TEAD4 role in trophectoderm commitment and development is not conserved in non-rodent mammals.

The first lineage differentiation in mammals gives rise to the inner cell mass (ICM) and the trophectoderm (TE). In mice, TEAD4 is a master regulation of TE commitment, as it regulates the expression of other TE-specific genes and its ablation prevents blastocyst formation, but its role in other mammals remains unclear. Herein, we have observed that TEAD4 ablation in two phylogenetically distant species (bovine and rabbits) does not impede TE differentiation, blastocyst formation and the expression of TE markers such as GATA3 and CDX2, although a reduced ICM cell number was observed in bovine TEAD4 KO blastocysts. Transcriptional analysis in bovine blastocysts revealed no major transcriptional effect of the ablation, although the expression of hypoblast and Hippo signalling-related genes tended to be decreased in KO embryos. Experiments were conducted in the bovine model to determine if TEAD4 was required for post-hatching development. TEAD4 KO spherical conceptuses showed normal development of the embryonic disc and TE, but hypoblast migration rate was reduced. At later stages of development (tubular conceptuses) no differences were observed between KO and WT conceptuses.

The tumor suppressor NF2 modulates TEAD4 stability and activity in Hippo signaling via direct interaction.

As an output effector of the Hippo signaling pathway, the TEAD transcription factor and co-activator YAP play crucial functions in promoting cell proliferation and organ size. The tumor suppressor NF2 has been shown to activate LATS1/2 kinases and interplay with the Hippo pathway to suppress the YAP-TEAD complex. However, whether and how NF2 could directly regulate TEAD remains unknown. We identified a direct link and physical interaction between NF2 and TEAD4. NF2 interacted with TEAD4 through its FERM domain and C-terminal tail and decreased the protein stability of TEAD4 independently of LATS1/2 and YAP. Furthermore, NF2 inhibited TEAD4 palmitoylation and induced the cytoplasmic translocation of TEAD4, resulting in ubiquitination and dysfunction of TEAD4. Moreover, the interaction with TEAD4 is required for NF2 function to suppress cell proliferation. These findings reveal an unanticipated role of NF2 as a binding partner and inhibitor of the transcription factor TEAD, shedding light on an alternative mechanism of how NF2 functions as a tumor suppressor through the Hippo signaling cascade.

Upregulation of CYR61 by TGF-ß and YAP signaling exerts a counter-suppression of hepatocellular carcinoma.

Transforming growth factor-ß (TGF-ß) and Hippo signaling are two critical pathways engaged in cancer progression by regulating both oncogenes and tumor suppressors, yet how the two pathways coordinately exert their functions in the development of hepatocellular carcinoma (HCC) remains elusive. In this study, we firstly conducted an integrated analysis of public liver cancer databases and our experimental TGF-ß target genes, identifying CYR61 as a pivotal candidate gene relating to HCC development. The expression of CYR61 is downregulated in clinical HCC tissues and cell lines than that in the normal counterparts. Evidence revealed that CYR61 is a direct target gene of TGF-ß in liver cancer cells. In addition, TGF-ß-stimulated Smad2/3 and the Hippo pathway downstream effectors YAP and TEAD4 can form a protein complex on the promoter of CYR61, thereby activating the promoter activity and stimulating CYR61 gene transcription in a collaborative manner. Functionally, depletion of CYR61 enhanced TGF-ß- or YAP-mediated growth and migration of liver cancer cells. Consistently, ectopic expression of CYR61 was capable of impeding TGF-ß- or YAP-induced malignant transformation of HCC cells in vitro and attenuating HCC xenograft growth in nude mice. Finally, transcriptomic analysis indicates that CYR61 can elicit an antitumor program in liver cancer cells. Together, these results add new evidence for the crosstalk between TGF-ß and Hippo signaling and unveil an important tumor suppressor function of CYR61 in liver cancer.

The YAP-TEAD4 complex promotes tumor lymphangiogenesis by transcriptionally upregulating CCBE1 in colorectal cancer.

The secreted protein collagen and calcium-binding EGF domain 1 (CCBE1) is critical for embryonic lymphatic development through its role in the proteolytic activation of mature vascular endothelial growth factor C (VEGFC). We previously reported that CCBE1 is overexpressed in colorectal cancer (CRC) and that its transcription is negatively regulated by the TGFß-SMAD pathway, but the transcriptional activation mechanism of CCBE1 in CRC remains unknown. Recent studies have revealed the vital role of the hippo effectors YAP/TAZ in lymphatic development; however, the role of YAP/TAZ in tumor lymphangiogenesis has not been clarified. In this study, we found that high nuclear expression of transcription factor TEAD4 is associated with lymph node metastasis and high lymphatic vessel density in patients with CRC. YAP/TAZ-TEAD4 complexes transcriptionally upregulated the expression of CCBE1 by directly binding to the enhancer region of CCBE1 in both CRC cells and cancer-associated fibroblasts, which resulted in enhanced VEGFC proteolysis and induced tube formation and migration of human lymphatic endothelial cells in vitro and lymphangiogenesis in a CRC cell-derived xenograft model in vivo. In addition, the bromodomain and extraterminal domain (BET) inhibitor JQ1 significantly inhibited the transcription of CCBE1, suppressed VEGFC proteolysis, and inhibited tumor lymphangiogenesis in vitro and in vivo. Collectively, our study reveals a new positive transcriptional regulatory mechanism of CCBE1 via YAP/TAZ-TEAD4-BRD4 complexes in CRC, which exposes the protumor lymphangiogenic role of YAP/TAZ and the potential inhibitory effect of BET inhibitors on tumor lymphangiogenesis.

Epigenetic drug screening for trophoblast syncytialization reveals a novel role for MLL1 in regulating fetoplacental growth.

BACKGROUND: Abnormal placental development is a significant factor contributing to perinatal morbidity and mortality, affecting approximately 5-7% of pregnant women. Trophoblast syncytialization plays a pivotal role in the establishment and maturation of the placenta, and its dysregulation is closely associated with several pregnancy-related disorders, including preeclampsia and intrauterine growth restriction. However, the underlying mechanisms and genetic determinants of syncytialization are largely unknown. METHODS: We conducted a systematic drug screen using an epigenetic compound library to systematically investigate the epigenetic mechanism essential for syncytialization, and identified mixed lineage leukemia 1 (MLL1), a histone 3 lysine 4 methyltransferase, as a crucial regulator of trophoblast syncytialization. BeWo cells were utilized to investigate the role of MLL1 during trophoblast syncytialization. RNA sequencing and CUT&Tag were further performed to search for potential target genes and the molecular pathways involved. Human placenta tissue was used to investigate the role of MLL1 in TEA domain transcription factor 4 (TEAD4) expression and the upstream signaling during syncytialization. A mouse model was used to examine whether inhibition of MLL1-mediated H3K4me3 regulated placental TEAD4 expression and fetoplacental growth. RESULTS: Genetic knockdown of MLL1 or pharmacological inhibition of the MLL1 methyltransferase complex (by MI-3454) markedly enhanced syncytialization, while overexpression of MLL1 inhibited forskolin (FSK)-induced syncytiotrophoblast formation. In human placental villous tissue, MLL1 was predominantly localized in the nuclei of cytotrophoblasts. Moreover, a notable upregulation in MLL1 expression was observed in the villus tissue of patients with preeclampsia compared with that in the control group. Based on RNA sequencing and CUT&Tag analyses, depletion of MLL1 inhibited the Hippo signaling pathway by suppressing TEAD4 expression by modulating H3K4me3 levels on the TEAD4 promoter region. TEAD4 overexpression significantly reversed the FSK-induced or MLL1 silencing-mediated trophoblast syncytialization. Additionally, decreased hypoxia-inducible factor 1A (HIF1A) enrichment at the MLL1 promoter was observed during syncytialization. Under hypoxic conditions, HIF1A could bind to and upregulate MLL1, leading to the activation of the MLL1/TEAD4 axis. In vivo studies demonstrated that the administration of MI-3454 significantly enhanced fetal vessel development and increased the thickness of the syncytial layer, thereby supporting fetoplacental growth. CONCLUSIONS: These results revealed a novel epigenetic mechanism underlying the progression of syncytialization with MLL1, and suggest potential avenues for identifying new therapeutic targets for pregnancy-related disorders.

PKP2 induced by YAP/TEAD4 promotes malignant progression of gastric cancer.

Gastric cancer (GC) exhibits significant heterogeneity and its prognosis remains dismal. Therefore, it is essential to investigate new approaches for diagnosing and treating GC. Desmosome proteins are crucial for the advancement and growth of cancer. Plakophilin-2 (PKP2), a member of the desmosome protein family, frequently exhibits aberrant expression and is strongly associated with many tumor types' progression. In this study, we found upregulation of PKP2 in GC. Further correlation analysis showed a notable association between increased PKP2 expression and both tumor stage and poor prognosis in individuals diagnosed with gastric adenocarcinoma. In addition, our research revealed that the Yes-associated protein1 (YAP1)/TEAD4 complex could stimulate the transcriptional expression of PKP2 in GC. Elevated PKP2 levels facilitate activation of the AKT/mammalian target of rapamycin signaling pathway, thereby promoting the malignant progression of GC. By constructing a mouse model, we ultimately validated the molecular mechanism and function of PKP2 in GC. Taken together, these discoveries suggest that PKP2, as a direct gene target of YAP/TEAD4 regulation, has the potential to be used as an indication of GC progression and prognosis. PKP2 is expected to be a promising therapeutic target for GC.

RBM8A, a new target of TEAD4, promotes breast cancer progression by regulating IGF1R and IRS-2.

BACKGROUND: Breast cancer (BC) is the most common malignant tumor in women worldwide, and further elucidation of the molecular mechanisms involved in BC pathogenesis is essential to improve the prognosis of BC patients. RNA Binding Motif Protein 8 A (RBM8A), with high affinity to a myriad of RNA transcripts, has been shown to play a crucial role in genesis and progression of multiple cancers. We attempted to explore its functional significance and molecular mechanisms in BC. METHODS: Bioinformatics analysis was performed on publicly available BC datasets. qRT-PCR was used to determine the expression of RBM8A in BC tissues. MTT assay, clone formation assay and flow cytometry were employed to examine BC cell proliferation and apoptosis in vitro. RNA immunoprecipitation (RIP) and RIP-seq were used to investigate the binding of RBM8A/EIF4A3 to the mRNA of IGF1R/IRS-2. RBM8A and EIF4A3 interactions were determined by co-immunoprecipitation (Co-IP) and immunofluorescence. Chromatin immunoprecipitation (Ch-IP) and dual-luciferase reporter assay were carried out to investigate the transcriptional regulation of RBM8A by TEAD4. Xenograft model was used to explore the effects of RBM8A and TEAD4 on BC cell growth in vivo. RESULTS: In this study, we showed that RBM8A is abnormally highly expressed in BC and knockdown of RBM8A inhibits BC cell proliferation and induces apoptosis in vitro. EIF4A3, which phenocopy RBM8A in BC, forms a complex with RBM8A in BC. Moreover, EIF4A3 and RBM8A complex regulate the expression of IGF1R and IRS-2 to activate the PI3K/AKT signaling pathway, thereby promoting BC progression. In addition, we identified TEAD4 as a transcriptional activator of RBM8A by Ch-IP, dual luciferase reporter gene and a series of functional rescue assays. Furthermore, we demonstrated the in vivo pro-carcinogenic effects of TEAD4 and RBM8A by xenograft tumor experiments in nude mice. CONCLUSION: Collectively, these findings suggest that TEAD4 novel transcriptional target RBM8A interacts with EIF4A3 to increase IGF1R and IRS-2 expression and activate PI3K/AKT signaling pathway, thereby further promoting the malignant phenotype of BC cells.

TEAD4 Activates PCSK9 to Promote Stomach Adenocarcinoma Cell Stemness through Fatty Acid Metabolism.

INTRODUCTION: This study attempted to investigate how proprotein convertase subtilisin/kexin type 9 (PCSK9) influences the stemness of stomach adenocarcinoma (STAD) cells. METHODS: CCK-8 and sphere-formation assays were used to detect cell viability and stemness. qRT-PCR and Western blot were used to detect PCSK9 and TEAD4 expression. The binding relationship was verified by dual-luciferase and chromatin immunoprecipitation assays. The effect of TEAD4 activating PCSK9 on the stemness of STAD cells was detected by bioinformatics, BODIPY 493/503, Oil red O, Western blot, and kits. In vivo experiments verified the role of the TEAD4/PCSK9 axis in tumor formation in nude mice. RESULTS: PCSK9 and TEAD4 were highly expressed in STAD. PCSK9 was enriched in the fatty acid metabolism (FAM) pathway. PCSK9 activated the fatty acid metabolism and promoted the proliferation and stemness of STAD cells. TEAD4 as a transcription factor upstream of PCSK9, cell experiments revealed that knockdown of PCSK9 inhibited STAD cell stemness, whereas further addition of fatty acid inhibitors could attenuate the promoting effect on STAD cell stemness brought by STAD overexpression. Rescue experiments showed overexpressed PCSK9 exerted an inhibitory effect on the stemness of STAD cells brought by TEAD4 knockdown. The hypothesis that TEAD4/PCSK9 axis can promote STAD cell growth was confirmed by in vivo experiments. CONCLUSION: Transcription factor TEAD4 could activate PCSK9 to promote the stemness of STAD cells through FAM. These results added weight to the assumption that TEAD4/PCSK9 axis has the potential to be the therapeutic target that inhibits cancer stem cell in STAD.

TEAD4 antagonizes cellular senescence by remodeling chromatin accessibility at enhancer regions.

Dramatic alterations in epigenetic landscapes are known to impact genome accessibility and transcription. Extensive evidence demonstrates that senescent cells undergo significant changes in chromatin structure; however, the mechanisms underlying the crosstalk between epigenetic parameters and gene expression profiles have not been fully elucidated. In the present study, we delineate the genome-wide redistribution of accessible chromatin regions that lead to broad transcriptome effects during senescence. We report that distinct senescence-activated accessibility regions (SAAs) are always distributed in H3K27ac-occupied enhancer regions, where they are responsible for elevated flanking senescence-associated secretory phenotype (SASP) expression and aberrant cellular signaling relevant to SASP secretion. Mechanistically, a single transcription factor, TEAD4, moves away from H3K27ac-labled SAAs to allow for prominent chromatin accessibility reconstruction during senescence. The enhanced SAAs signal driven by TEAD4 suppression subsequently induces a robust increase in the expression of adjacent SASP genes and the secretion of downstream factors, which contribute to the progression of senescence. Our findings illustrate a dynamic landscape of chromatin accessibility following senescence entry, and further reveal an insightful function for TEAD4 in regulating the broad chromatin state that modulates the overall transcriptional program of SASP genes.

Integrative Epigenetic and Molecular Analysis Reveals a Novel Promoter for a New Isoform of the Transcription Factor TEAD4.

TEAD4 is a transcription factor that plays a crucial role in the Hippo pathway by regulating the expression of genes related to proliferation and apoptosis. It is also involved in the maintenance and differentiation of the trophectoderm during pre- and post-implantation embryonic development. An alternative promoter for the TEAD4 gene was identified through epigenetic profile analysis, and a new transcript from the intronic region of TEAD4 was discovered using the 5'RACE method. The transcript of the novel promoter encodes a TEAD4 isoform (TEAD4-ΔN) that lacks the DNA-binding domain but retains the C-terminal protein-protein interaction domain. Gene expression studies, including end-point PCR and Western blotting, showed that full-length TEAD4 was present in all investigated tissues. However, TEAD4-ΔN was only detectable in certain cell types. The TEAD4-ΔN promoter is conserved throughout evolution and demonstrates transcriptional activity in transient-expression experiments. Our study reveals that TEAD4 interacts with the alternative promoter and increases the expression of the truncated isoform. DNA methylation plays a crucial function in the restricted expression of the TEAD4-ΔN isoform in specific tissues, including the umbilical cord and the placenta. The data presented indicate that the DNA-methylation status of the TEAD4-ΔN promoter plays a critical role in regulating organ size, cancer development, and placenta differentiation.

Dual function of VGLL4 in muscle regeneration.

VGLL4 has previously been identified as a negative regulator of YAP. Here we show that VGLL4 regulates muscle regeneration in both YAP-dependent and YAP-independent manners at different stages. Knockout of VGLL4 in mice leads to smaller myofiber size and defective muscle contraction force. Furthermore, our studies reveal that knockout of VGLL4 results in increased muscle satellite cells proliferation and impaired myoblast differentiation, which ultimately leads to delayed muscle regeneration. Mechanistically, the results show that VGLL4 works as a conventional repressor of YAP at the proliferation stage of muscle regeneration. At the differentiation stage, VGLL4 acts as a co-activator of TEAD4 to promote MyoG transactivation and facilitate the initiation of differentiation in a YAP-independent manner. Moreover, VGLL4 stabilizes the protein-protein interactions between MyoD and TEAD4 to achieve efficient MyoG transactivation. Our findings define the dual roles of VGLL4 in regulating muscle regeneration at different stages and may open novel therapeutic perspectives for muscle regeneration.

A role of TRIM59 in pulmonary hypertension: modulating the protein ubiquitylation modification.

BACKGROUND: Pulmonary hypertension (PH), an infrequent disease, is characterized by excessive pulmonary vascular remodeling and proliferation of pulmonary artery smooth muscle cells (PASMCs). However, its underlying molecular mechanisms remain unclear. Uncovering its molecular mechanisms will be beneficial to the treatment of PH. METHODS: Differently expressed genes (DEGs) in the lung tissues of PH patients were analyzed with a GEO dataset GSE113439. From these DEGs, we focused on TRIM59 which was highly expressed in PH patients. Subsequently, the expression of TRIM59 in the pulmonary arteries of PH patients, lung tissues of PH rat model and PASMCs cultured in a hypoxic condition was verified by quantitative real-time PCR (qPCR), western blot and immunohistochemistry. Furthermore, the role of TRIM59 in PAMSC proliferation and pathological changes in PH rats was assessed via gain-of-function and loss-of-function experiments. In addition, the transcriptional regulation of YAP1/TEAD4 on TRIM59 was confirmed by qPCR, western blot, luciferase reporter assay, ChIP and DNA pull-down. In order to uncover the underlying mechanisms of TRIM59, a protein ubiquitomics and a CoIP- HPLC-MS/MS were companied to identify the direct targets of TRIM59. RESULTS: TRIM59 was highly expressed in the pulmonary arteries of PH patients and lung tissues of PH rats. Over-expression of TRIM59 accelerated the proliferation of PASMCs, while TRIM59 silencing resulted in the opposite results. Moreover, TRIM59 silencing mitigated the injuries in heart and lung and attenuated pulmonary vascular remodeling during PH. In addition, its transcription was positively regulated by YAP1/TEAD4. Then we further explored the underlying mechanisms of TRIM59 and found that TRIM59 overexpression resulted in an altered ubiquitylation of proteins. Accompanied with the results of CoIP- HPLC-MS/MS, 34 proteins were identified as the direct targets of TRIM59. CONCLUSION: TRIM59 was highly expressed in PH patients and promoted the proliferation of PASMCs and pulmonary vascular remodeling, thus contributing to the pathogenesis of PH. It is indicated that TRIM59 may become a potential target for PH treatment.

Transcription factor TEAD4 facilitates glycolysis and proliferation of gastric cancer cells by activating PKMYT1.

BACKGROUND: Gastric cancer (GC) ranks third for cancer deaths worldwide, and glycolysis is a hallmark of several cancers, including GC. TEAD4 plays a role in establishing an oncogenic cascade in cancers, including GC. Whether TEAD4 can influence the glycolysis of GC cells remains uncovered. Hence, this study attempted to investigate the impact on glycolysis of GC cells by TEAD4. METHODS: By using bioinformatics analysis, differentially expressed mRNAs were screened, and downstream regulatory genes were predicted. Expression levels of TEAD4 and PKMYT1 were assessed by qRT-PCR. The binding sites between TEAD4 and PKMYT1 were predicted by the JASPAR database, meanwhile their modulatory relationship was confirmed through dual-luciferase assay and chromatin Immunoprecipitation (ChIP). Cell viability and proliferation were assayed via CCK-8 and colony formation assays. Glycolysis was measured by assaying extracellular acidification rate, oxygen consumption rate, and production of pyruvic acid, lactate, citrate, and malate. Expression levels of proteins (HK-2 and PKM2) related to glycolysis were assessed by Western blot. RESULTS: TEAD4 was upregulated in GC tissues and cells. TEAD4 knockdown substantially repressed glycolysis and proliferation of GC cells. PKMYT1, the target gene downstream of TEAD4, was identified via bioinformatics prediction, and its expression was elevated in GC. Dual-luciferase and ChIP assay validated the targeted relationship between the promoter region of PKMYT1 and TEAD4. As revealed by rescue experiments, the knockdown of TEAD4 reversed the stimulative effect on GC cell glycolysis and proliferation by forced expression of PKMYT1. CONCLUSION: TEAD4 activated PKMYT1 to facilitate the proliferation and glycolysis of GC cells. TEAD4 and PKMYT1 may be possible therapeutic targets for GC.

TEAD4 ensures postimplantation development by promoting trophoblast self-renewal: An implication in early human pregnancy loss.

Early pregnancy loss affects ∼15% of all implantation-confirmed human conceptions. However, evolutionarily conserved molecular mechanisms that regulate self-renewal of trophoblast progenitors and their association with early pregnancy loss are poorly understood. Here, we provide evidence that transcription factor TEAD4 ensures survival of postimplantation mouse and human embryos by controlling self-renewal and stemness of trophoblast progenitors within the placenta primordium. In an early postimplantation mouse embryo, TEAD4 is selectively expressed in trophoblast stem cell-like progenitor cells (TSPCs), and loss of Tead4 in postimplantation mouse TSPCs impairs their self-renewal, leading to embryonic lethality before embryonic day 9.0, a developmental stage equivalent to the first trimester of human gestation. Both TEAD4 and its cofactor, yes-associated protein 1 (YAP1), are specifically expressed in cytotrophoblast (CTB) progenitors of a first-trimester human placenta. We also show that a subset of unexplained recurrent pregnancy losses (idiopathic RPLs) is associated with impaired TEAD4 expression in CTB progenitors. Furthermore, by establishing idiopathic RPL patient-specific human trophoblast stem cells (RPL-TSCs), we show that loss of TEAD4 is associated with defective self-renewal in RPL-TSCs and rescue of TEAD4 expression restores their self-renewal ability. Unbiased genomics studies revealed that TEAD4 directly regulates expression of key cell cycle genes in both mouse and human TSCs and establishes a conserved transcriptional program. Our findings show that TEAD4, an effector of the Hippo signaling pathway, is essential for the establishment of pregnancy in a postimplantation mammalian embryo and indicate that impairment of the Hippo signaling pathway could be a molecular cause for early human pregnancy loss.

TFAP2C exacerbates psoriasis-like inflammation by promoting Th17 and Th1 cells activation through regulating TEAD4 transcription.

BACKGROUND: Psoriasis is one of the chronic and autoimmune skin diseases. It is important to uncover the mechanisms underlying the psoriasis. Transcription factor activator protein (TFAP-2) gamma, also known as AP2-gamma, is a protein encoded by the TFAP2C gene. Immune-mediated pathophysiological processes could be linked to psoriasis, but the mechanism is still unclear. Therefore, to date the cause of psoriasis has not been understood completely. MATERIALS AND METHODS: Psoriasis is a complex disease triggered by genetic, immunological, and environmental stimuli. Keratinocytes play an important role in both initiation and maintenance phases of psoriasis. A psoriatic keratinocyte model was established by stimulating high sensitivity of human epidermal keratinocytes (HaCaT) to topoisomerase inhibitor cell lines using the accumulation of M5 cytokines comprising interleukin (IL)-17A, IL-22, oncostatin M, IL-1α, and tumor necrosis factor-α (TNF-α). The TFAP2C and transcriptional enhanced associate domain 4 (TEAD4) genes expression was evaluated by reverse transcription-quantitative polymerase chain reaction. Western blot analysis was used to examine protein expression. Cell viability (quantitative) of keratinocytes, including cytotoxicity, proliferation, and cell activation, was evaluated by the MTT assay. The relative percentage values of interleukin (IL)-17a, interferon gamma, and IL-4+ cells were measured by flow cytometry. Accordingly, chromatin immunoprecipitation and luciferase reporter assays were applied to evaluate the binding affinity of TFAP2C and TEAD4 promoter. RESULTS: Level of the TFAP2C gene was elevated in the lesional skin of psoriasis patients. On the other hand, silencing of the TFAP2C gene suppressed the proliferation and inflammatory response in M5-induced keratinocytes. In addition, inhibition of TFAP2C alleviated imiquimod (IMQ)-induced skin injury in mice model. We also observed that suppression of TFAP2C inhibited the activation of T-helper 17 (Th17) and Th1 cells in IMQ-induced mice model. Mechanically, TFAP2C promoted TEAD4 transcriptional activation. CONCLUSION: TFAP2C exacerbated psoriasis-like inflammation by increasing the activation of Th17 and Th1 cells by regulating TEAD4 transcription. This finding clearly indicated that TFAP2C could be considered a valuable biomarker for the prevention and treatment for psoriasis.

Novel Allosteric Effectors Targeting Human Transcription Factor TEAD.

The Hippo pathway is an evolutionary conserved signaling network involved in several cellular regulatory processes. Dephosphorylation and overexpression of Yes-associated proteins (YAPs) in the Hippo-off state are common in several types of solid tumors. YAP overexpression results in its nuclear translocation and interaction with transcriptional enhanced associate domain 1-4 (TEAD1-4) transcription factors. Covalent and non-covalent inhibitors have been developed to target several interaction sites between TEAD and YAP. The most targeted and effective site for these developed inhibitors is the palmitate-binding pocket in the TEAD1-4 proteins. Screening of a DNA-encoded library against the TEAD central pocket was performed experimentally to identify six new allosteric inhibitors. Inspired by the structure of the TED-347 inhibitor, chemical modification was performed on the original inhibitors by replacing secondary methyl amide with a chloromethyl ketone moiety. Various computational tools, including molecular dynamics, free energy perturbation, and Markov state model analysis, were employed to study the effect of ligand binding on the protein conformational space. Four of the six modified ligands were associated with enhanced allosteric communication between the TEAD4 and YAP1 domains indicated by the relative free energy perturbation to original molecules. Phe229, Thr332, Ile374, and Ile395 residues were revealed to be essential for the effective binding of the inhibitors.

TEAD4 regulates trophectoderm differentiation upstream of CDX2 in a GATA3-independent manner in the human preimplantation embryo.

STUDY QUESTION: What is the role of transcriptional-enhanced associate (TEA) domain family member 4 (TEAD4) in trophectoderm (TE) differentiation during human embryo preimplantation development in comparison to mouse? SUMMARY ANSWER: TEAD4 regulates TE lineage differentiation in the human preimplantation embryo acting upstream of caudal-type homeobox protein 2 (CDX2), but in contrast to the mouse in a GATA-binding protein 3 (GATA3)-independent manner. WHAT IS KNOWN ALREADY: Tead4 is one of the earliest transcription factors expressed during mouse embryo preimplantation development and is required for the expression of TE-associated genes. Functional knock-out studies in mouse, inactivating Tead4 by site-specific recombination, have shown that Tead4-targeted embryos have compromised development and expression of the TE-specific Cdx2 and Gata3 is downregulated. Cdx2 and Gata3 act in parallel pathways downstream of Tead4 to induce successful TE differentiation. Downstream loss of Cdx2 expression, compromises TE differentiation and subsequent blastocoel formation and leads to the ectopic expression of inner cell mass (ICM) genes, including POU Class 5 homeobox 1 (Pou5f1) and SRY-box transcription factor (Sox2). Cdx2 is a more potent regulator of TE fate in mouse as loss of Cdx2 expression induces more severe phenotypes compared with loss of Gata3 expression. The role of TEAD4 and its downstream effectors during human preimplantation embryo development has not been investigated yet. STUDY DESIGN, SIZE, DURATION: The clustered regularly interspaced short palindromic repeats-clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes (CRISPR-Cas9) system was first introduced in pronuclei (PN)-stage mouse zygotes aiming to identify a guide RNA (gRNA), yielding high editing efficiency and effective disruption of the Tead4 locus. Three guides were tested (gRNA1-3), each time targeting a distinct region of Exon 2 of Tead4. The effects of targeting on developmental capacity were studied in Tead4-targeted embryos (n = 164-summarized data from gRNA1-3) and were compared with two control groups; sham-injected embryos (n = 26) and non-injected media-control embryos (n = 51). The editing efficiency was determined by next-generation sequencing (NGS). In total, n = 55 (summarized data from gRNA1-3) targeted mouse embryos were analysed by NGS. Immunofluorescence analysis to confirm successful targeting by gRNA1 was performed in Tead4-targeted embryos, and non-injected media-control embryos. The downregulation of secondary TE-associated markers Cdx2 and Gata3 was used as an indirect confirmation of successful Tead4-targeting (previously shown to be expressed downstream of Tead4). Additional groups of gRNA1 Tead4-targeted (n = 45) and media control (n = 36) embryos were cultured for an extended period of 8.5 days, to further assess the developmental capacity of the Tead4-targeted group to develop beyond implantation stages. Following the mouse investigation, human metaphase-II (MII) oocytes obtained by IVM were microinjected with gRNA-Cas9 during ICSI (n = 74) to target TEAD4 or used as media-control (n = 33). The editing efficiency was successfully assessed in n = 25 TEAD4-targeted human embryos. Finally, immunofluorescence analysis for TEAD4, CDX2, GATA3 and the ICM marker SOX2 was performed in TEAD4-targeted (n = 10) and non-injected media-control embryos (n = 29). PARTICIPANTS/MATERIALS, SETTING, METHODS: A ribonucleoprotein complex consisting of a gRNA-Cas9 mixture, designed to target Exon 2 of Tead4/TEAD4, was microinjected in mouse PN stage zygotes or human IVM MII oocytes along with sperm. Generated embryos were cultured in vitro for 4 days in mouse or 6.5 days in human. In mouse, an additional group of Tead4-targeted and media-control embryos was cultured in vitro for an extended period of 8.5 days. Embryonic development and morphology were assessed daily, during culture in vitro of mouse and human embryos and was followed by a detailed scoring at late blastocyst stage. Targeting efficiency following gRNA-Cas9 introduction was assessed via immunostaining and NGS analysis. MAIN RESULTS AND THE ROLE OF CHANCE: NGS analysis of the Tead4-targeted locus revealed very high editing efficiencies for all three guides, with 100% of the mouse embryos (55 out of 55) carrying genetic modifications resulting from CRISPR-Cas9 genome editing. More specifically, 65.22% (15 out 23) of the PN zygotes microinjected with gRNA1-Cas9, which exhibited the highest efficiency, carried exclusively mutated alleles. The developmental capacity of targeted embryos was significantly reduced (data from gRNA1), as 44.17% of the embryos arrested at the morula stage (2.5 days post coitum), coincident with the initiation of TE lineage differentiation, compared with 8.51% in control and 12.50% in sham control groups. High-quality blastocyst formation rates (Grade 3) were 8.97% in the gRNA1-targeted group, compared with 87.23% in the media-control and 87.50% in the sham group. Immunofluorescence analysis in targeted embryos confirmed downregulation of Tead4, Cdx2, and Gata3 expression, which resulted from successful targeting of the Tead4 locus. Tead4-targeted mouse embryos stained positive for the ICM markers Pou5f1 and Sox2, indicating that expression of ICM lineage markers is not affected. Tead4-targeted embryos were able to cavitate and form a blastocoel without being able to hatch. Extended embryo culture following zona pellucida removal, revealed that the targeted embryos can attach and form egg-cylinder-like structures in the absence of trophoblast giant cells. In human embryos, Exon 2 of TEAD4 was successfully targeted by CRISPR-Cas9 (n = 74). In total, 25 embryos from various developmental stages were analysed by NGS and 96.00% (24 out of 25) of the embryos carried genetic modifications because of gRNA-Cas9 editing. In the subgroup of the 24 edited embryos, 17 (70.83%) carried only mutant alleles and 11 out of these 17 (64.70%) carried exclusively frameshift mutations. Six out of 11 embryos reached the blastocyst stage. In contrast to mice, human-targeted embryos formed blastocysts at a rate (25.00%) that did not differ significantly from the control group (23.81%). However, blastocyst morphology and TE quality were significantly compromised following TEAD4-targeting, showing grade C TE scores, with TE containing very few cells. Immunofluorescence analysis of TEAD4-targeted embryos (n = 10) confirmed successful editing by the complete absence of TEAD4 and its downstream TE marker CDX2, but the embryos generated retained expression of GATA3, which is in contrast to what we have observed and has previously been reported in mouse. In this regard, our results indicate that GATA3 acts in parallel with TEAD4/CDX2 towards TE differentiation in human. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: CRISPR-Cas9 germline genome editing, in some cases, induces mosaic genotypes. These genotypes are a result of inefficient and delayed editing, and complicate the phenotypic analysis and developmental assessment of the injected embryos. We cannot exclude the possibility that the observed differences between mouse and human are the result of variable effects triggered by the culture conditions, which were however similar for both mouse and human embryos in this study. Furthermore, this study utilized human oocytes obtained by IVM, which may not fully recapitulate the developmental behaviour of in vivo matured oocytes. WIDER IMPLICATIONS OF THE FINDINGS: Elucidation of the evolutionary conservation of molecular mechanisms that regulate the differentiation and formation of the trophoblast lineage can give us fundamental insights into early implantation failure, which accounts for ∼15% of human conceptions. STUDY FUNDING/COMPETING INTEREST(S): The research was funded by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01) and Ghent University (BOF.BAS.2018.0018.01). G.C. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 11L8822N). A.B. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 1298722 N). We further thank Ferring Pharmaceuticals (Aalst, Belgium) for their unrestricted educational grant. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Upregulated YAP promotes oncogenic CTNNB1 expression contributing to molecular pathology of hepatoblastoma.

BACKGROUND: Hepatoblastoma (HB) is one of the most common cancers in children. Recent studies have shown that the occurrence of nuclear accumulation of ß-catenin reaches 90%-100% because of the anomalous activation of the Wnt pathway in HB patients. Furthermore, emerging studies have shown that concomitant activated forms of YAP and ß-catenin trigger the formation and progression of HB. YAP might play a vital role in ß-catenin-mediated HB development. However, the molecular mechanisms by which YAP/TEAD4 transcription factor regulates CTNNB1 underlying HB pathogenesis are still unclear. PROCEDURE: YAP and CTNNB1 expression and correlation were analyzed by a combination of network enrichment analysis and gene set enrichment analysis of the public microarray datasets (GSE131329 and GSE81928). The protein levels of YAP and ß-catenin were further validated by Western blotting in paired patients' samples. The direct interplay between YAP/TEAD4 and the promoter region of CTNNB1 was proven by the combination of dual-luciferase report assay and chromatin immunoprecipitation assay. RESULTS: YAP-conserved signature and WNT signaling pathway were significantly enriched in HB patients, with upregulated expression of YAP and ß-catenin compared to non-HB patients. Further functional assays demonstrated that YAP/TEAD4 transcription factor complex could bind to the CTNNB1 promoter region directly to promote ß-catenin expression and cell proliferation. Targeting the YAP/TEAD4 complex with a specific small-molecule compound markedly suppressed HepaG2 cell proliferation. CONCLUSIONS: As the upstream transcription factor of CTNNB1, YAP/TEAD4 is a promising target for the treatment of HB patients with high levels of YAP and ß-catenin.

TAZ promotes vasculogenic mimicry in gastric cancer through the upregulation of TEAD4.

BACKGROUND AND AIM: Vasculogenic mimicry (VM) is a unique blood supply pattern in malignant tumors that is closely associated with metastasis and poor prognosis. The Hippo signaling effector TAZ is upregulated in several cancers, promoting cancer proliferation and metastasis. This study aimed to identify the function of TAZ and its regulatory mechanism in promoting VM in gastric cancer (GC). METHODS: The expression of TAZ and TEAD4 and their correlations with overall survival and VM-related markers were analyzed in 228 cases of GC. The regulatory mechanism of TAZ and its interaction with TEAD4 in epithelial-mesenchymal transition (EMT) and VM were investigated in vitro and in vivo. RESULTS: TAZ was highly expressed in GC samples and was associated with shorter patient survival time. TAZ expression was positively correlated with TEAD4 and VM in patients with GC. TAZ enhanced the migration and invasion capacity of GC cells through EMT in vitro and upregulated the expression of VM-associated proteins, including VE-cadherin, MMP2, and MMP9, thus promoting VM formation. Overexpression of TAZ accelerated the growth of subcutaneous xenograft and promoted VM formation in vivo. Co-immunoprecipitation assays showed that TAZ can directly bind to TEAD4, and in vitro experiments showed that this binding mediates the function of TAZ in regulating EMT and VM formation in GC. CONCLUSIONS: TAZ promotes GC metastasis and VM by upregulating TEAD4 expression. Our findings expand the role of TAZ in VM and provide new theoretical support for the use of antiangiogenic therapy in the treatment of advanced GC.

NT5DC2 promotes leiomyosarcoma tumour cell growth via stabilizing unpalmitoylated TEAD4 and generating a positive feedback loop.

5'-Nucleotidase Domain Containing 2 (NT5DC2) is a novel oncoprotein, the regulatory effects of which have not been well characterized. This study aimed to investigate the expression profile and functional regulation of NT5DC2 and its potential interplay with TEAD4 in leiomyosarcoma (LMS). Bioinformatic analysis was conducted using data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) program. LMS cell lines SK-LMS-1 and SK-UT-1 were used for both in vitro and in vivo analysis. Results showed that NT5DC2 is aberrantly upregulated in LMS. Its overexpression was associated with unfavourable survival. Deletion of NT5DC2 significantly reduced the expression of cyclin B1, cyclin A2, cyclin E1 and CDK1 and increased G1 phase arrest in LMS cell lines, and suppressed their proliferation both in vitro and in vivo. NT5DC2 interacted with unpalmitoylated TEAD4, and this association reduced TEAD4 degradation via the ubiquitin-proteasome pathway. TRIM27 is a novel E3 ubiquitin ligase that induces K27/48-linked ubiquitination of unpalmitoylated TEAD4 at Lys278. TEAD4 inhibition significantly suppressed LMS cell growth both in vitro and in vivo. Dual-luciferase assay demonstrated that TEAD4 could bind to the NT5DC2 promoter and activate its transcription. Based on these findings, we infer that the NT5DC2-TEAD4 positive feedback loop plays an important role in LMS development and might serve as a potential therapeutic target.