TEAD family transcription factors in development and disease

Members of the TEAD transcription factor family, also known as transcriptional enhancer factors (TEFs), have emerged as important regulators of development. In humans, there are four TEAD proteins (TEAD1-4), which are broadly expressed in embryonic and adult tissues, although their expression patterns differs throughout development (Yasunami et al., 1996). TEAD proteins have a TEA DNA-binding domain, and TEAD1 (TEF-1) was a founding member of the TEA domain family (Bürglin, 1991). As transcription factors, TEADs orchestrate gene transcription to guide development (Landin-Malt et al., 2016). The most well-known role of TEADs involves the regulation of progenitor proliferation, stem cell identity, and lineage specification. TEAD factors also control tissue size and function by coupling external cytoarchitectural signals to cell growth and specification.

TEAD proteins require co-factors to exert their transcriptional activity, the most well-known of which are YAP (also known as YAP1) and TAZ (also known as WWTR1). YAP and TAZ are part of the Hippo signalling pathway and are key regulators of cell proliferation, stemness and differentiation (Ma et al., 2019). Vestigial-like proteins (VGLLs) have emerged as another major group of TEAD co-factors (Simon et al., 2016). Compared with YAP and TAZ, less is known about the signalling pathways and targets downstream of VGLLs. However, VGLLs are known to regulate proliferation, differentiation and development in several types of tissues, such as skeletal muscle (Pobbati and Hong, 2013).

In addition to YAP, TAZ and VGLLs, there are a number of other proteins that have been implicated as TEAD co-factors. For instance, TEAD2 has been shown to interact with the p160 family of proteins (Belandia and Parker, 2000). More recently, FAM181A and FAM181B were suggested to interact with TEAD at the same binding region as YAP and TAZ (Bokhovchuk et al., 2020). Together, these findings suggest that the interaction of TEADs with different co-factors can drive distinct transcriptional and morphological outcomes.

Here, we provide an overview of the TEAD family and discuss its interaction with YAP, TAZ and VGLLs. As we focus on TEAD and its co-factors, we do not cover the Hippo pathway in detail; instead, we refer the reader to several recent reviews (Davis and Tapon, 2019; Zheng and Pan, 2019; Manning et al., 2020). We discuss current knowledge of the role of TEADs throughout development, from the blastocyst to the adult, as well as the involvement of this family in tissue homeostasis and regeneration. Moreover, the disease implications of TEAD dysregulation are discussed. Finally, the interaction between TEAD family proteins and their co-factors is discussed, providing insight into how TEADs function both with and without Hippo signalling.

The earliest studies on the Hippo pathway and TEAD were performed in Drosophila melanogaster (hereafter Drosophila). Drosophila have a single TEAD family gene, scalloped (sd), which was identified in 1929 as a result of the wing phenotype of sd mutants (Grunberg, 1929). The sd gene was cloned some 60 years later (Campbell et al., 1991), and was found to encode a TEAD family protein (Campbell et al., 1992; Jacquemin and Davidson, 1997; Anbanandam et al., 2006). Similar to its mammalian orthologues, Sd can bind to DNA in a sequence-specific manner (Guss et al., 2001; Halder and Carroll, 2001). It controls neural progenitor proliferation in the central nervous system (Rohith and Shyamala, 2017) and, together with nerfin-1 (INSM1 in humans), controls medulla neuron fate (Vissers et al., 2018). sd is also important for embryonic motor neuron axon projections (Guss et al., 2013), and for taste behaviour (Inamdar et al., 1993).

Sd interacts with both members of the Drosophila VGLL co-factor family: Vestigial (Vg) and Tondu-domain-containing Growth Inhibitor (Tgi, also known as Sd-Binding-Protein, SdBP,) (reviewed by Simon et al., 2016). vg was identified as a nuclear factor involved in haltere and wing development (Williams et al., 1991), and was found to function via its interaction with Sd (Halder et al., 1998). Sd controls wing disc growth (Liu et al., 2000), and Sd and Vg physically interact to control wing formation (Halder et al., 1998; Paumard-Rigal et al., 1998; Simmonds et al., 1998; Garg et al., 2007), together acting as a selector (Mann and Carroll, 2002) for wing identity (Kim et al., 1996; Simmonds et al., 1998; Halder and Carroll, 2001). The Vg-Sd dimer regulates a number of genes in the wing, including sd and vg themselves (Halder et al., 1998; Guss et al., 2001; Halder and Carroll, 2001; Lunde et al., 2003). The Vg-Sd dimer also regulates cell cycle progression by activating expression of the E2F1 cell cycle gene (Lunde et al., 2003; Wu et al., 2008). Interestingly, E2F1 shows reciprocal control of Sd-Vg activity (Legent et al., 2006). sd and vg are also important for embryonic muscle development (Deng et al., 2009).

The second Drosophila VGLL family member, Tgi, was first identified as a hypothetical protein with two Tondu domains, homologous to mammalian Vgll4 (Chen et al., 2004). Tgi has been found to physically interact with Sd, and acts as a co-factor for the repression of some Hippo pathway target genes (Guo et al., 2013; Koontz et al., 2013).

Similar to its mammalian TEAD orthologues, Drosophila Sd interacts with a second co-factor – the YAP/TAZ orthologue Yorkie (Yki) (Huang et al., 2005). The Sd-Yki interaction is a major driver of growth and proliferation, and is regulated by the Hippo signalling pathway (reviewed by Davis and Tapon, 2019; Zheng and Pan, 2019; Manning et al., 2020). Sd binds Yki and acts downstream of the Hippo pathway during eye and wing development (Goulev et al., 2008; Wu et al., 2008; Zhang et al., 2008). Yki and Tgi compete for Sd binding in the eye, wing and ovarian follicle cells, in which Sd-Yki promotes growth and Sd-Tgi supresses growth. (Koontz et al., 2013). Sd and Yki also regulate haematopoiesis in Drosophila (Ferguson and Martinez-Agosto, 2014).

These studies in Drosophila have provided the foundation for understanding TEAD functions in mammals and other vertebrates. Below, we discuss the role of TEADs in vertebrate species, such as zebrafish, frogs, mice and humans.

There are four TEAD members in vertebrate species, including humans (TEAD1-4), mice (Tead1-4) and zebrafish (Tead1a, Tead1b, Tead3a, and Tead3b). From Drosophila to humans, TEADs are highly conserved, with mammalian TEADs sharing approximately 98% homology with Sd in the DNA-binding domain, and 50% homology in the co-factor-binding domain. They are so well conserved that human TEAD1 is able to substitute for Sd in wing development (Deshpande et al., 1997). Amongst themselves, the four mammalian TEADs share approximately 87.9% homology in the DNA-binding domain and 72% in the co-factor-binding domain (Holden and Cunningham, 2018).

As is the case in Drosophila, the activity and function of TEAD family proteins in vertebrates is largely regulated by the availability and activity of TEAD co-factors. This is exemplified by the canonical Hippo pathway members YAP and TAZ, activity of which is regulated by their phosphorylation status (Fig. 1) (Davis and Tapon, 2019; Zheng and Pan, 2019; Manning et al., 2020). When the Hippo pathway is ‘off’, YAP and TAZ are non-phosphorylated and are able to translocate to the nucleus to form a complex with TEAD. The TEAD-YAP/TAZ complex primarily activates target gene transcription; however, it can also play a repressor role (Kim et al., 2015). However, when Hippo signalling is ‘on’, upstream signalling causes phosphorylation of the Hippo kinases MST1 and MST2 (STK3), which phosphorylate LATS1 and LATS2, which in turn phosphorylate YAP and TAZ. This causes YAP and TAZ to be retained in the cytoplasm and undergo E3 ubiquitin ligase-mediated degradation (Liu et al., 2010; Zhao et al., 2010), rendering them unable to interact with TEADs. YAP and TAZ are paralogues, sharing approximately 50% amino acid sequence identity and common domain structures, including the TEAD-binding domain, WW domains and a transactivation domain (Fig. 2) (Kanai et al., 2000; Gibault et al., 2018). Unsurprisingly, they often have similar or redundant roles to each other.

In addition to interacting with YAP and TAZ, vertebrate TEAD1-4 can interact with VGLL family proteins (VGLL1-4). VGLLs are expressed in various tissues, such as cardiac and skeletal muscle, where they regulate cell processes such as proliferation and differentiation via TEADs. However, compared with YAP and TAZ, little is known about their upstream regulation. It has been shown that VGLL4 can be regulated by microRNAs (Liu et al., 2018), phosphorylation (Zeng et al., 2017), acetylation (Lin et al., 2016) and ubiquitylation (Zhang et al., 2016). However, less is known about the regulation of VGLL1-3. VGLL1-3 each have one TEAD-binding domain (TDU), whereas VGLL4 has two (Fig. 2). Consequently, VGLL4 is thought to have distinct functions from VGLL1-3 (Yamaguchi, 2020). Moreover, the VGLL-binding site overlaps with the YAP/TAZ binding site on TEAD. As such, when VGLL and YAP/TAZ are both present in the nucleus, they are likely to compete for TEAD binding (Fig. 1) (Yamaguchi, 2020). Interestingly, TEAD1-4 have approximately the same affinity for YAP, TAZ and VGLL (Bokhovchuk et al., 2019).

Tead1 and Tead2 can also interact with the p160 family of proteins, which consists of Src1 (Ncoa1), Src2 (Ncoa2; also known as Tif2, Grip1) and Src3 (Ncoa3; also known as Rac3, Aib1, pCIP, Actr) (Belandia and Parker, 2000). The p160 proteins are co-activators that bind to nuclear receptors and promote transcription in a hormone-dependent manner. In addition to nuclear receptors, p160 proteins can act as co-activators for transcription factors such as AP1, β-catenin and Smad3 (Xu et al., 2009). In a yeast two-hybrid screen, Tead1 and Tead2 were identified as binding partners of Src1. Further analysis indicated that Src1, Src2 and Src3 may promote TEAD-mediated transcription (Belandia and Parker, 2000). However, since this initial study, no further analysis has been reported on the TEAD-p160 interaction, or the role it plays in development. Moreover, it is unclear whether p160 proteins interact with TEADs individually, or if they interact with the TEAD-YAP/TAZ or TEAD-VGLL complex.

Recently, two novel proteins have been shown to interact with TEAD: FAM181A and FAM181B. These bind to TEAD4 via an Ω loop, which is highly homologous to the TEAD-binding domain of YAP (Bokhovchuk et al., 2020). Little is known about the function of FAM181 proteins; however, one study showed that, in mice, Fam181a/b are expressed in neural tissues, such as the forebrain, midbrain, hindbrain and neural tube. However, knockout of Fam181b does not result in an obvious phenotype (Marks et al., 2016). Therefore, further work is required to determine the role of FAM181A/B in development in order to determine the significance of its interaction with TEAD.

When bound to a co-factor, TEAD exerts its transcriptional activity by binding to promotor (Tamm et al., 2011) and enhancer (Ribas et al., 2011; Cebola et al., 2015; Zhu et al., 2019) regions. Moreover, TEAD-YAP/TAZ can recruit epigenetic modifiers, such as the Trithorax-related protein (TRR), the Switch/sucrose nonfermentable (SWI/SNF) complex, the nucleosome-remodelling and deacetylase (NuRD) complex, GAGA factor (GAF; Trithorax-like, Trl in Drosophila), and the Mediator complex (Hillmer and Link, 2019). TEADs have also been shown to form ternary complexes with other transcription factors to coordinate target gene transcription. For example, the TEAD4-YAP/TAZ complex interacts with activator protein 1 (AP1) at enhancer regions that harbour motifs for both TEAD4 and AP1. Together, they coordinate the transcription of genes involved in the cell cycle, mitosis and migration (Zanconato et al., 2015; Liu et al., 2016). Similarly, TEAD4-VGLL4 and the C-terminal binding protein (CtBP2) interact to form a transcriptional repressor complex during adipogenesis (Zhang et al., 2018). Other transcription factors that TEAD1 can interact with include myocyte enhancer factor 2 (MEF2), serum response factor (SRF) and the nuclear phosphoprotein MAX (Gupta et al., 2000, 2001; Maeda et al., 2002b).

Cell density signals play a large role in regulating the interaction between TEADs and their co-factors, and hence TEAD transcriptional activity. Cell morphology, cytoskeletal tension, cell-cell adhesion, stiffness and attachment to the extracellular matrix, metabolic stress, and numerous other factors all contribute to the regulation of YAP/TAZ subcellular localisation, and therefore TEAD activity (Low et al., 2014; Panciera et al., 2017; Zhang et al., 2020). Furthermore, other major signalling pathways such as the Wnt/β-catenin, TGFβ and EGFR pathways may cross-talk with Hippo signalling to influence TEAD activity (Huh et al., 2019).

In addition to being regulated by differential co-factor interactions, TEAD proteins themselves can be regulated by cytoplasmic translocation and post-translational modifications. To begin with, under osmotic stress, but not high-density stress, P38 MAPK drives translocation of TEAD to the cytoplasm, eliminating YAP/TAZ regulation of target genes (Lin et al., 2017). TEAD cytoplasmic translocation can occur in Mst1/2 and Lats1/2 knockout cells, indicating that P38 MAPK regulates TEAD activity independent of Hippo signalling (Lin et al., 2017).

TEADs are also regulated by post-translational modifications, such as phosphorylation and palmitoylation. For instance, phosphorylation of TEAD1 by protein kinase A (PKA) represses its DNA-binding ability, but not its ability to interact with other proteins (Gupta et al., 2000). Similarly, protein kinase C (PKC)-mediated phosphorylation of TEAD1 also reduces its DNA-binding ability (Jiang et al., 2001). TEAD1-4 can also undergo auto-palmitoylation, which regulates their stability (Noland et al., 2016) and binding affinity to Yap/Taz, but not Vgll4 (Chan et al., 2016). At high cell density, activation of the membrane-associated protein NF2 results in decreased expression of the palmitate-synthesising enzymes, FASN and ACC. Consequently, decreased palmitate levels result in decreased TEAD auto-palmitoylation (Kim and Gumbiner, 2019). Moreover, TEAD is regulated by depalmitoylases, such as APT2 (LYPLA2), and palmitoylation-deficient TEAD4 is degraded by E3 ubiquitin ligase (Kim and Gumbiner, 2019). Interestingly, the effect that palmitoylation has on protein levels and stability may differ slightly for each TEAD member (Kim and Gumbiner, 2019). Notably, TEAD palmitoylation is independent of LATS1/2 activity, showing that it regulates TEAD independent of upstream Hippo signalling (Kim and Gumbiner, 2019).

At a systemic level, this complex array of signals guides tissue growth to the correct size and shape. For instance, high mechanical forces, such as stretching induced at the curvatures of an epithelial sheet, have been suggested to pattern the proliferation and shape of the tissue (Aragona et al., 2013). In addition to controlling tissue shape and size, TEADs spatially regulate progenitor cell differentiation based on surrounding signals. For instance, during segmentation of the zebrafish hindbrain, boundary cells respond to mechanical cues through TEAD-YAP/TAZ activity, which guides the balance between progenitor self-renewal and differentiation into neurons (Voltes et al., 2019).

TEADs play a multitude of functions during development (summarised in Table 1). In mice, the role of TEADs first emerges during the preimplantation stages of development, in which Tead4-Yap is required for the specification of trophectoderm. Specifically, the TEAD4-YAP complex is involved in the specification of the trophectoderm via the activation of Cdx2 and Gata3 (Yagi et al., 2007; Nishioka et al., 2009; Ralston et al., 2010), and the inactivation of pluripotency factors such as Sox2 (Frum et al., 2019). Shortly after the trophectoderm is formed, Tead1-Yap is involved in specification of the epiblast (Hashimoto and Sasaki, 2019).

Subsequent to this, during early post-implantation development, Tead1 and Tead2 are involved in the development of the notochord, neural tube and neural crest (Kaneko et al., 2007; Cao et al., 2008; Sawada et al., 2008). Deletion of Tead2 results in high rates of exencephaly in mice due to failure of neural tube closure (Kaneko et al., 2007). In mouse, zebrafish and frog embryos, Tead1/2-Yap1 have been shown to promote the expansion of Pax3⁺ neural crest progenitors (Kaneko et al., 2007; Gee et al., 2011; Cao et al., 2014). Vgll3 has also been shown to promote neural crest migration and Pax3 expression in the frog embryo via its interaction with Tead1 and Ets1 (Simon et al., 2017). Defects in neural crest development can cause defects in anterior-posterior axis specification, as well as in somite and head morphology, phenotypes commonly shared across TEAD, YAP and VGLL knockout animal models (Sawada et al., 2008; Jiang et al., 2009; Gee et al., 2011; Johnson et al., 2011; Simon et al., 2017). Interestingly, Tead1a, Tead3a, Yap, Taz, Vgll4l and Vgll4b are also involved in the formation of Kupffer's vesicle, which establishes left-right asymmetry in zebrafish (Fillatre et al., 2019). RNA-seq analysis revealed that the majority of genes regulated by Yap/Taz in this context are similarly regulated by Vgll4l (either up- or downregulated). This suggests that, in contrast to other systems where YAP/TAZ and VGLL4 have antagonistic roles (Zhang et al., 2014), VGLL4 is not antagonistic to YAP/TAZ function in the Kupffer's vesicle, but instead may regulate similar pathways (Fillatre et al., 2019).

TEADs are also involved in the development of various organs. One of the most well-known roles of TEADs is in heart morphogenesis, where TEAD1 is required for cardiomyocyte proliferation (Liu et al., 2019; Wen et al., 2019). Ubiquitous deletion of mouse Tead1 causes embryonic lethality with heart defects (Chen et al., 1994; Wen et al., 2017), and cardiomyocyte-specific deletion of Tead1 results in perinatal lethality (Liu et al., 2019). Conversely, striated muscle-specific overexpression of Tead1 causes cardiac dysfunction (Tsika et al., 2010). Intriguingly, Tead1 appears to switch co-factors in the developing postnatal mouse heart. In the newborn heart, Tead1-Yap is the predominant interaction and promotes cardiomyocyte proliferation, whereas the Tead1-Vgll4 interaction is predominant in the adult heart and inhibits cardiomyocyte proliferation (Lin et al., 2016). This not only suggests opposing roles for Yap and Vgll4 in the heart, but also suggests that one co-factor could be therapeutically targeted without affecting the other. In addition to regulating cardiomyocytes, Tead1 regulates blood vessel development by promoting the proliferation of vascular smooth muscle cells (Wen et al., 2019).

TEADs are also involved in the development of skeletal muscle, where they act by regulating the proliferation and differentiation of satellite cells. During the early stages of mouse embryogenesis, TEADs bind to the Myf5 promotor ECR111 and drive Myf5 expression in the ventrocaudal and ventrorostral somatic compartments of the embryo; however, it is unclear which specific TEAD members are involved (Ribas et al., 2011). During the initial stages of myogenesis in mice, both Tead1-Yap and Taz promote satellite cell proliferation (Tremblay et al., 2014; Sun et al., 2017). However, during later stages of myogenesis, Tead4-Taz promotes differentiation, whereas Yap inhibits it (Sun et al., 2017). Moreover, Tead4-Vgll4 represses Yap binding to Tead4 during the proliferation stage, but then promotes the expression of lineage-specific genes, such as MyoG, during differentiation (Feng et al., 2019). Similarly, TEAD1-Vgll2 promotes the differentiation of satellite cells (Maeda et al., 2002a). These studies show that, as seen in the heart, different co-factors can alter the function of TEADs considerably, with YAP, TAZ and VGLL all having distinct roles in skeletal muscle. In future studies, it would be interesting to investigate how these co-factors differentially affect TEAD function at the molecular or transcriptional level.

In the developing mouse brain, Tead1/2/3, Yap and Taz promote the expansion of neural stem cells (Han et al., 2015; Saito et al., 2018; Mukhtar et al., 2020). Individual TEAD members can also play different roles in cortical development. For example, it was found that Tead1 and Tead3 promote the formation of Tbr1⁺ neurons and Ctip2 (Bcl11b)⁺ neurons in the cortical plate, whereas Tead2 has the opposite effect (Mukhtar et al., 2020). TEAD-Yap/Taz also contribute to rhombomere development in the zebrafish hindbrain, although it is unclear which TEAD members are involved as the reporter used in this study broadly reflects Yap/Taz-Tead activity (Voltes et al., 2019). Looking forwards, the analysis of individual TEAD knockout animal models could provide clarity on which TEAD members are involved in hindbrain development. In the mouse peripheral nervous system, Tead1 regulates Schwann cell development (Lopez-Anido et al., 2016). Tead1/2-Yap are also involved with regulating the S phase of retinal glial cells in the adult frog (Cabochette et al., 2015), and Tead1-Yap regulates the response of Müller glia to photoreceptor degeneration in the adult mouse (Hamon et al., 2017). Moreover, Tead1a-Yap/Taz promotes retinal pigment cell lineage formation in zebrafish (Miesfeld et al., 2015).

TEADs also influence the differentiation of a number of other cell types during development. For example, Tead4 forms a ternary complex with Vgll4 and CtBP2 to repress adipogenesis in mice (Zhang et al., 2018). In addition, the interaction between Yap and TEADs (Tead1-4) inhibits the differentiation of mouse mesenchymal stem cells into osteoblasts, whereas TEAD-Vgll4 promotes differentiation by inhibiting TEAD-YAP interactions (Suo et al., 2020). Finally, TEAD-YAP also promote the proliferation and stem-like characteristics of various other progenitor cells, such as progenitor cells of the inner ear (Gnedeva et al., 2020), intestine (Imajo et al., 2015), pancreas (Cebola et al., 2015) and epidermis (Schlegelmilch et al., 2011; Zhang et al., 2011). To the best of our knowledge, there has been limited investigation into the role of VGLL proteins in these tissues.

After birth, TEADs continue to play several important roles in maintaining the homeostasis of various tissues, as well as contributing to tissue responses to injury and mechanical stimuli. To begin with, TEADs are required for maintaining function in some populations of cells, including both progenitor and differentiated cells. For example, Tead1/2-Yap regulates the proliferation and differentiation of postnatal mouse epidermal progenitor cells (Beverdam et al., 2013). Moreover, Tead1 is involved in regulating the function of differentiated cells, such as adult mouse cardiomyocytes (Liu et al., 2017).

In some tissues, TEAD activity levels are low but are upregulated following injury to promote regeneration. In the mouse liver, TEAD4-YAP promotes the proliferation of hepatic progenitors during regeneration (Yimlamai et al., 2014; Su et al., 2015). TEAD-YAP and Tead1/3/4-Vgll3 promote skeletal muscle healing and hypertrophy in mice following injury to motor nerves (Watt et al., 2015; Figeac et al., 2019). In murine vascular smooth muscle cells, TEAD-YAP promotes the ‘phenotypic switching’ from a contractile to a proliferative phenotype in response to damage, thus facilitating vascular remodelling (Liu et al., 2014; Osman et al., 2019; Kimura et al., 2020). Finally, Tead1-Yap1 promotes angiogenesis and alveolar regeneration in the mouse lung following pneumonectomy (Mammoto et al., 2019).

Intriguingly, TEADs have also been shown to regulate tissue growth or morphology in response to mechanical stimuli. This has been particularly well demonstrated in adult skeletal muscle. Both TEAD-YAP and Tead1/3/4-Vgll3 regulate skeletal muscle mass and promote hypertrophy following mechanical overload (Goodman et al., 2015; Figeac et al., 2019). Moreover, TEAD1/4-Vgll2 contributes to normal muscle fibre distribution by increasing the number of slow-twitch type I fibres in response to mechanical overload (Tsika et al., 2008; Honda et al., 2019). TEAD1-YAP has also been shown to regulate alveolar bone remodelling by promoting osteoclastogenesis, and its activity decreases in response to compressive forces. Inhibition of Mst1/2 increases TEAD-mediated transcription, indicating that the canonical Hippo pathway regulates osteoclastogenesis in response to mechanical force (Li et al., 2019). Finally, Tead2-Yap is involved in blood vessel maintenance in response to shear stress (Nakajima et al., 2017). In contrast to alveolar bone remodelling, Mst1/2 and Lats1/2 are not affected by shear stress, suggesting that the canonical Hippo pathway is not involved. Instead, Yap nuclear localisation is regulated by the actin skeleton and AMOT (Nakajima et al., 2017). Altogether, these studies provide interesting insight into how mechanical signals can affect TEAD-mediated transcription, consequently altering the function of a tissue. Some of these mechanical signals may be Hippo dependent, whereas other may be Hippo independent. Further investigation into how TEADs are regulated by mechanical stimuli in vivo would provide greater understanding of the role of TEADs in development and homeostasis.

Because TEADs play crucial roles in the balance between progenitor cell proliferation and differentiation, it is not surprising that TEAD dysregulation can result in disease. Hypoactivation of TEADs can result in organ dysgenesis, whereas hyperactivation can cause proliferative diseases such as cancer (Fig. 3).

In humans, there have been no reports of homozygous TEAD mutations. This is most likely due to spontaneous abortion, considering the essential role of TEADs during early development. Indeed, TEADs are required early in human development, such as in the formation of extra-embryonic tissue (Jacquemin et al., 1998; Saha et al., 2020). Extensive genome and exome sequencing indicate that TEAD1 and TEAD3 are haploinsufficient, whereas TEAD2 and TEAD4 are not (Karczewski et al., 2020). This predicted haploinsufficiency for TEAD1 fits with the finding that a heterozygous missense mutation of TEAD1 is causative for Sveinsson chorioretinal atrophy (Fossdal et al., 2004; Kitagawa, 2007). This missense mutation (Y421H) destabilises the interaction of TEAD1 with YAP and TAZ, but not VGLL1-3 (Bokhovchuk et al., 2019). Interestingly, individuals with this mutation have no other disorders or signs of co-morbidity (Jonasson et al., 2007). It is unclear why only the eye is affected, despite the major role that TEAD1 plays in the rest of the body. Perhaps TEAD1, or the TEAD1^Y421H mutation, is redundant in other regions of the body, although this seems unlikely due to the requirement for TEAD1 in various tissues, including the heart (Liu et al., 2017). In future studies, work on patient-derived cell lines or organoids may shine light on the pathological effects of this mutation and why it only affects retinal tissue. Alternatively, it may be beneficial to generate an animal model with a homologous mutation, allowing the disorder to be thoroughly investigated.

In addition to Sveinsson chorioretinal atrophy, several other developmental disorders have been associated with mutations in TEAD genes or co-factors (Table 2). For instance, TEAD1 variants may be a rare cause of Aicardi syndrome, a congenital disorder characterised by agenesis of the corpus callosum, neuronal migration defects, chorioretinal lacunae and infantile spasms. A study identified a TEAD1 variant in one individual with Aicardi syndrome, out of a cohort of ten (Schrauwen et al., 2015). In contrast, a second study consisting of 38 individuals did not identify any TEAD1 variants (Wong et al., 2017). Modelling this mutation in rodent models could enable researchers to determine whether this TEAD1 variant contributes to Aicardi-related phenotypes in mice.

The co-factors of TEADs, particularly YAP and TAZ, have also been implicated in numerous other diseases, such as coloboma (Williamson et al., 2014), Alexander disease (Wang et al., 2018), Huntington disease (Mueller et al., 2018) and disorders of the immune system (Hong et al., 2018). Although TEADs have not yet been directly investigated in these diseases, it is highly likely that they are involved, owing to their close relationship with YAP/TAZ. It will therefore be important to investigate the role of TEADs in these diseases to gain a better understanding of the molecular mechanisms of the disease, and to uncover potential targets for therapy.

As they are pro-proliferative genes, it is not surprising that TEADs play major roles in cancer. Indeed, dysregulation of TEADs has been linked to numerous types of cancer, including medulloblastoma, breast, gastric, renal, prostate, colorectal, hepatocellular, and squamous cell carcinoma (Holden and Cunningham, 2018; Huh et al., 2019). VGLL1-3 are also associated with cancer and tumorigenesis (Yamaguchi, 2020). In contrast to VGLL1-3, VGLL4 has been shown to act as a tumour suppressor by inhibiting YAP binding and promoting apoptosis (Yamaguchi, 2020). A greater understanding the transcriptional programmes exerted by TEADs during development will help to shed light on cancer pathologies that occur when TEADs are dysregulated.

Owing to their major role in cancer, the TEAD-YAP/TAZ complex has gained significant interest as a therapeutic target for cancer or other disorders (Liu-Chittenden et al., 2012; Zhou et al., 2015; Kaan et al., 2017). Indeed, numerous in vivo and in vitro studies show that targeting TEAD can inhibit cancer growth (Liu-Chittenden et al., 2012; Zhang et al., 2014; Lin et al., 2017). Although no compounds targeting TEADs have progressed to the clinical stage, various recent studies have identified molecules that target the co-factor-binding domains, the DNA-binding domain or the stabilizing lipid pocket of TEADs (Fig. 2) (Gibault et al., 2018; Calses et al., 2019; Holden et al., 2020).

In addition to being a target for drug treatment, TEADs have applications in other therapies. For instance, the TEAD-YAP/TAZ complex is involved in maintaining the pluripotency and self-renewal of embryonic stem cells (ESCs), and therefore could have applications in stem cell-related research (Tamm et al., 2011; Beyer et al., 2013; Chung et al., 2016). YAP and TAZ have also been shown to promote the efficiency of induced pluripotent stem cell (iPSC) generation (Lian et al., 2010; Qin et al., 2012; Zhao et al., 2017). Moreover, induced expression of YAP or TAZ can facilitate dedifferentiation of mature cells into progenitor-like cells. This has been shown in mammary gland, neuronal, pancreatic exocrine, and liver cells (Yimlamai et al., 2014; Panciera et al., 2016). Considering that TEADs are major transcription factors for YAP and TAZ, it is likely that they regulate transcription via TEADs. Because ESCs and iPSCs and have valuable applications in research and therapies, it would be beneficial to gain a clearer understanding of the role that TEADs play in these cells.

Finally, TEADs may have potential applications in gene therapy owing to their role in regeneration and homeostasis. For example, viral gene delivery of activated Yap in postnatal inner ear sensory epithelia promotes cell cycle re-entry after hair cell loss in a TEAD-dependant manner (Gnedeva et al., 2020). Further work is required to investigate the therapeutic potential of TEADs in other tissues, as well as to elucidate safety considerations that may arise, such as an increased risk of cancer.

One theme arising from the studies described above is that the interaction of TEADs with different co-factors can drive distinct transcriptional and morphological outcomes. In some studies, such as those of the neural crest and Kupffer's vesicle, VGLL plays a similar role to YAP/TAZ, often by regulating the same genes (Simon et al., 2017; Fillatre et al., 2019). However, at other times VGLLs play a distinct and antagonistic role to YAP/TAZ, such as the role of Vgll2 in skeletal muscle, or of Vgll4 in the heart (Lin et al., 2016; Feng et al., 2019). Similarly, although YAP and TAZ are redundant in some developmental contexts (Sawada et al., 2008), this is not always the case (Sun et al., 2017; Hagenbeek et al., 2018; Plouffe et al., 2018). For example, in satellite cells, Yap and Taz regulate many common genes and promote proliferation; however, Taz may also regulate distinct genes and promote myogenic differentiation (Sun et al., 2017). Taken together, these studies show that the relationships between TEADs and their co-factors are highly context specific. This poses an interesting question: how do YAP, TAZ and VGLL differentially alter the function of TEAD in various developmental stages or tissues? Do different co-factors elicit distinct binding events to promoters and enhancers, and/or recruit discrete epigenetic modifiers? Understanding the different roles of each TEAD co-factor is a crucial step towards understanding how TEADs guide development and homeostasis across numerous contexts.

In addition to their co-factors, individual TEAD members also elicit unique functions. This is foremost demonstrated by their unique expression patterns (Yasunami et al., 1996) and the distinct effects that loss of function of different TEAD members have. For instance, ubiquitous knockout of Tead4 causes early embryonic lethality due to trophectoderm defects (Yagi et al., 2007), whereas ubiquitous knockout of Tead1 causes embryonic lethality at a later stage, primarily as a result of heart defects (Chen et al., 1994). However, the unique roles of TEADs are not only due to their different expression patterns. There is evidence that TEADs may also have unique functions even when they are expressed in the same tissue. This has been demonstrated in the forebrain, where Tead1 and Tead3 promote the formation of Tbr1⁺ neurons and Ctip2⁺ neurons; however, Tead2 inhibits the formation of such neurons (Mukhtar et al., 2020). Nonetheless, despite the evidence that individual TEAD members have unique functions, it is unclear how they function differentially, particularly considering their high homology with each other in the DNA-binding domain. Moreover, each TEAD member is able to interact with YAP, TAZ and VGLL1 with approximately the same affinity (Bokhovchuk et al., 2019). Perhaps different TEAD members form different tertiary complexes with other transcription factors, thus affecting their transcriptional output? Determining how TEADs elicit unique functions will be highly valuable for understanding how these transcription factors function in development, homeostasis and disease.

A number of studies have revealed that TEADs play a major role in development. However, many questions remain to understand fully how TEADs function on a transcriptional, cellular and systemic level, as well as in the context of disease. In the past, YAP has been considered to be the main co-factor of TEADs, and many studies have focused exclusively on this interaction. However, with the increasing evidence of the major roles that TEAD-VGLL interactions play during development, as well as the unique roles of TEAD-TAZ, it is important to consider these interactions with equal weight. Crucially, there is still much to learn about the VGLL protein family, particularly in terms of its upstream regulation. Moreover, other potential co-factors of TEADs, such as the p160 family and FAM181A/B, require further investigation (Belandia and Parker, 2000; Bokhovchuk et al., 2020).

Another important question pertains to how exactly the functions of TEADs differ when bound to different co-factors. Do they recognise different promotor or enhancer regions, or interact with other transcription factors? It is important to elucidate similarities and differences of each co-factor to understand fully the roles of TEADs at a transcriptional level. Similarly, how do TEAD members function differentially, especially when they are expressed in the same tissue? Further work investigating transcriptional output (e.g. RNA-seq), protein-DNA interaction (e.g. ChIP-seq) or chromatin accessibility (e.g. ATAC-seq), of different TEAD-co-factor combinations would be highly useful in determining how different co-factors and TEAD members influence transcription.

Although there are numerous diseases in which Hippo signalling is implicated, the role of TEADs in disease has been investigated to a lesser degree. As YAP and TAZ can interact with transcription factors other than TEADs (Kim et al., 2018), it will be important to confirm whether TEADs are involved in Hippo pathway diseases. This may lead to new insights into the pathology, as well as potential molecular targets for drug therapy. Finally, although TEADs could be a promising target for cancer therapy, there is still much to learn about the role of this family of transcription factors, particularly in late stages of development and in the adult. This will determine whether targeting TEADs therapeutically will have any deleterious side effects, such as impairment of healing or homeostasis in certain tissues.

Figures in this Primer were generated in BioRender.