively. These bidirectional effects indicate that MEF2 is a key regulator of plasticity and that relieving the suppressive effects of MEF2-mediated transcription permits memory formation.

Neurotoxin-induced selective ubiquitination and regulation of MEF2A isoform in neuronal stress response.

The myocyte enhancer factor 2A-D (MEF2) proteins are members of the MCM1-agamous-deficiens-serum response factor family of transcription factors. Various MEF2 isoform proteins are enriched in neurons and exhibit distinct patterns of expression in different regions of the brain. In neurons, MEF2 functions as a converging factor to regulate many neuronal functions including survival. MEF2 activities are tightly controlled in neurons in response to stress. Whether stress signal may differentially regulate MEF2s remains largely unknown. In this work, we showed that MEF2A, but not MEF2C or MEF2D, was modified by ubiquitination in dopaminergic neuronal cell line SN4741 cells. MEF2A was ubiquitinated at its N'-terminus, and the ubiquitination of MEF2A was first detectable in the nuclear compartment and later in the cytoplasm. Ubiquitination of MEF2A correlated with reduced DNA-binding activity and transcriptional activity. More importantly, disturbing the degradation of ubiquitinated MEF2A through proteasome pathway by neurotoxins known to induce Parkinson's disease features in model animals caused accumulation of ubiquitinated MEF2A, reduced MEF2 activity, and impaired cellular viability. Our work thus provides the first evidence to demonstrate an isoforms-specific regulation of MEF2s by ubiquitination-proteasome pathway in dopaminergic neuronal cell by neurotoxins, suggesting that stress signal and cellular context-dependent dysregulation of MEF2s may underlie the loss of neuronal viability.

Notch and Mef2 synergize to promote proliferation and metastasis through JNK signal activation in Drosophila.

Genetic analyses in Drosophila revealed a synergy between Notch and the pleiotropic transcription factor Mef2 (myocyte enhancer factor 2), which profoundly influences proliferation and metastasis. We show that these hyperproliferative and invasive Drosophila phenotypes are attributed to upregulation of eiger, a member of the tumour necrosis factor superfamily of ligands, and the consequent activation of Jun N-terminal kinase signalling, which in turn triggers the expression of the invasive marker MMP1. Expression studies in human breast tumour samples demonstrate correlation between Notch and Mef2 paralogues and support the notion that Notch-MEF2 synergy may be significant for modulating human mammary oncogenesis.

Functional and mechanistic exploration of an adult neurogenesis-promoting small molecule.

Adult neurogenesis occurs throughout life in the mammalian hippocampus and is essential for memory and mood control. There is significant interest in identifying ways to promote neurogenesis and ensure maintenance of these hippocampal functions. Previous work with a synthetic small molecule, isoxazole 9 (Isx-9), highlighted its neuronal-differentiating properties in vitro. However, the ability of Isx-9 to drive neurogenesis in vivo or improve hippocampal function was unknown. Here we show that Isx-9 promotes neurogenesis in vivo, enhancing the proliferation and differentiation of hippocampal subgranular zone (SGZ) neuroblasts, and the dendritic arborization of adult-generated dentate gyrus neurons. Isx-9 also improves hippocampal function, enhancing memory in the Morris water maze. Notably, Isx-9 enhances neurogenesis and memory without detectable increases in cellular or animal activity or vascularization. Molecular exploration of Isx-9-induced regulation of neurogenesis (via FACS and microarray of SGZ stem and progenitor cells) suggested the involvement of the myocyte-enhancer family of proteins (Mef2). Indeed, transgenic-mediated inducible knockout of all brain-enriched Mef2 isoforms (Mef2a/c/d) specifically from neural stem cells and their progeny confirmed Mef2's requirement for Isx-9-induced increase in hippocampal neurogenesis. Thus, Isx-9 enhances hippocampal neurogenesis and memory in vivo, and its effects are reliant on Mef2, revealing a novel cell-intrinsic molecular pathway regulating adult neurogenesis.

Myh7b/miR-499 gene expression is transcriptionally regulated by MRFs and Eos.

The sarcomeric myosin gene, Myh7b, encodes an intronic microRNA, miR-499, which regulates cardiac and skeletal muscle biology, yet little is known about its transcriptional regulation. To identify the transcription factors involved in regulating Myh7b/miR-499 gene expression, we have mapped the transcriptional start sites and identified an upstream 6.2 kb region of the mouse Myh7b gene whose activity mimics the expression pattern of the endogenous Myh7b gene both in vitro and in vivo. Through promoter deletion analysis, we have mapped a distal E-box element and a proximal Ikaros site that are essential for Myh7b promoter activity in muscle cells. We show that the myogenic regulatory factors, MyoD, Myf5 and Myogenin, bind to the E-box, while a lymphoid transcription factor, Ikaros 4 (Eos), binds to the Ikaros motif. Further, we show that through physical interaction, MyoD and Eos form an active transcriptional complex on the chromatin to regulate the expression of the endogenous Myh7b/miR-499 gene in muscle cells. We also provide the first evidence that Eos can regulate expression of additional myosin genes (Myosin 1 and ß-Myosin) via the miR-499/Sox6 pathway. Therefore, our results indicate a novel role for Eos in the regulation of the myofiber gene program.