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1.  Vgll2a is required for neural crest cell survival during zebrafish craniofacial development 
Developmental biology  2011;357(1):269-281.
Invertebrate and vertebrate vestigial (vg) and vestigial-like (vgl) genes are involved in embryonic patterning and cell fate determination. These genes encode cofactors that interact with members of the TEAD/Scalloped family of transcription factors and modulate their activity. We have previously shown that, in mice, Vgll2 is differentially expressed in the developing facial prominences. In this study, we show that the zebrafish ortholog vgll2a is expressed in the pharyngeal endoderm and ectoderm surrounding the neural crest derived mesenchyme of the pharyngeal arches. Moreover, both the FGF and retinoic acid (RA) signaling pathways, which are critical components of the hierarchy controlling craniofacial patterning, regulate this domain of vgll2a expression. Consistent with these observations, vgll2a is required within the pharyngeal endoderm for NCC survival and pharyngeal cartilage development. Specifically, knockdown of Vgll2a in zebrafish embryos using Morpholino injection results in increased cell death within the pharyngeal arches, aberrant endodermal pouch morphogenesis, and hypoplastic cranial cartilages. Overall, our data reveal a novel non-cell autonomous role for Vgll2a in development of the NCC-derived vertebrate craniofacial skeleton.
PMCID: PMC3519931  PMID: 21741961
Vestigial-like; Vgl-2; VITO-1; craniofacial; zebrafish; FGF; Retinoic Acid; Cell Death
2.  Redundant Roles of Tead1 and Tead2 in Notochord Development and the Regulation of Cell Proliferation and Survival▿  
Molecular and Cellular Biology  2008;28(10):3177-3189.
Four members of the TEAD/TEF family of transcription factors are expressed widely in mouse embryos and adult tissues. Although in vitro studies have suggested various roles for TEAD proteins, their in vivo functions remain poorly understood. Here we examined the role of Tead genes by generating mouse mutants for Tead1 and Tead2. Tead2−/− mice appeared normal, but Tead1−/−; Tead2−/− embryos died at embryonic day 9.5 (E9.5) with severe growth defects and morphological abnormalities. At E8.5, Tead1−/−; Tead2−/− embryos were already small and lacked characteristic structures such as a closed neural tube, a notochord, and somites. Despite these overt abnormalities, differentiation and patterning of the neural plate and endoderm were relatively normal. In contrast, the paraxial mesoderm and lateral plate mesoderm were displaced laterally, and a differentiated notochord was not maintained. These abnormalities and defects in yolk sac vasculature organization resemble those of mutants for Yap, which encodes a coactivator of TEAD proteins. Moreover, we demonstrated genetic interactions between Tead1 and Tead2 and Yap. Finally, Tead1−/−; Tead2−/− embryos showed reduced cell proliferation and increased apoptosis. These results suggest that Tead1 and Tead2 are functionally redundant, use YAP as a major coactivator, and support notochord maintenance as well as cell proliferation and survival in mouse development.
PMCID: PMC2423158  PMID: 18332127
3.  Transcription factor TEAD4 regulates expression of Myogenin and the unfolded protein response genes during C2C12 cell differentiation 
Cell Death and Differentiation  2011;19(2):220-231.
The TEAD (1–4) transcription factors comprise the conserved TEA/ATTS DNA-binding domain recognising the MCAT element in the promoters of muscle-specific genes. Despite extensive genetic analysis, the function of TEAD factors in muscle differentiation has proved elusive due to redundancy among the family members. Expression of the TEA/ATTS DNA-binding domain that acts as a dominant negative repressor of TEAD factors in C2C12 myoblasts inhibits their differentiation, whereas selective shRNA knockdown of TEAD4 results in abnormal differentiation characterised by the formation of shortened myotubes. Chromatin immunoprecipitation coupled to array hybridisation shows that TEAD4 occupies 867 promoters including those of myogenic miRNAs. We show that TEAD factors directly induce Myogenin, CDKN1A and Caveolin 3 expression to promote myoblast differentiation. RNA-seq identifies a set of genes whose expression is strongly reduced upon TEAD4 knockdown among which are structural and regulatory proteins and those required for the unfolded protein response. In contrast, TEAD4 represses expression of the growth factor CTGF (connective tissue growth factor) to promote differentiation. Together these results show that TEAD factor activity is essential for normal C2C12 cell differentiation and suggest a role for TEAD4 in regulating expression of the unfolded protein response genes.
PMCID: PMC3263497  PMID: 21701496
MYOD1; ER-stress; myoblast fusion; Chromatin immunoprecipitation; RNA-seq
4.  TEAD1-dependent expression of the FoxO3a gene in mouse skeletal muscle 
TEAD1 (TEA domain family member 1) is constitutively expressed in cardiac and skeletal muscles. It acts as a key molecule of muscle development, and trans-activates multiple target genes involved in cell proliferation and differentiation pathways. However, its target genes in skeletal muscles, regulatory mechanisms and networks are unknown.
In this paper, we have identified 136 target genes regulated directly by TEAD1 in skeletal muscle using integrated analyses of ChIP-on-chip. Most of the targets take part in the cell process, physiology process, biological regulation metabolism and development process. The targets also play an important role in MAPK, mTOR, T cell receptor, JAK-STAT, calcineurin and insulin signaling pathways. TEAD1 regulates foxo3a transcription through binding to the M-CAT element in foxo3a promoter, demonstrated with independent ChIP-PCR, EMSA and luciferase reporter system assay. In addition, results of over-expression and inhibition experiments suggest that foxo3a is positively regulated by TEAD1.
Our present data suggests that TEAD1 plays an important role in the regulation of gene expression and different signaling pathways may co-operate with each other mediated by TEAD1. We have preliminarily concluded that TEAD1 may regulate FoxO3a expression through calcineurin/MEF2/NFAT and IGF-1/PI3K/AKT signaling pathways in skeletal muscles. These findings provide important clues for further analysis of the role of FoxO3a gene in the formation and transformation of skeletal muscle fiber types.
PMCID: PMC3025863  PMID: 21211055
5.  Essential Role of TEA Domain Transcription Factors in the Negative Regulation of the MYH 7 Gene by Thyroid Hormone and Its Receptors 
PLoS ONE  2014;9(4):e88610.
MYH7 (also referred to as cardiac myosin heavy chain β) gene expression is known to be repressed by thyroid hormone (T3). However, the molecular mechanism by which T3 inhibits the transcription of its target genes (negative regulation) remains to be clarified, whereas those of transcriptional activation by T3 (positive regulation) have been elucidated in detail. Two MCAT (muscle C, A, and T) sites and an A/T-rich region in the MYH7 gene have been shown to play a critical role in the expression of this gene and are known to be recognized by the TEAD/TEF family of transcription factors (TEADs). Using a reconstitution system with CV-1 cells, which has been utilized in the analysis of positive as well as negative regulation, we demonstrate that both T3 receptor (TR) β1 and α1 inhibit TEAD-dependent activation of the MYH7 promoter in a T3 dose-dependent manner. TRβ1 bound with GC-1, a TRβ-selective T3 analog, also repressed TEAD-induced activity. Although T3-dependent inhibition required the DNA-binding domain (DBD) of TRβ1, it remained after the putative negative T3-responsive elements were mutated. A co-immunoprecipitation study demonstrated the in vivo association of TRβ1 with TEAD-1, and the interaction surfaces were mapped to the DBD of the TRβ1 and TEA domains of TEAD-1, both of which are highly conserved among TRs and TEADs, respectively. The importance of TEADs in MYH7 expression was also validated with RNA interference using rat embryonic cardiomyocyte H9c2 cells. These results indicate that T3-bound TRs interfere with transactivation by TEADs via protein-protein interactions, resulting in the negative regulation of MYH7 promoter activity.
PMCID: PMC4004540  PMID: 24781449
6.  Alteration of TEAD1 Expression Levels Confers Apoptotic Resistance through the Transcriptional Up-Regulation of Livin 
PLoS ONE  2012;7(9):e45498.
TEA domain (TEAD) proteins are highly conserved transcription factors involved in embryonic development and differentiation of various tissues. More recently, emerging evidences for a contribution of these proteins towards apoptosis and cell proliferation regulation have also been proposed. These effects appear to be mediated by the interaction between TEAD and its co-activator Yes-Associated Protein (YAP), the downstream effector of the Hippo tumour suppressor pathway.
Methodology/Principal Findings
We further investigated the mechanisms underlying TEAD-mediated apoptosis regulation and showed that overexpression or RNAi-mediated silencing of the TEAD1 protein is sufficient to protect mammalian cell lines from induced apoptosis, suggesting a proapoptotic function for TEAD1 and a non physiological cytoprotective effect for overexpressed TEAD1. Moreover we show that the apoptotic resistance conferred by altered TEAD1 expression is mediated by the transcriptional up-regulation of Livin, a member of the Inhibitor of Apoptosis Protein (IAP) family. In addition, we show that overexpression of a repressive form of TEAD1 can induce Livin up-regulation, indicating that the effect of TEAD1 on Livin expression is indirect and favoring a model in which TEAD1 activates a repressor of Livin by interacting with a limiting cofactor that gets titrated upon TEAD1 up-regulation. Interestingly, we show that overexpression of a mutated form of TEAD1 (Y421H) implicated in Sveinsson's chorioretinal atrophy that strongly reduces its interaction with YAP as well as its activation, can induce Livin expression and protect cells from induced apoptosis, suggesting that YAP is not the cofactor involved in this process.
Taken together our data reveal a new, Livin-dependent, apoptotic role for TEAD1 in mammals and provide mechanistic insight downstream of TEAD1 deregulation in cancers.
PMCID: PMC3454436  PMID: 23029054
7.  Myofiber-specific TEAD1 overexpression drives satellite cell hyperplasia and counters pathological effects of dystrophin deficiency 
eLife  null;5:e15461.
When unperturbed, somatic stem cells are poised to affect immediate tissue restoration upon trauma. Yet, little is known regarding the mechanistic basis controlling initial and homeostatic ‘scaling’ of stem cell pool sizes relative to their target tissues for effective regeneration. Here, we show that TEAD1-expressing skeletal muscle of transgenic mice features a dramatic hyperplasia of muscle stem cells (i.e. satellite cells, SCs) but surprisingly without affecting muscle tissue size. Super-numeral SCs attain a ‘normal’ quiescent state, accelerate regeneration, and maintain regenerative capacity over several injury-induced regeneration bouts. In dystrophic muscle, the TEAD1 transgene also ameliorated the pathology. We further demonstrate that hyperplastic SCs accumulate non-cell-autonomously via signal(s) from the TEAD1-expressing myofiber, suggesting that myofiber-specific TEAD1 overexpression activates a physiological signaling pathway(s) that determines initial and homeostatic SC pool size. We propose that TEAD1 and its downstream effectors are medically relevant targets for enhancing muscle regeneration and ameliorating muscle pathology.
eLife digest
Skeletal muscles are primarily composed of cells called muscle fibers, which attach to bones via tendons. These muscle fibers contract to help move the body. Muscle also contains a population of muscle stem cells that repair injured tissue. Normally, in adult skeletal muscle, these stems cells are in a resting state. However, upon injury, the stem cells become activated, divide to increase in number and then develop into new muscle fibers to replace those that were damaged. The balance between the number of stem cells and the size of the muscle must be tightly regulated to ensure that there are enough stem cells to fully regenerate the tissue after injury. However, little is known about how tissues keep their number of stem cells in proportion with their overall size.
Previous attempts to make mice with more muscle stem cells invariably also created mice with larger muscles overall. This raised the question: is it possible to increase the numbers of stem cells without changing the size of the muscle?
Now, Southard, Kim et al. show it is possible and report that mice engineered to overproduce a protein called Tead1 in their muscle fibers have up to 6-times more stem cells yet normally sized muscles. Tead1 is a transcription factor that controls the activity of a number of genes as part of a major signaling pathway.
The stem cells in mice that overproduce Tead1 began to increase in number two weeks after the mice were born because they went through additional rounds of cell division before they entered the resting state. Further experiments then showed that having more stem cells meant that the muscles were repaired more quickly after an injury. Additionally, when mice with extra Tead1 had a mutation that normally leads to muscle wasting, experiments showed that the progression of the disease was stunted.
Southard, Kim et al. also show that the muscle fibers that are directly attached to the muscle stem cells are needed for the stem cells to increase in number in the Tead1-overexpressing mice. Together these findings suggest that a signal from the muscle fiber to its stem cells regulates the size of the stem cell population in the tissue. The next challenge is to uncover the molecule (or molecules) that signals from the muscle fiber to the stem cells and to gain deeper insight into how the Tead1 protein can counteract the effects of a muscle wasting disease.
PMCID: PMC5059137  PMID: 27725085
skeletal muscle; satellite cell; regeneration; muscular dystrophy; Mouse
8.  VITO-1 is an essential cofactor of TEF1-dependent muscle-specific gene regulation 
Nucleic Acids Research  2004;32(2):791-802.
The expression of several muscle-specific genes is partially or completely regulated by MCAT elements, which bind members of the TEF family of transcription factors. TEF1 itself is unable to activate reporter plasmids bearing TEF1-binding sites, suggesting that additional bridging or co-activating factors are necessary to allow interaction of TEF1 with the transcriptional machinery. In addition, none of the known TEF genes are exclusively expressed in the cardiac or skeletal muscle lineage to account for the muscle-specific expression of MCAT-dependent genes. Here we describe that VITO-1, a new SID (scalloped interaction domain)-containing protein, binds to TEF1 in vitro and strongly stimulates transcription of a MCAT reporter plasmid together with TEF-1. Since VITO-1 is predominantly expressed in the skeletal muscle lineage, it might serve as an essential transcriptional intermediary factor to promote muscle-specific expression via MCAT cis-regulatory elements. Although VITO-1 alone is not sufficient to initiate myogenic conversion of 10T1/2 fibroblastic cells, it enhanced MyoD-mediated myogenic conversion. In addition, interference with VITO-1 expression by siRNA attenuated differentiation of C2C12 muscle cells and MyoD-dependent myogenesis in 10T1/2 cells. We conclude that VITO-1 is a crucial new cofactor of the muscle regulatory programme.
PMCID: PMC373362  PMID: 14762206
9.  Members of the TEAD family of transcription factors regulate the expression of Myf5 in ventral somitic compartments 
Developmental Biology  2011;355(2-2):372-380.
The transcriptional regulation of the Mrf4/Myf5 locus depends on a multitude of enhancers that, in equilibria with transcription balancing sequences and the promoters, regulate the expression of the two genes throughout embryonic development and in the adult. Transcription in a particular set of muscle progenitors can be driven by the combined outputs of several enhancers that are not able to recapitulate the entire expression pattern in isolation, or by the action of a single enhancer the activity of which in isolation is equivalent to that within the context of the locus. We identified a new enhancer element of this second class, ECR111, which is highly conserved in all vertebrate species and is necessary and sufficient to drive Myf5 expression in ventro-caudal and ventro-rostral somitic compartments in the mouse embryo. EMSA analyses and data obtained from binding-site mutations in transgenic embryos show that a binding site for a TEA Domain (TEAD) transcription factor is essential for the function of this new enhancer, while ChIP assays show that at least two members of the family of transcription factors bind to it in vivo.
► The ECR111 enhancer controls Myf5 expression in ventral somitic compartments in early development. ► A TEAD binding site is essential for the activity of the ECR111 enhancer. ► ChIP analyses show that TEAD factors bind in vivo to the ECR111 enhancer. ► TEAD proteins localise to the myotome and the dermomyotome during early development.
PMCID: PMC3123743  PMID: 21527258
Myf5; Mrf4; TEAD; Myogenic regulatory factor; Myogenesis; Somite; Dermomyotome; Transcription regulation
10.  Thyroid hormone regulates muscle fiber type conversion via miR-133a1 
The Journal of Cell Biology  2014;207(6):753-766.
Thyroid hormone promotes slow-to-fast muscle fiber type conversion by inducing miR-133a1 and thereby repressing the expression of the slow muscle determinant TEAD1.
It is known that thyroid hormone (TH) is a major determinant of muscle fiber composition, but the molecular mechanism by which it does so remains unclear. Here, we demonstrated that miR-133a1 is a direct target gene of TH in muscle. Intriguingly, miR-133a, which is enriched in fast-twitch muscle, regulates slow-to-fast muscle fiber type conversion by targeting TEA domain family member 1 (TEAD1), a key regulator of slow muscle gene expression. Inhibition of miR-133a in vivo abrogated TH action on muscle fiber type conversion. Moreover, TEAD1 overexpression antagonized the effect of miR-133a as well as TH on muscle fiber type switch. Additionally, we demonstrate that TH negatively regulates the transcription of myosin heavy chain I indirectly via miR-133a/TEAD1. Collectively, we propose that TH inhibits the slow muscle phenotype through a novel epigenetic mechanism involving repression of TEAD1 expression via targeting by miR-133a1. This identification of a TH-regulated microRNA therefore sheds new light on how TH achieves its diverse biological activities.
PMCID: PMC4274265  PMID: 25512392
11.  The TEAD Family and Its Oncogenic Role in Promoting Tumorigenesis 
The TEAD family of transcription factors is necessary for developmental processes. The family members contain a TEA domain for the binding with DNA elements and a transactivation domain for the interaction with transcription coactivators. TEAD proteins are required for the participation of coactivators to transmit the signal of pathways for the downstream signaling processes. TEADs also play an important role in tumor initiation and facilitate cancer progression via activating a series of progression-inducing genes, such as CTGF, Cyr61, Myc and Gli2. Recent studies have highlighted that TEADs, together with their coactivators, promote or even act as the crucial parts in the development of various malignancies, such as liver, ovarian, breast and prostate cancers. Furthermore, TEADs are proposed to be useful prognostic biomarkers due to the ideal correlation between high expression and clinicopathological parameters in gastric, breast, ovarian and prostate cancers. In this review, we summarize the functional role of TEAD proteins in tumorigenesis and discuss the key role of TEAD transcription factors in the linking of signal cascade transductions. Improved knowledge of the TEAD proteins will be helpful for deep understanding of the molecular mechanisms of tumorigenesis and identifying ideal predictive or prognostic biomarkers, even providing clinical translation for anticancer therapy in human cancers.
PMCID: PMC4730377  PMID: 26805820
TEAD proteins; transcription factor; Hippo pathway; YAP; TAZ; vgll
12.  Alternative Requirements for Vestigial, Scalloped, and Dmef2 during Muscle Differentiation in Drosophila melanogaster 
Molecular Biology of the Cell  2009;20(1):256-269.
Vertebrate development requires the activity of the myocyte enhancer factor 2 (mef2) gene family for muscle cell specification and subsequent differentiation. Additionally, several muscle-specific functions of MEF2 family proteins require binding additional cofactors including members of the Transcription Enhancing Factor-1 (TEF-1) and Vestigial-like protein families. In Drosophila there is a single mef2 (Dmef2) gene as well single homologues of TEF-1 and vestigial-like, scalloped (sd), and vestigial (vg), respectively. To clarify the role(s) of these factors, we examined the requirements for Vg and Sd during Drosophila muscle specification. We found that both are required for muscle differentiation as loss of sd or vg leads to a reproducible loss of a subset of either cardiac or somatic muscle cells in developing embryos. This muscle requirement for Sd or Vg is cell specific, as ubiquitous overexpression of either or both of these proteins in muscle cells has a deleterious effect on muscle differentiation. Finally, using both in vitro and in vivo binding assays, we determined that Sd, Vg, and Dmef2 can interact directly. Thus, the muscle-specific phenotypes we have associated with Vg or Sd may be a consequence of alternative binding of Vg and/or Sd to Dmef2 forming alternative protein complexes that modify Dmef2 activity.
PMCID: PMC2613084  PMID: 18987343
13.  Establishment of transgenic lines to monitor and manipulate Yap/Taz-Tead activity in zebrafish reveals both evolutionarily conserved and divergent functions of the Hippo pathway 
Mechanisms of development  2014;0:177-188.
To investigate the role of Hippo pathway signaling during vertebrate development transgenic zebrafish lines were generated and validated to dynamically monitor and manipulate Yap/Taz-Tead activity. Spatial and temporal analysis of Yap/Taz-Tead activity suggested the importance of Hippo signaling during cardiac precursor migration and other developmental processes. When the transcriptional co-activators, Yap and Taz were restricted from interacting with DNA-binding Tead transcription factors through expression of a dominant negative transgene, cardiac precursors failed to migrate completely to the midline resulting in strong cardia bifida. Yap/Taz-Tead activity reporters also allowed us to investigate upstream and downstream factors known to regulate Hippo signaling output in Drosophila. While Crumbs mutations in Drosophila eye disc epithelia increase nuclear translocation and activity of Yorkie (the fly homolog of Yap/Taz), zebrafish crb2a mutants lacked nuclear Yap positive cells and down-regulated Yap/Taz-Tead activity reporters in the eye epithelia, despite the loss of apical-basal cell polarity in those cells. However, as an example of evolutionary conservation, the Tondu-domain containing protein Vestigial-like 4b (Vgll4b) was found to down-regulate endogenous Yap/Taz-Tead activity in the retinal pigment epithelium, similar to Drosophila Tgi in imaginal discs. In conclusion, the Yap/Taz-Tead activity reporters revealed the dynamics of Yap/Taz-Tead signaling and novel insights into Hippo pathway regulation for vertebrates. These studies highlight the utility of this transgenic tool-suite for ongoing analysis into the mechanisms of Hippo pathway regulation and the consequences of signaling output.
PMCID: PMC4138299  PMID: 24560909
Hippo signaling; fluorescent reporter; heart development; eye development; in vivo imaging
14.  Validation of chemical compound library screening for transcriptional co‐activator with PDZ‐binding motif inhibitors using GFP‐fused transcriptional co‐activator with PDZ‐binding motif 
Cancer Science  2016;107(6):791-802.
Transcriptional co‐activator with PDZ‐binding motif (TAZ) plays versatile roles in cell proliferation and differentiation. It is phosphorylated by large tumor suppressor kinases, the core kinases of the tumor‐suppressive Hippo pathway. Phosphorylation induces the cytoplasmic accumulation of TAZ and its degradation. In human cancers, the deregulation of the Hippo pathway and gene amplification enhance TAZ activity. TAZ interacts with TEA domain family members (TEAD), and upregulates genes implicated in epithelial–mesenchymal transition. It also confers stemness to cancer cells. Thus, TAZ activation provides cancer cells with malignant properties and worsens the clinical prognosis. Therefore, TAZ attracts attention as a therapeutic target in cancer therapy. We applied 18 606 small chemical compounds to human osteosarcoma U2OS cells expressing GFP‐fused TAZ (GFP‐TAZ), monitored the subcellular localization of GFP‐TAZ, and selected 33 compounds that shifted GFP‐TAZ to the cytoplasm. Unexpectedly, only a limited number of compounds suppressed TAZ‐mediated enhancement of TEAD‐responsive reporter activity. Moreover, the compounds that weakened TEAD reporter activity did not necessarily decrease the unphosphorylated TAZ. In this study, we focused on three compounds that decreased both TEAD reporter activity and unphosphorylated TAZ, and treated several human cancer cells with these compounds. One compound did not show a remarkable effect, whereas the other two compounds compromised the cell viability in certain cancer cells. In conclusion, the GFP‐TAZ‐based assay can be used as the first screening for compounds that inhibit TAZ and show anticancer properties. To develop anticancer drugs, we need additional assays to select the compounds.
PMCID: PMC4968592  PMID: 27009852
Drug screening; GFP; Hippo pathway; phosphorylation; TAZ
15.  Fgfr4 is required for effective muscle regeneration in vivo: Delineation of a MyoD-Tead2-Fgfr4 transcriptional pathway 
The Journal of biological chemistry  2005;281(1):429-438.
Fgfr4 has been shown to be important for appropriate muscle development in chick limb buds, however, Fgfr4 null mice show no phenotype. Here, we show that staged induction of muscle regeneration in Fgfr4 null mice becomes highly abnormal at the time point when Fgfr4 is normally expressed. By 7 days of regeneration, differentiation of myotubes became poorly coordinated and delayed by both histology and embryonic myosin heavy chain staining. By 14 days, much of the muscle was replaced by fat and calcifications. To begin to dissect the molecular pathways involving Fgfr4, we queried the promoter sequences for transcriptional factor binding sites, and tested candidate regulators in a 27 time point regeneration series. The Fgfr4 promoter region contained a Tead protein binding site (M-CAT 5′-CATTCCT-3′), and Tead2 showed induction during regeneration commensurate with Fgfr4 regulation. Co-transfection of Tead2 and Fgfr4 promoter reporter constructs into C2C12 myotubes showed Tead2 to activate Fgfr4, and mutation of the M-CAT motif in the Fgfr4 promoter abolished these effects. Immunostaining for Tead2 showed timed expression in myotube nuclei consistent with the mRNA data. Query of the expression timing and genomic sequences of Tead2 suggested direct regulation by MyoD, and, consistent with this, MyoD directly bound to two strong E-boxes in the first intron of Tead2 by chromatin immunoprecipitation assay. Moreover, co-transfection of MyoD and Tead2 intron reporter constructs into 10T1/2 cells activated reporter activity in a dose dependent manner. This activation was greatly reduced when the two E-boxes were mutated. Our data suggest a novel MyoD-Tead2-Fgfr4 pathway important for effective muscle regeneration.
PMCID: PMC1892582  PMID: 16267055
Muscle regeneration; Tead; TEF; Fgfr; MyoD; Microarray
16.  The Hippo pathway mediates inhibition of vascular smooth muscle cell proliferation by cAMP 
Inhibition of vascular smooth muscle cell (VSMC) proliferation by intracellular cAMP prevents excessive neointima formation and hence angioplasty restenosis and vein-graft failure. These protective effects are mediated via actin-cytoskeleton remodelling and subsequent regulation of gene expression by mechanisms that are incompletely understood. Here we investigated the role of components of the growth-regulatory Hippo pathway, specifically the transcription factor TEAD and its co-factors YAP and TAZ in VSMC.
Methods and results
Elevation of cAMP using forskolin, dibutyryl-cAMP or the physiological agonists, Cicaprost or adenosine, significantly increased phosphorylation and nuclear export YAP and TAZ and inhibited TEAD-luciferase report gene activity. Similar effects were obtained by inhibiting RhoA activity with C3-transferase, its downstream kinase, ROCK, with Y27632, or actin-polymerisation with Latrunculin-B. Conversely, expression of constitutively-active RhoA reversed the inhibitory effects of forskolin on TEAD-luciferase. Forskolin significantly inhibited the mRNA expression of the pro-mitogenic genes, CCN1, CTGF, c-MYC and TGFB2 and this was reversed by expression of constitutively-active YAP or TAZ phospho-mutants. Inhibition of YAP and TAZ function with RNAi or Verteporfin significantly reduced VSMC proliferation. Furthermore, the anti-mitogenic effects of forskolin were reversed by overexpression of constitutively-active YAP or TAZ.
Taken together, these data demonstrate that cAMP-induced actin-cytoskeleton remodelling inhibits YAP/TAZ–TEAD dependent expression of pro-mitogenic genes in VSMC. This mechanism contributes novel insight into the anti-mitogenic effects of cAMP in VSMC and suggests a new target for intervention.
Graphical abstract
Elevated cAMP inhibits the activity of Rho GTPases and thus antagonises actin polymerisation. Impaired actin polymerisation induces the phosphorylation and nuclear export of YAP and TAZ. The nuclear export of YAP and TAZ reduces the activity of TEAD transcription factors and the transcription of TEAD-dependent cell-cycle genes. This mechanism underlies the anti-mitogenic effects of cAMP in vascular smooth muscle cells.
•Elevated cAMP induces phosphorylation and nuclear export of YAP and TAZ in VSMC.•Elevated cAMP inhibits TEAD-dependent transcription of proliferation genes in VSMC.•YAP and TAZ are essential for VSMC proliferation.•The anti-mitogenic effects of cAMP in VSMC are dependent on inhibition of YAP/TAZ–TEAD mediated gene expression.
PMCID: PMC4727789  PMID: 26625714
YAP; TAZ; TEAD; cAMP; 3′-5′-Cyclic adenosine monophosphate; VSMC
17.  Tead and AP1 coordinate transcription and motility 
Cell reports  2016;14(5):1169-1180.
The Tead family transcription factors are the major intracellular mediators of the Hippo-Yap pathway. Despite the importance of Hippo signaling in tumorigenesis, Tead-dependent downstream oncogenic programs and target genes in cancer cells remain poorly understood. Here we characterize Tead4-mediated transcriptional networks in a diverse range of cancer cells, including neuroblastoma, colorectal, lung, and endometrial carcinomas. By intersecting genome-wide chromatin occupancy analyses of Tead4, JunD and Fra1/2, we find that Tead4 cooperates with AP1 transcription factors to coordinate target gene transcription. We find that Tead-AP1 interaction is JNK independent, but engages the SRC1-3 coactivators to promote downstream transcription. Furthermore we show that Tead-AP1 cooperation regulates the activity of the Dock-Rac/CDC42 module and drives the expression of a unique core set of target genes, thereby directing cell migration and invasion. Together, our data unveil a critical regulatory mechanism underlying Tead- and AP1-controlled transcriptional and functional outputs in cancer cells.
PMCID: PMC4749442  PMID: 26832411
Tead; AP1; transcriptional co-regulation; invasion
Neuro-Oncology  2014;16(Suppl 5):v55.
Glioblastomas are characterized by their ability to disseminate into the local brain parenchyma; thus, confounding surgical excision and radiotherapy. Hence, it is imperative to identify and decipher the signaling networks that drive invasion. Glioblastoma cells utilize molecular transporters at both their leading (inward facing) and lagging (outward facing) edge to modulate cell volume and invade the confined microenvironment of the brain. These transporters include solute transporters as well as the aquaporins, and collectively behave as an osmotic engine for cellular invasion. However, the transcriptional regulators of these transporters have not been fully identified. Here, we report that TEAD4, a transcription factor implicated in neural development, is a potent regulator of the osmotic engine through transcriptional control of solute and water transporters. In particular, our data demonstrates that loss of TEAD4 decreases glioblastoma cells ability to migrate and invade through small pores (Boyden chamber and Matrigel transwell, respectively) mimicking the confined microenvironment of the brain (p < 0.05). Additionally, we uncover the role of TEAD4 in regulating members of the Na + /H+ exchanger, chloride co-transporter and aquaporin families, as well as the volume regulated anion channel to enable water permeation (p < 0.05). Apart from regulating cell dispersal, our data also shows that TEAD4 regulates glioblastoma proliferation (p < 0.05). Using the TCGA and REMBRANDT datasets, we observed TEAD4 is selectively overexpressed and hyperactive in glioblastomas compared to non-cancer cortex and lower grade gliomas (p < 0.05). Furthermore, we found that patients with elevated TEAD4 expression have a significantly shorter progression free survival and overall survival (p < 0.05). Taken together, our results show that TEAD4 is a potent regulator of cell dispersal through transcriptional control of the osmotic engine and has relevance to clinical outcomes of glioblastoma patients. Hence, TEAD4 may be a selective and potent therapeutic target that warrants further exploration.
PMCID: PMC4217977
19.  Nuclear localization of TEF3-1 promotes cell cycle progression and angiogenesis in cancer 
Oncotarget  2016;7(12):13827-13841.
TEF3-1 (transcriptional enhancer factor 3 isoform 1), also known as TEAD4 (TEA domain family member 4), was recently revealed as an oncogenic character in cancer development. However, the underlying molecular pathogenic mechanisms remain undefined. In this paper, we investigated nuclear TEF3-1 could promote G1/S transition in HUVECs, and the expression levels of cyclins and CDKs were upregulated. Additionally, if TEF3-1 was knocked down, the expression of cyclins and CDKs was downregulated while the expression of P21, a negative regulator of the cell cycle, was upregulated. A microarray analysis also confirmed that TEF3-1 overexpression upregulates genes that are related to cell cycle progression and the promotion of angiogenesis. Moreover, we observed that nuclear TEF3-1 was highly expressed during the formation of vascular structures in gastric cancer (GC). Finally, tumor xenograft experiments indicated that, when TEF3-1 was knocked down, tumor growth and angiogenesis were also suppressed. Taken together, these results demonstrate for the first time that TEF3-1 localization to the nucleus stimulates the cell cycle progression in HUVECs and specifically contributes to tumor angiogenesis. Nuclear TEF3-1 in HUVECs may serve as an oncogenic biomarker, and the suppression of TEF3-1 may be a potential target in anti-tumor therapy.
PMCID: PMC4924681  PMID: 26885617
transcription enhancer factor 3 isoform 1; angiogenesis; cell cycle; HUVEC; cancer therapy
20.  Yes-Associated Protein 65 (YAP) Expands Neural Progenitors and Regulates Pax3 Expression in the Neural Plate Border Zone 
PLoS ONE  2011;6(6):e20309.
Yes-associated protein 65 (YAP) contains multiple protein-protein interaction domains and functions as both a transcriptional co-activator and as a scaffolding protein. Mouse embryos lacking YAP did not survive past embryonic day 8.5 and showed signs of defective yolk sac vasculogenesis, chorioallantoic fusion, and anterior-posterior (A-P) axis elongation. Given that the YAP knockout mouse defects might be due in part to nutritional deficiencies, we sought to better characterize a role for YAP during early development using embryos that develop externally. YAP morpholino (MO)-mediated loss-of-function in both frog and fish resulted in incomplete epiboly at gastrulation and impaired axis formation, similar to the mouse phenotype. In frog, germ layer specific genes were expressed, but they were temporally delayed. YAP MO-mediated partial knockdown in frog allowed a shortened axis to form. YAP gain-of-function in Xenopus expanded the progenitor populations in the neural plate (sox2+) and neural plate border zone (pax3+), while inhibiting the expression of later markers of tissues derived from the neural plate border zone (neural crest, pre-placodal ectoderm, hatching gland), as well as epidermis and somitic muscle. YAP directly regulates pax3 expression via association with TEAD1 (N-TEF) at a highly conserved, previously undescribed, TEAD-binding site within the 5′ regulatory region of pax3. Structure/function analyses revealed that the PDZ-binding motif of YAP contributes to the inhibition of epidermal and somitic muscle differentiation, but a complete, intact YAP protein is required for expansion of the neural plate and neural plate border zone progenitor pools. These results provide a thorough analysis of YAP mediated gene expression changes in loss- and gain-of-function experiments. Furthermore, this is the first report to use YAP structure-function analyzes to determine which portion of YAP is involved in specific gene expression changes and the first to show direct in vivo evidence of YAP's role in regulating pax3 neural crest expression.
PMCID: PMC3110623  PMID: 21687713
21.  The interplay between TEAD4 and KLF5 promotes breast cancer partially through inhibiting the transcription of p27Kip1 
Oncotarget  2015;6(19):17685-17697.
Growing evidence suggests that YAP/TAZ are mediators of the Hippo pathway and promote breast cancer. However, the roles of YAP/TAZ transcription factor partners TEADs in breast cancer remain unclear. Here we found that TEAD4 was expressed in breast cancer cell lines, especially in triple negative breast cancers (TNBC) cell lines. TEAD4 binds to KLF5. Knockdown of either TEAD4 or KLF5 in HCC1937 and HCC1806 cells induced the expression of CDK inhibitor p27. Depletion of either TEAD4 or KLF5 activated the p27 gene promoter and increased the p27 mRNA levels. Depletion of p27 partially prevents growth inhibition caused by TEAD4 and KLF5 knockdown. TEAD4 overexpression stimulated proliferation in vitro and tumor growth in mice, while stable knockdown of TEAD4 inhibited proliferation in vitro and tumor growth in mice. Thus TEAD4 and KLF5, in collaboration, promoted TNBC cell proliferation and tumor growth in part by inhibiting p27 gene transcription. TEAD4 is a potential target and biomarker for the development of novel therapeutics for breast cancer.
PMCID: PMC4627338  PMID: 25970772
TEAD4; KLF5; p27; hippo pathway; TNBC
22.  Shaping Leg Muscles in Drosophila: Role of ladybird, a Conserved Regulator of Appendicular Myogenesis 
PLoS ONE  2006;1(1):e122.
Legs are locomotor appendages used by a variety of evolutionarily distant vertebrates and invertebrates. The primary biological leg function, locomotion, requires the formation of a specialised appendicular musculature. Here we report evidence that ladybird, an orthologue of the Lbx1 gene recognised as a hallmark of appendicular myogenesis in vertebrates, is expressed in leg myoblasts, and regulates the shape, ultrastructure and functional properties of leg muscles in Drosophila. ladybird expression is progressively activated in myoblasts associated with the imaginal leg disc and precedes that of the founder cell marker dumbfounded. The RNAi-mediated attenuation of ladybird expression alters properties of developing myotubes, impairing their ability to grow and interact with the internal tendons and epithelial attachment sites. It also affects sarcomeric ultrastructure, resulting in reduced leg muscle performance and impaired mobility in surviving flies. The over-expression of ladybird also results in an abnormal pattern of dorsally located leg muscles, indicating different requirements for ladybird in dorsal versus ventral muscles. This differential effect is consistent with the higher level of Ladybird in ventrally located myoblasts and with positive ladybird regulation by extrinsic Wingless signalling from the ventral epithelium. In addition, ladybird expression correlates with that of FGF receptor Heartless and the read-out of FGF signalling downstream of FGF. FGF signals regulate the number of leg disc associated myoblasts and are able to accelerate myogenic differentiation by activating ladybird, leading to ectopic muscle fibre formation. A key role for ladybird in leg myogenesis is further supported by its capacity to repress vestigial and to down-regulate the vestigial-governed flight muscle developmental programme. Thus in Drosophila like in vertebrates, appendicular muscles develop from a specialised pool of myoblasts expressing ladybird/Lbx1. The ladybird/Lbx1 gene family appears as a part of an ancient genetic circuitry determining leg-specific properties of myoblasts and making an appendage adapted for locomotion.
PMCID: PMC1762424  PMID: 17205126
23.  Developmental expression of lineage specific genes in porcine embryos of different origins 
This study compared the expression of genes involved in pluripotency, segregation of inner cell mass (ICM) and trophectoderm (TE), and primitive endoderm (PE) formation in porcine embryos produced by in vitro fertilization (IVF), parthenogenetic activation (PA), and nuclear transfer (NT) using either fetal fibroblasts (FF-NT) or mesenchymal stem cells (MSC-NT).
Blastocyst formation and total cell number were analyzed. The expression patterns of transcripts, including SRY-related HMG-box gene 2 (SOX2), reduced expression gene 1 (REX1/ZFP42), LIN28, caudal type homeobox 2 (CDX2), TEA domain family member 4 (TEAD4), integrin beta 1 (ITGB1) and GATA6 were assessed at the 4–8 cell and blastocyst stage embryos by real-time PCR.
Developmental rates to blastocyst stage and total cell number were higher in IVF and PA embryos than in NT embryos. But MSC-NT embryos had increased blastocyst formation and higher total cell number compared to FF-NT embryos. The relative expressions of transcripts were higher in blastocysts than in 4–8 cell stage embryos. The mRNA expression levels of SOX2 and REX1 were largely similar in embryos of different origins. However, the genes such as LIN28, CDX2, TEAD4, ITGB1 and GATA6 showed the differential expression pattern in PA and NT embryos compared to IVF embryos. Importantly, the transcript levels in MSC-NT embryos were relatively less variable to IVF than those in FF-NT embryos.
MSCs seem to be better donors for porcine NT as they improved the developmental competency, and influenced the expression pattern of genes quite similar with IVF embryos than that of FFs.
PMCID: PMC3430790  PMID: 22639061
Gene expression; Preimplantation embryos; Nuclear transfer; Porcine
24.  Dystrobrevin and dystrophin family gene expression in zebrafish 
Dystrophin/dystrobrevin superfamily proteins play structural and signalling roles at the plasma membrane of many cell types. Defects in them or the associated multiprotein complex cause a range of neuromuscular disorders. Members of the dystrophin branch of the family form heterodimers with members of the dystrobrevin branch, mediated by their coiled-coil domains. To determine which combinations of these proteins might interact during embryonic development, we set out to characterise the gene expression pattern of dystrophin and dystrobrevin family members in zebrafish. γ-dystrobrevin (dtng), a novel dystrobrevin recently identified in fish, is the predominant form of dystrobrevin in embryonic development. Dtng and dmd (dystrophin) have similar spatial and temporal expression patterns in muscle, where transcripts are localized to the ends of differentiated fibres at the somite borders. Dtng is expressed in the notochord while dmd is expressed in the chordo-neural hinge and then in floor plate and hypochord. In addition, dtng is dynamically expressed in rhombomeres 2 and 4-6 of the hindbrain and in the ventral midbrain. α-dystrobrevin (dtna) is expressed widely in the brain with particularly strong expression in the hypothalamus and the telencephalon; drp2 is also expressed widely in the brain. Utrophin expression is found in early pronephros and lateral line development and utrophin and dystrophin are both expressed later in the gut. β-dystrobrevin (dtnb) is expressed in the pronephric duct and widely at low levels. In summary, we find clear instances of co-expression of dystrophin and dystrobrevin family members in muscle, brain and pronephric duct development and many examples of strong and specific expression of members of one family but not the other, an intriguing finding given the presumed heterodimeric state of these molecules.
PMCID: PMC3360968  PMID: 18042440
muscle; zebrafish; notochord; midbrain; rhombomere; gene expression; utrophin; dystrophin; dystrobrevin; drp2; dystrotelin
25.  Soggy, a spermatocyte-specific gene, lies 3.8 kb upstream of and antipodal to TEAD-2, a transcription factor expressed at the beginning of mouse development 
Nucleic Acids Research  2000;28(20):3982-3990.
Investigation of the regulatory region of mTEAD-2, a gene expressed at the beginning of mouse pre-implantation development, led to the surprising discovery of another gene only 3.8 kb upstream of mTEAD-2. Here we show that this new gene is a single copy, testis-specific gene called Soggy (mSgy) that produces a single, dominant mRNA ∼1.3 kb in length. It is transcribed in the direction opposite to mTEAD-2, thus placing the regulatory elements of these two genes in close proximity. mSgy contains three methionine codons that could potentially act as translation start sites, but most mSGY protein synthesis in vitro was initiated from the first Met codon to produce a full-length protein, suggesting that mSGY normally consists of 230 amino acids (26.7 kDa). Transcription began at a cluster of nucleotides ∼150 bp upstream of the first Met codon using a TATA-less promoter contained within the first 0.9 kb upstream. The activity of this promoter was repressed by upstream sequences between –0.9 and –2.5 kb in cells that did not express mSgy, but this repression was relieved in cells that did express mSgy. mSgy mRNA was detected in embryos only after day 15 and in adult tissues only in the developing spermatocytes of seminiferous tubules, suggesting that mSgy is a spermatocyte-specific gene. Since mTEAD-2 and mSgy were not expressed in the same cells, the mSgy/mTEAD-2 locus provides a unique paradigm for differential regulation of gene expression during mammalian development.
PMCID: PMC110775  PMID: 11024178

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