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The emerging roles of TCF4 in disease and development Marc P. Forrest1, Matthew J. Hill1, Andrew J. Quantock2, Enca Martin-Rendon3,4, and Derek J. Blake1 1

Institute of Psychological Medicine and Clinical Neurosciences, MRC Centre for Neuropsychiatric Genetics and Genomics, School of Medicine, Cardiff University, Cardiff, UK 2 Structural Biophysics Group, School of Optometry and Vision Sciences, Cardiff University, Cardiff, UK 3 Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, Oxford, UK 4 Stem Cell Research Laboratory, NHS Blood and Transplant, John Radcliffe Hospital, Oxford, UK

Genome-wide association studies have identified common variants in transcription factor 4 (TCF4) as susceptibility loci for schizophrenia, Fuchs’ endothelial corneal dystrophy, and primary sclerosing cholangitis. By contrast, rare TCF4 mutations cause Pitt–Hopkins syndrome, a disorder characterized by intellectual disability and developmental delay, and have also been described in patients with other neurodevelopmental disorders. TCF4 therefore sits at the nexus between common and rare disorders. TCF4 interacts with other basic helix–loop– helix proteins, forming transcriptional networks that regulate the differentiation of several distinct cell types. Here, we review the role of TCF4 in these seemingly diverse disorders and discuss recent data implicating TCF4 as an important regulator of neurodevelopment and epithelial– mesenchymal transition. TCF4 involvement in common and rare disorders Attempts to identify risk genes for common disorders in humans have started to yield impressive results. Genetic variation in many new and hitherto unexplored loci has been associated with a broad range of common diseases including schizophrenia and intellectual disability (ID). One of the most promising of these risk genes is transcription factor 4 (TCF4) on human chromosome 18 (Figure 1A). TCF4 was one of the first genes to reach genome-wide significance in large-scale genetic association studies of schizophrenia [1]. Intriguingly, common TCF4 variants (see Glossary) are also associated with increased risk of Fuchs’corneal endothelial dystrophy (FECD) and primary sclerosing cholangitis (PSC), whereas rare TCF4 mutations cause Pitt–Hopkins syndrome (PTHS), a genetic disorder characterized by ID, distinctive facial features, developmental delay, and autonomic dysfunction. Although PTHSassociated mutations result in haploinsufficiency, the Corresponding author: Blake, D.J. ([email protected]). Keywords: transcription; intellectual disability; epithelial–mesenchymal transition; schizophrenia; Fuchs’ endothelial corneal dystrophy; Pitt–Hopkins syndrome. 1471-4914/$ – see front matter ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molmed.2014.01.010

mechanism where common TCF4 variants confer risk to these seemingly diverse disorders is currently unknown. However, recent studies have shown that TCF4 regulates several fundamental developmental processes in a variety of different cell types. In this article, we briefly review the developmental functions of TCF4 before focusing on the role of TCF4 in a range of different disorders. TCF4 in general transcription TCF4 is a member of the basic helix–loop–helix (bHLH) family of transcription factors that have an important role in a number of developmental processes (Box 1). Although TCF4 (GeneID: 6925) is the official Human Genome Organization (HUGO) symbol, in the literature it is often referred to as E2-2, immunoglobulin transcription factor 2 (ITF2), or SL3-3 enhancer factor 2 (SEF2). Importantly, transcription factor 4 should not be confused with transcription factor 7-like 2 (TCF7L2), a gene on chromosome 10q25.3 that is also referred to as T cell factor 4, and therefore shares the TCF4 acronym. The seemingly confusing nomenclature that surrounds TCF4 partially reflects the different contexts in which the gene was first described. The TCF4 (ITF2) cDNA was initially discovered as its cognate protein was able to bind the mE5 heavy chain and kE2 light chain immunoglobulin enhancers [2]. The mE5 and kE2 enhancer sequences both contain an Ephrussi box (E-Box) DNA element (CACCTG) that was recognized as an important TCF4-binding site [2,3]. Independently, two TCF4 isoforms (SEF2-1A or TCF4-A, and SEF2-1B or TCF4-B) isolated from human thymocytes were found to bind an enhancer in the murine leukemia virus SL3-3 genome [4]. TCF4 was also found to modulate other E-box-containing regulatory regions such as the rat tyrosine hydroxylase enhancer and the human somatostatin receptor-2 promoter [5,6]. Together, these studies established TCF4 as a conserved bHLH transcription factor that binds E-box sequences in the promoters and enhancers of certain genes. In common with the other E-proteins, the modular domain structure of TCF4 allows it to interact with numerous transcriptional regulators and to bind directly to DNA via the bHLH domain [7]. The human TCF4 gene is predicted to encode at least 18 different proteins with unique N-terminal sequences [8]. Several alternatively spliced TCF4 variants have also been Trends in Molecular Medicine xx (2014) 1–10

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Review Glossary

Box 1. The bHLH protein family

Common variants: such as SNPs and microsatellites, are polymorphic loci dispersed throughout the genome. They are found with high frequency (minor allele frequency >1%) and can affect gene function by altering amino acid sequence (nonsynonymous polymorphism) or by affecting gene expression or RNA processing. E-proteins: members of the class I bHLH family of transcription factors. E-proteins form homodimers or heterodimerize with other classes of bHLH transcription factors. The three E-protein genes in humans are TCF4, TCF3, and TCF12. Endophenotype: a concept in genetic epidemiology used to describe phenotypes positioned between genotype and disease. They should be heritable and associated with the illness but state-independent. They are assumed to have simpler genetic underpinnings than the associated diseases. Epithelial–mesenchymal transition (EMT): reversible differentiation process where cells lose their epithelial characteristics and become motile and invasive (see Box 2). Fuchs’ endothelial corneal dystrophy (FECD): a slowly progressing eye disease caused by degradation of the corneal endothelium resulting in corneal edema and loss of vision. Genome-wide association studies (GWAS): a technique in genetic epidemiology where SNPs in a genome are scanned for association with a particular disease or phenotype. A typical study design genotypes millions of SNPs in large numbers of cases compared with controls for association with a specific trait. Haploinsufficiency: occurs in diploid organisms (where there are two copies of each chromosome) that have only a single functional copy of a gene. Haploinsufficiency arises when the remaining functional copy of the gene does not produce a sufficient gene product (often a protein) to maintain the wild type phenotype. Haploinsufficiency is the cause of many, but not all, autosomal dominant disorders. Intellectual disability (ID): the World Health Organization defines ID as a significantly reduced ability to understand new or complex information and to learn and apply new skills. ID is present before adulthood and is often categorized by an intelligence quotient of less than 70. Odds ratio (OR): a measurement of the association effect size used in case– control genetic association studies. It is defined as the ratio between the proportion of individuals in the case group having a specific allele and the proportion of individuals in the control group having the same allele. Pitt–Hopkins syndrome (PTHS): is a rare genetic disorder characterized by ID, distinctive facial features, developmental delay, breathing abnormalities, and autonomic dysfunction. Autosomal dominant TCF4 mutations appear to cause the most frequent form of PTHS. Plasmacytoid dendritic cell (pDC): a specific cell type of the immune system that secretes high levels of interferon in response to viral stimulation. Pontine nucleus: a region in the brainstem that connects cortical structures to the cerebellum. Primary sclerosing cholangitis (PSC): a relatively rare form of cholestatic liver disease caused by inflammation and scarring of the bile ducts. Rare variants: in the context of human genetic disease, rare variants can be defined as having a minor allele frequency less than 1%. These variants are often highly penetrant, meaning that when they are present, they frequently cause the disease phenotype. For example, mutations that cause Mendelian genetic diseases in humans (e.g., PTHS) are considered rare variants and may affect the function or synthesis of a protein. Other rare variants include deletions and duplications of genes or parts of genes. These mutations are also known as CNVs. Schizophrenia: a mental disorder characterized by disorganized thought and abnormal emotional responses. Symptoms include paranoia, delusions, hallucinations, and emotional disturbances. It has a prevalence of approximately 1% with a typical age of onset in the early twenties. Although highly heritable, the underlying genetics is complex. Ulcerative colitis: a form of inflammatory bowel disease where inflammation develops in the large intestine. Verbal memory: a concept in cognitive psychology concerned with the memory and recall of verbal information and other linguistic traits.

The bHLH family is organized into seven functional classes according to expression, dimerization capability, and DNA-binding specificity [94]. The class I bHLH proteins, or E-proteins, are related to the Drosophila daughterless gene [3,95]. In addition to TCF4, humans express two other E-proteins, TCF3 (E2A) and TCF12 (HEB), each also encoding two major isoforms. E-proteins are widely expressed in different tissues and are able to form stable homo- and heterodimers with other bHLH proteins that bind to E-boxes with the consensus sequence ‘CANNTG’ in genomic DNA [21]. The class I bHLH proteins can heterodimerize with class II (such as the products of the proneural genes ASCL1 and ATOH1), class V (ID proteins), and class VI (HES1) members of the bHLH family. Class II transcription factors have tissue-restricted expression patterns and are potent inducers of cell type specification [51]. To efficiently bind DNA, class II bHLH transcription factors must heterodimerize with an E-protein, making E-proteins key regulators of biological activity [22,96]. Conversely, class V and class VI transcription factors are characterized by their inhibitory or repressive activities. The ID proteins are orthologous to the Drosophila extramacrochaetae protein and lack the basic region of the bHLH sequence, rendering them unable to bind DNA [97]. ID proteins heterodimerize with E-proteins and sequester them into inactive complexes that limit their availability for class II transcription factors [98]. The expression of ID proteins therefore inhibits the activity of the bHLH transcription factor network, affecting cell proliferation and differentiation [99]. The hairy and enhancer of split (HES) proteins (bHLH class VI) are unique as they contain a proline residue in their basic region. This unique structure enables them to bind N-boxes (consensus sequence ‘CACNAG’) rather than E-boxes [100,101]. HES proteins also heterodimerize with class I factors and repress their transcriptional activity [100,102]. HES proteins play a central role in maintaining progenitor cells in an undifferentiated state [103]. The class VII proteins contain Per–ARNT–Sim (PAS) domains that sense oxygen tension, redox potential, light, and some other stimuli.

documented, further expanding the potential repertoire of distinct TCF4 isoforms in humans [8,9]. However, only two major TCF4 isoforms, TCF4-A and TCF4-B (Figure 1B), have been extensively studied [10,11]. Full-length TCF4 (TCF4-B), in common with the other E-proteins, has two activation domains (AD1 and AD2) that are thought to modulate transcriptional activity, and a nuclear localization signal (NLS) that controls subcellular localization. By contrast, TCF4-A lacks AD1 and the NLS, and is thought to traffic to the nucleus when heterodimerized with another bHLH transcription factor [8]. 2

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TCF4-B has been described as a transcriptional repressor in different cell types [12–14]. However, it has been demonstrated that this isoform can also activate transcription in vitro, and its function is therefore highly contextdependent [5,15,16]. Part of its contrasting effects on transcription may depend on its interaction with transcriptional regulators. For example, AD1 is proposed to interact with the transcriptional activator p300 (EP300), a protein with histone acetyltransferase activity that can activate transcription [17,18]. It has been proposed that the transcriptional co-repressor, runt-related transcription factor 1 (RUNX1T1) must compete with p300 for the same AD1binding site [19]. The N-terminal region of the bHLH domain contains a highly conserved group of positively charged amino acids that make up the basic region that interacts with DNA [20,21]. The remainder of the bHLH motif forms a surface that favors homo- and heterodimerization through the assembly of a stable four-helix bundle with a defined hydrophobic core [21,22]. The structure of E-protein dimers exploits the central twofold symmetry of the hexanucleotide E-box with each monomer asymmetrically contacting an E-box half site [21]. Directly C-terminal to the bHLH domain is a highly conserved set of amino acids termed domain C. This domain is exclusively present in Eproteins and is critical for dimerization in physiological contexts [23]. The bHLH domain thus permits TCF4 to interact with a potentially vast array of transcription factors [24]. Although the exact repertoire of TCF4-binding

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(TGC)n

rs1452787 rs613872

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rs1261117

rs17512836

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3′ Human TCF4

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TCF4-B

p300 (EP300) RUNX1T1

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Figure 1. The TCF4 gene and protein. (A) The schematic shows the genomic organization of the TCF4 gene and the location of common variants associated with schizophrenia (red), Fuchs’ endothelial corneal dystrophy (FECD; blue), and primary sclerosing cholangitis (PSC; green). The horizontal line represents genomic DNA, whereas the boxes represent the location of the coding (unshaded) and noncoding (gray) exons. (B) Conceptual organization of TCF4 protein domains and their functions. The transcriptional activity and subcellular location of TCF4 is tightly controlled by interactions with multiple proteins. Activation domains 1 and 2 (AD1 and AD2) interact with the p300 transcriptional coactivator protein, whereas only AD1 can recruit the RUNX1T1 co-repressor. The CE repressor (CE) is thought to modulate the activity of AD1 to attenuate the transcriptional activity of TCF4. By contrast, a second repressor domain (Rep) can repress the activities of both AD1 and AD2 and is thought to maintain Eprotein homodimers in an inactive state on certain enhancers. The basic domain (b) mediates DNA binding to genomic E-box sequences. The helix–hoop–helix (HLH) domain is an interaction surface for basic HLH (bHLH) homo- and heterodimerization. Domain C is required for in vivo homodimerization. The HLH domain of TCF4 can interact with a large repertoire bHLH, including the proneural proteins ATOH1 and NGN2 and the dominant negative antagonist ID2. Whereas heterodimers formed between ATOH1 or NGN2 and TCF4 can act as transcriptional activators or repressors, interactions with ID2 sequester TCF4 into an inactive complex that cannot bind to DNA. Ca2+dependent proteins such as calmodulin (CaM) interact directly with the basic region of the bHLH domain to selectively inhibit the DNA binding of E-protein homodimers. In addition to interactions with other bHLH proteins described above, a nuclear localization signal (NLS) also controls the subcellular location of TCF4. Abbreviations: AD1, activation domain 1; AD2, activation domain 2; ATOH1, atonal homolog 1; b, basic domain; CaM, calmodulin; CE, CE repressor domain; C, domain C; EP300, E1A-binding protein p300; FECD, Fuchs’ endothelial corneal dystrophy; HLH, helix–loop–helix; ID2, inhibitor of DNA binding 2; NGN2, neurogenin-2; NLS, nuclear localization signal; PSC, primary sclerosing cholangitis; Rep, repressor domain; RUNX1T1, runt-related transcription factor 1; SZ, schizophrenia.

partners has yet to be determined, the related E-protein TCF3 has been shown to interact with numerous transcriptional and regulatory proteins [25]. The DNA-binding properties of TCF4 and the other Eproteins are also regulated by Ca2+-binding proteins such as calmodulin (CaM), S100a, and S100b [26–29]. In the presence of Ca2+, CaM can selectively inhibit the DNA binding of E-protein homodimers, whereas E-protein heterodimers are much less sensitive [26]. Ca2+-dependent proteins interact directly with the basic region of the bHLH domain, involved in DNA binding [27]. Consistent with these interactions, altering levels of CaM or Ca2+ in cells has a direct effect on E-protein-mediated transcription [30,31]. Requirement for TCF4 in lymphoid development E-proteins and their bHLH-binding partners are critical regulators of the transcriptional networks controlling many aspects of lymphoid development in the immune

system [32]. Although it is beyond the scope of this article to provide a detailed synopsis of this area, the reader is referred to the following review articles [32,33]. Studies in transgenic mice have shown that Tcf4 can affect both B and T cell development [34,35]. Although Tcf4 is not absolutely required for B cell development, Tcf4 knockout mice display a reduced number of pro-B cells, demonstrating a role for Tcf4 in early B cell development, albeit in cells with reduced levels of the other E-proteins [34]. By contrast, Tcf4 plays a crucial role in plasmacytoid dendritic cell (pDC) development. pDC cells are a distinct immune cell type specialized in type I interferon secretion in response to viral nucleic acids [36]. Tcf4 is highly expressed in pDCs and is essential for the development and maintenance of the pDC phenotype [37,38]. Accordingly, Tcf4 controls the expression of several pDC-specific genes that are not expressed in uncommitted precursors [38]. Tcf4+/ mutant mice and patients with PTHS (see below) have immature 3

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Review pDCs characterized by impaired interferon secretion and altered expression of pDC-specific genes [38]. To add another layer of complexity to this process, TCF4 is negatively regulated by Id2 during DC differentiation [39]. Expression of each of these genes is further regulated by cytokine-induced signal transducer and activator of transcription (STAT) signaling that controls the balance of Id2 and Tcf4 expression during pDC development [40]. Taken together, these data suggest that Tcf4 and Id2 coordinate the gene expression programs that control pDC development in vivo. Whether TCF4 variants are associated with an increased risk of infection has yet to be determined; however, hyperactivation of pDCs has been described in a range of autoimmune disorders [41]. TCF4 induces epithelial–mesenchymal transition In certain cell types, TCF4 is emerging as an important regulator of epithelial–mesenchymal transition (EMT; Box 2) [42]. EMT is a complex development process where epithelial cells depolarize and acquire motile and invasive properties. Accordingly, EMT plays an important role during embryonic development, in tissue repair, and in cancer metastasis. Epithelial cells undergoing EMT lose their apico-basal polarity and intercellular junctions to become migratory mesenchymal cells [43]. Although the differential expression of multiple epithelial and mesenchymal markers are used to characterize the EMT process, a hallmark of EMT is the loss of epithelial cadherin (E-cadherin; CDH1) expression [44]. The overexpression of TCF4 in epithelial Madin–Darby canine kidney cells leads to a potent induction of EMT [11]. Cells overexpressing TCF4 acquire a motile and highly invasive phenotype that is associated with change in a number of EMT markers [11]. CDH1 expression is downregulated with other epithelial markers, whereas mesenchymal markers such as neuronal cadherin (N-cadherin; CDH2), vimentin, and fibronectin are upregulated in these cells [11]. The phenotype is similar to epithelial cells overexpressing the transcription factors SNAIL1 (SNAI1), SNAIL2 (SNAI2), and TCF3 (E47) that are also important regulators of EMT [45–47]. However, TCF4 appears to be acting downstream

Box 2. Epithelial–mesenchymal transition Epithelial–mesenchymal transition (EMT) is a reversible developmental process where epithelial cells lose their adhesive properties and apico-basal polarity and gain a migratory and invasive phenotype. EMT is an important process in cancer metastasis but is also a fundamental property of many cell populations during early embryonic development. For example, migratory neural crest cells delaminate from the neuroectoderm to generate mesenchymal tissue that will give rise to neurons of the peripheral nervous system and glia cells [104]. Several EMT regulators have been identified. These factors can be broadly divided into three groups: growth factors TGF-b superfamily members, transcriptional regulators SNAIL1 and SNAIL2 (SNAI1 and SNAI2), and matrix proteins and receptors (E-cadherin). Repression of E-cadherin (CDH1) is considered to be one of the cardinal features of cells undergoing EMT. Accordingly, growth factor signaling activates the EMT program by induction of transcription factors such as SNAI1, ZEB2, and TCF4 that directly or indirectly repress CDH1. Repression of epithelial cell markers is accompanied by the increased synthesis and deposition of extracellular matrix proteins in cells undergoing EMT. 4

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of these transcription factors and is not required to maintain the mesenchymal fate [11,48]. Pathway analysis of differentially expressed genes in TCF4-depleted human neuroblastoma cells showed that multiple signaling pathways and genes involved in EMT were altered by knockdown of TCF4 [49]. Notably, knockdown of TCF4 affected several genes in the transforming growth factor b (TGF-b) signaling pathway that is known to be important for inducing EMT [49,50]. TCF4 knockdown was also associated with differential expression of the EMT regulators, SNAI2 and zinc finger E-box binding homeobox 2 (ZEB2). Thus, several lines of convergent evidence support a role for TCF4 as an EMT regulator in different cells. Proneural genes and neurodevelopment bHLH proteins have an essential role during development of the nervous system [51,52]. Studies in Drosophila and vertebrate models have been instrumental in uncovering the complex transcription factor networks that regulate the neurogenic differentiation process [51,52]. Proneural transcription factors belonging to the bHLH family, such as atonal homolog 1 (ATOH1) and NEUROG2 (Boxes 1 and 3), are expressed in neural progenitors and coordinate the differentiation programs that specify different neuronal populations. Many proneural factors form heterodimers with TCF4 to regulate gene expression during neurodevelopment. These interactions are of clinical interest because human TCF4 variants have been associated with ID and schizophrenia. Homozygous Tcf4 knockout (Tcf4/) mice die within 24 h of birth indicating that Tcf4 is a crucial transcription factor required for normal developmental [34,53]. Tcf4/ mice have no gross anatomical defects; however, detailed analysis of their hindbrain has shown that these mice have disrupted pontine nucleus development [53]. Tcf4 deletion in mice causes a reduction in the number of neurons

Box 3. Neurodevelopment In the vertebrate embryo, neurodevelopment begins with the creation of a single layer of highly polarized cells, known as the neuroepithelium, that contains neural stem cells with self-renewing properties [105]. Neuroepithelial cells proliferate and give rise to additional classes of neural progenitor cells with distinct morphological and proliferative properties such as radial glia and intermediate progenitors [105]. Neural progenitors eventually differentiate into the different types of mature neurons and glia that populate the brain [105]. The onset of neurogenesis is marked by the loss of epithelial features that characterize the neuroepithelium, cellular detachment from the basement membrane (delamination), and the onset of neuronal migration [106]. The process of neurogenesis is dependent on the expression of the proneural bHLH transcription factors related to the Drosophila atonal (ato) and achaete-scute (ac/sc) families [51]. Genes with proneural functions in vertebrates include Ascl1, Atoh1, Atoh7, and Neurog1–3 [107]. Proneural genes have been studied for their capacity to program the neuronal lineage; however, they are also recognized for their more precise roles in defining neural subtypes in the brain [51]. Proneural genes are expressed throughout neurogenesis and are important for coordinating cell cycle exit, delamination, neuronal migration, and the final stages of neuronal differentiation [108–111]. As class II bHLH transcription factors, the proneural proteins are thought to require an E-protein such as Tcf4 in order to heterodimerize and become transcriptionally active.

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Review forming the pontine nucleus and an accumulation of ectopic neurons outside this region that fail to migrate to their correct location. Importantly, these deficits are highly specific to Tcf4, as the development of this hindbrain nucleus in other E-protein knockout mice (Tcf3/ and Tcf12/) is normal [53]. The development of the pontine nucleus is also dependent on the expression of the proneural gene Atoh1 that can heterodimerize with Tcf4 [53]. Interestingly, Atoh1+// Tcf4+/ mutant mice also display defects in pontine nucleus development, whereas Tcf4+/ have no abnormalities in this region [53]. The abundance of specific Tcf4 heterodimers may therefore be important for the development of specific neuronal subpopulations in the brain. This study elegantly demonstrates the requirement for Tcf4 and particular Tcf4/Atoh1 heterodimers in brain development. Rare TCF4 variants and neurodevelopmental disorders Rare TCF4 mutations cause autosomal dominant PTHS, a severe form of ID associated with a constellation of other developmental abnormalities [54]. PTHS was first described in two unrelated children with severe ID, similar facial features, and episodes of hyperventilation followed by apnea and cyanosis [55]. Subsequently, heterozygous TCF4 mutations were found in PTHS patients that result in haploinsufficiency [54,56]. The identification of TCF4 as the genetic cause of PTHS has led to a detailed characterization of the disorder. In addition to the symptoms described above, visual and ocular abnormalities, epilepsy, and digestive complications are also frequently reported. Although a range of different mutations have been described in PTHS patients, most missense mutations are clustered in the bHLH domain of the protein [15,16,54,56–58]. Many of these mutations impair the transcriptional activity of the mutant protein suggesting that TCF4 must regulate the expression of several critical neurodevelopmental genes [16,57]. One clue to the identity of these genes originates from the discovery of patients with a PTHS-like disorder that have mutations in contactin associated protein-like 2 (CNTNAP2) and neurexin 1 (NRXN1) that encode related neuronal adhesion receptors [59]. Intriguingly, CNTNAP2 and NRXN1 mutations are associated with a range of neurodevelopmental disorders, including schizophrenia and autism [24,60,61]. Indeed, TCF4 has been shown to regulate the CNTNAP2 and NRXN1 promoters in vitro, suggesting a functional association among the three genes implicated in PTHS [16]. Chromosomal abnormalities such as deletions and translocations that disrupt TCF4 have also been reported in patients with autism and idiopathic neurodevelopmental disorders [62]. These rare chromosomal variants are either balanced chromosomal translocations or deletions (copy number variants, CNVs) that disrupt one TCF4 allele and therefore lead to haploinsufficiency. Whether these cases represent a distinct disorder or are undiagnosed PTHS remains to be determined [62]. Finally, TCF4 mutations have also been described in children with nonsyndromic ID [63,64].

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with an increased risk of many common diseases, including some psychiatric disorders such as schizophrenia [65]. In addition to the rare mutations that cause PTHS, common TCF4 variants are associated with a small but robust increase in risk for schizophrenia [1,66]. Schizophrenia is a severe, life-long, psychiatric disorder that usually manifests in early adulthood, and is characterized by hallucinations, delusions, cognitive deficits, and affective retraction. The prevailing hypothesis is that schizophrenia is a neurodevelopmental disorder with a strong genetic component [67]. The neurodevelopmental hypothesis is supported by lack of neurodegenerative processes during the course of the disease, and by the fact that affected individuals show cognitive and social impairment before the first episode of the disease [67]. Early neurodevelopmental defects caused by genetic and/or environmental insults may be responsible for an altered developmental trajectory causing brain dysfunction [68]. As mentioned above, TCF4 is one of the few genes robustly associated with schizophrenia through common genetic variation [65]. Several independent genetic loci in or near TCF4 have been associated with schizophrenia (Figure 1A). The initial discovery identified single nucleotide polymorphism (SNP) rs9960767, located in intron three of TCF4, as genome-wide significant in a large meta-analysis of 12 945 schizophrenia cases and 34 591 controls [1]. In addition to rs9960767, several other SNPs in and around TCF4 have also been associated with increased risk of schizophrenia (Figure 1A, Table 1). TCF4 may also be regulated by another schizophrenia risk gene, the microRNA miR-137 [69]. MicroRNAs act posttranscriptionally to silence gene expression of their mRNA targets. These data suggest that the two schizophrenia risk genes may interact to modulate TCF4 levels in the brain. Although genetic association studies identify genes or genomic regions that harbor disease risk loci, they rarely pinpoint the true functional variant. Genome-wide association studies (GWAS) are confounded by linkage disequilibrium, where an associated SNP may be tagging a variant in a distinct, physically linked region of the genome. It is therefore important to determine whether functional variants exist at the locus of interest in order to gain additional insight into disease pathophysiology. Attempts to find common, nonsynonymous coding variants Table 1. Common TCF4 variants and disease riska SNP rs613872 rs9960767 rs4309482 c rs17512836 rs12966547 c rs1261117 rs17594526 rs1452787

P-value 2.3  1026 4.1  109 7.8  109 2.4  108 2.6  109 2.5  1010 1.3  107 2.6  108

Odds ratio 5.5/30 b 1.23 1.09 1.40 1.09 1.60 1.33–1.44 0.75 d

Refs [87] [1] [112] [66] [66,112] [113] [114] [84]

a

Abbreviations: FECD, Fuchs’ endothelial corneal dystrophy; PSC, primary sclerosing cholangitis; SZ, schizophrenia; SNP, single nucleotide polymorphism.

b c

TCF4 variants confer risk of schizophrenia Recent genetics-led studies have been instrumental in uncovering variants in the human genome that are associated

Disease FECD SZ SZ SZ SZ SZ SZ PSC

Odds ratio for heterozygotes and homozygotes.

Between coiled-coiled domain containing 68 (CCDC68) and TCF4.

d

By convention the odds ratios are reported for the minor allele. This is normally the risk allele, but in the case of PSC the major allele is associated with disease susceptibility.

5

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Review in the TCF4 gene were initially unsuccessful and are unlikely to explain the association of TCF4 with schizophrenia [70]. However, rare functional TCF4 variants have been identified by deep sequencing in a relatively small number of schizophrenia cases (versus controls) [71]. Interestingly, these heterozygous missense mutations identified in this study were distinct from those that cause PTHS [16,57]. Taken together, these data suggest that TCF4 variants may alter the function of the protein(s) in some patients with schizophrenia. Another approach to understanding how common variants contribute to disease risk is to examine the effects of individual SNPs on expression of the gene of interest. Cisregulatory elements in or near a gene may directly control its expression [72]. SNPs can therefore be used to study cis regulation and may contribute directly to the phenotypic diversity of gene expression. It is also possible to use SNPs to uncover allelic imbalances in gene expression that may be attributable to polymorphisms in cis-regulatory elements. This paradigm has been used to show that allelic expression of TCF4 differs between brain regions and in individuals [73]. Although this study did not look at patients with schizophrenia or explicitly those with schizophrenia risk alleles, the data show that common cis-acting variants may regulate TCF4 expression in the brain. Furthermore, schizophrenia-associated SNPs do not appear to mediate cis-acting effects on TCF4 expression in patients carrying the rs9960767 risk allele in adult brain [70]. This finding does not negate the possibility that TCF4 risk SNPs may have regulatory effects at earlier stages of development or in specific brain regions as shown for other schizophrenia risk genes [74]. TCF4 variants affect behavior and cognition The impact of schizophrenia-associated TCF4 risk variants on cognition and information processing has been analyzed in several different paradigms. In these studies, cognitive performance is assessed and stratified according to TCF4 genotype, in either patients or healthy volunteers carrying the different common risk alleles. These studies have shown statistically significant effects of TCF4 variants on some schizophrenia endophenotypes and cognition [75]. Impaired verbal memory is among the most prominent cognitive deficits in schizophrenia patients [76]. In a sample of 401 schizophrenia patients, the TCF4 variant rs9960767 was shown to influence verbal memory in the Rey Auditory Verbal Learning Test [77]. Contrary to expectations, schizophrenia patients carrying the TCF4 risk allele (the C-allele of rs9960767) showed better recognition in verbal memory, suggesting a role for this variant in the development of memory-related brain structures. TCF4 genotype did not impact on other cognitive tests measuring the domains of attention and executive function [77]. By contrast, a recent study that included 255 schizophrenia patients found that some TCF4 risk SNPs were associated with poorer verbal fluency in cases compared with controls [78]. The effect of the TCF4 variant rs9960767 on sensorimotor gating has also been assessed in healthy volunteers and schizophrenia patients [79]. Sensory gating is an essential psychological process that allows the filtering 6

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of sensory information during cognitive tasks. This mechanism is disrupted in several psychiatric disorders and is considered a reliable schizophrenia endophenotype [80]. Pre-pulse inhibition (PPI) is a translational task that can be used to measure sensorimotor gating in humans and animals [80]. Individuals presented with a weak pre-stimulus (pre-pulse) are able to inhibit their reaction to a strong stimulus. Carriers of the schizophrenia risk variant (the C-allele of rs9960767) in both the healthy control and patient groups produced a statistically significant reduction in PPI, indicative of an impaired inhibitory response [79]. Remarkably, PPI is also reduced in transgenic mice that overexpress Tcf4 [81]. A follow-up study measured auditory sensory gating assessed by P50 suppression of the auditory evoked potential [82]. P50 suppression has been related to attentional performance, working memory, and behavioral inhibition and is another measure of gating function [83]. In this large multicenter study, 1821 healthy volunteers were genotyped for 21 different TCF4 polymorphisms. This study found that schizophrenia risk alleles at four different TCF4 SNPs were associated with a highly significant reduction in P50 suppression. In addition to rs9960767, two of three TCF4 SNPs (rs17512836 and rs17597926) associated with P50 suppression were also the most significantly associated with schizophrenia risk in one of the largest GWAS published to date [66,82]. This decrease in P50 suppression was more pronounced in smokers than in nonsmokers, suggesting an interaction between TCF4 risk alleles and smoking behavior on cognitive functioning [82]. These studies indicate that TCF4 may influence key mechanisms regulating information processing that could contribute to the cognitive deficits and other endophenotypes observed in schizophrenia. TCF4 is a risk factor for liver disease PSC is a chronic liver disease that results from inflammation of the hepatic bile ducts. A large proportion of patients with PSC also have concurrent ulcerative colitis (UC). As is the case with other disorders described here, PSC is a complex disease with genetic, environmental, and infectious contributory factors. Recent GWAS on a cohort of patients with PSC found that TCF4 variants are associated with a modest increase in disease susceptibility (Figure 1A, Table 1) [84]. TCF4 variants were not associated with UC, suggesting that the disorders are genetically dissociable. These data are particularly interesting because TCF4 is required for T cell differentiation, whereas recent genetic findings have implicated the innate immune response, autoimmunity, and genes involved in T cell development in PSC [85,86]. TCF4 variants and Fuchs’ endothelial corneal dystrophy Common variation in TCF4 is also associated with an increased risk of FECD, a condition characterized by a progressive loss of vision that affects one in 20 Americans over the age of 40 years [87]. FECD is caused by a progressive degeneration of the corneal endothelium, a monolayer of cells with limited replicative ability in humans, which lines the inner surface of the cornea separating it from the aqueous humor of the inner eye. Hallmarks

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Review of FECD include the deposition of abnormal extracellular matrix material to form guttae in the basement membrane of the corneal endothelium (Descemet’s membrane), an increased expression of clusterin/apolipoprotein J [88], and an increase in light scattering in the collagen-rich corneal stroma, which becomes edematous due to the endothelial dysfunction. Vision loss is treatable via graft surgery, traditionally via a full thickness transplant of a donor cornea, but more often nowadays by a posterior lamellar keratoplasty that replaces only the diseased inner aspect of the cornea. Such is the medical burden of FECD that it is a major indication of corneal graft surgery worldwide. A GWAS identified TCF4 as a highly significant risk factor in typical FECD (Figure 1A, Table 1) [87]. Importantly, this association increased the odds of developing FECD by a factor of 5.5 for one risk allele and by 30 for patients with two risk alleles. These odds ratios allow cases to be predicted from controls with 76% accuracy, suggesting that TCF4 is a major contributor to FECD. The TCF4 risk allele (rs613872) is also associated with FECD disease severity and central corneal thickness, enforcing its role in the endothelial organization of the cornea [89]. In addition to the rs613872 allele, a separate study identified a trinucleotide expansion (CTG) in intron three of TCF4 that is present in a high proportion of FECD cases [90]. In a small cohort of 66 patients with severe FECD, repeat lengths of 50 or more trinucleotides were found in 79% of cases compared with only 3% of controls. This initial study has been replicated and extended to families where there were more than one affected family member [91]. In over half of the families studied, the expanded trinucleotide repeat co-segregated with FECD with complete penetrance. These data support the notion that the expanded repeat is the true functional variant in some FECD cases. Although the mechanism by which this expansion causes susceptibility to FECD is unknown, it is possible that the expanded alleles interfere with transcription and splicing of TCF4 in corneal endothelial cells. In addition to TCF4, other genes associated with FECD include collagen type 8 alpha-2 (COL8A2) in the less common early-onset form of the disease, Zn2+ finger E-box binding homeobox 1 (ZEB1), solute carrier family 4, sodium borate transporter, member 11 (SLC4A11), and ATP/GTP binding protein like-1 (AGBL1) [92,93]. Interestingly, genetic data suggest the involvement of EMTrelated processes in FECD. In common with TCF4, overexpression of ZEB1 has been shown to drive EMT in various cell types (Box 2). ZEB1 and TCF4 both bind to E-boxes suggesting that they both may repress the expression of the same or a similar subset of genes in corneal endothelial cells. It is therefore possible that TCF4 and ZEB1 affect EMT-related processes in the corneal endothelium. Recently, mutations in AGBL1 have been found to cause dominant, late-onset FECD [93]. AGBL1 encodes a glutamate decarboxylase found in the corneal endothelium that has been shown to bind to TCF4. AGBL1 mutations that cause FECD are reported to impair binding to TCF4, suggesting that the two FECD proteins may functionally interact in the cornea [93].

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Concluding remarks Recent advances in genetic epidemiology have started to identify variants associated with complex diseases in humans. The genetic association of common and rare TCF4 variants with neurodevelopmental disorders underscores its importance for brain development and function. In addition, TCF4 is a risk factor for two other common disorders, FECD and PSC that prima facie have very few similarities to brain disorders. How TCF4 variants influence the genetic susceptibility to each of these disorders is largely unknown (Box 4). This problem is also confounded by the inherent limitations in the GWAS methodology to pinpoint the causal or functional variant at a specific locus. However, studies on the rare alleles that cause PTHS suggest that TCF4 dosage is important. Missense mutations and whole gene deletions that cause PTHS essentially result in the same highly penetrant phenotype: ID and various comorbid disorders affecting a range or organs. Thus, subtle changes

Box 4. Outstanding questions  Understanding the pleiotropic effects of TCF4 in disease TCF4 variants are associated with seemingly diverse disorders. Evidence from PTHS patients and transgenic animals would suggest that TCF4 dosage is important and likely to influence transcriptional programs in several cell types. Psychiatric disorders can be viewed as a continuum of severities, often with the same gene contributing to multiple disorders [65]. This appears to be true for TCF4 where allelic variation is associated with ID and schizophrenia. Furthermore, the symptoms of PTHS, arguably the most extreme of all the disorders involving TCF4, suggest that TCF4 has multiple roles in different tissues. These observations would seem to suggest that TCF4 variants might have cell type specific effects in a range of disorders. Perhaps one explanation for this apparent paradox resides in the diversity of isoforms encoded by the TCF4 gene. Studies of TCF4 expression have revealed that the transcripts encoding some isoforms are expressed in a tissue-specific manner from multiple promoters [8]. Disease-associated variants are likely to have differential effects on the cis regulation and processing of individual transcripts in a tissue-specific manner. It will therefore be important to determine the repertoire of TCF4 isoforms expressed in a specific cell type such as corneal endothelial cells for FECD.  Identifying functional TCF4 variants as risk factors in common diseases In addition to the rare TCF4 variants associated with PTHS, ID and schizophrenia, the function of common TCF4 variants is unknown. Multiple TCF4 SNPs have been linked to schizophrenia susceptibility but their impact on TCF4 function is currently unresolved. It is possible that common TCF4 variants may affect the expression or processing of the gene. By contrast, recent studies in several neurodegenerative disorders have shown that expanded trinucleotide repeats sequester RNA-binding proteins leading to selective cell death through a toxic gain-of-function mechanism [115]. Although the CTG repeat in FECD may interfere with transcription and expression of TCF4 in corneal endothelial cells, it is a distinct possibility that RNA stress may contribute directly to the molecular pathogenesis of the disorder.  Defining physiologically relevant TCF4 targets Although the role of TCF4 as a transcriptional activator or repressor is well established, very few physiological TCF4 targets have been described. The identity of TCF4-regulated genes will greatly improve our understanding of both common and rare disorders associated with the different TCF4 alleles. Furthermore, pathway analysis of the TCF4 target genes will contribute to our understanding of the range of cellular processes regulated by this transcription factor. 7

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Acknowledgments Immune cell fate

ID2 Epithelialmesenchymal transion

PSC?

TCF4

ATOH1

SNAI1 FECD

PTHS/ID

SZ Neurodevelopment TRENDS in Molecular Medicine

Figure 2. TCF4 involvement in disease processes. The schematic summarizes the involvement of TCF4 in common and rare disorders. The circles represent the biological processes involving TCF4, whereas the triangles map the disease associations. Representative heterodimerization partners for each process are also shown. The overlaps between the different processes and diseases represent the potential for shared mechanisms in each domain. Abbreviations: ATOH1, atonal homolog 1; FECD, Fuchs’ endothelial corneal dystrophy; ID, intellectual disability; ID2, inhibitor of DNA binding 2; PTHS, Pitt–Hopkins syndrome; PSC, primary sclerosing cholangitis; SZ, schizophrenia; SNAI1, SNAIL1.

in the temporal and spatial expression of TCF4 may result in less severe phenotypes than PTHS, such as schizophrenia. The evidence that TCF4 associates with other bHLH transcription factors to regulate gene expression is compelling. The establishment of a TCF4-dependent gene expression network in part orchestrates differentiation of some neural progenitors, pDCs, and EMT induction (Figure 2). Although the identity of TCF4 targets may vary between cell types, the underlying principle appears to be the same: differentiated cells acquire a new molecular and physiological phenotype and in some cases become migratory. It is therefore tempting to speculate that some of the defects seen in PTHS patients, Tcf4deficient mice, and even FECD are due to impaired EMT-like processes in the developing nervous system and corneal endothelium. The presence of other E-proteins in the same cell suggests a degree of redundancy that may obscure the function of TCF4. For example, the role of TCF4 in B cell development is only apparent when other E-proteins have been depleted in transgenic mouse models. Similarly, the presence of high levels of ID proteins could sequester TCF4 into inactive heterodimers thereby attenuating its activity. It is also noteworthy that TCF4 is the only Eprotein expressed throughout the adult mouse brain [81]. Taken together, these caveats provide a conceptual framework to begin to delineate the function of TCF4 in development and disease. The genetic discoveries described herein therefore offer a unique opportunity to understand the pleiotropic roles of TCF4 in human disease. Advancing our knowledge of the fundamental biology of TCF4 will be an essential part of deciphering disease mechanisms related to common and rare disorders affecting the brain and other tissues. 8

This work was funded by a Medical Research Council (MRC) studentship (M.P.F.) and an MRC Centenary Award (D.J.B., M.P.F.).

References 1 Stefansson, H. et al. (2009) Common variants conferring risk of schizophrenia. Nature 460, 744–747 2 Henthorn, P. et al. (1990) Two distinct transcription factors that bind the immunoglobulin enhancer mE5/k2 motif. Science 247, 467–470 3 Murre, C. et al. (1989) A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell 56, 777–783 4 Corneliussen, B. et al. (1991) Helix–loop–helix transcriptional activators bind to a sequence in glucocorticoid response elements of retrovirus enhancers. J. Virol. 65, 6084–6093 5 Pscherer, A. et al. (1996) The helix–loop–helix transcription factor SEF-2 regulates the activity of a novel initiator element in the promoter of the human somatostatin receptor II gene. EMBO J. 15, 6680–6690 6 Yoon, S.O. and Chikaraishi, D.M. (1994) Isolation of two E-box binding factors that interact with the rat tyrosine hydroxylase enhancer. J. Biol. Chem. 269, 18453–18462 7 Phillips, S.E. (1994) Built by association: structure and function of helix–loop–helix DNA-binding proteins. Structure 2, 1–4 8 Sepp, M. et al. (2011) Functional diversity of human basic helix–loop– helix transcription factor TCF4 isoforms generated by alternative 50 exon usage and splicing. PLoS ONE 6, e22138 9 Liu, Y. et al. (1998) A splice variant of E2-2 basic helix–loop–helix protein represses the brain-specific fibroblast growth factor 1 promoter through the binding to an imperfect E-box. J. Biol. Chem. 273, 19269–19276 10 Skerjanc, I.S. et al. (1996) A splice variant of the ITF-2 transcript encodes a transcription factor that inhibits MyoD activity. J. Biol. Chem. 271, 3555–3561 11 Sobrado, V.R. et al. (2009) The class I bHLH factors E2-2A and E2-2B regulate EMT. J. Cell Sci. 122, 1014–1024 12 Petropoulos, H. and Skerjanc, I.S. (2000) Analysis of the inhibition of MyoD activity by ITF-2B and full-length E12/E47. J. Biol. Chem. 275, 25095–25101 13 Lu, Y. et al. (2005) A negative regulatory element-dependent inhibitory role of ITF2B on IL-2 receptor a gene. Biochem. Biophys. Res. Commun. 336, 142–149 14 Furumura, M. et al. (2001) Involvement of ITF2 in the transcriptional regulation of melanogenic genes. J. Biol. Chem. 276, 28147–28154 15 de Pontual, L. et al. (2009) Mutational, functional, and expression studies of the TCF4 gene in Pitt–Hopkins syndrome. Hum. Mutat. 30, 669–676 16 Forrest, M. et al. (2012) Functional analysis of TCF4 missense mutations that cause Pitt–Hopkins syndrome. Hum. Mutat. 33, 1676–1686 17 Massari, M.E. et al. (1999) A conserved motif present in a class of helix–loop–helix proteins activates transcription by direct recruitment of the SAGA complex. Mol. Cell 4, 63–73 18 Bayly, R. et al. (2004) E2A–PBX1 interacts directly with the KIX domain of CBP/p300 in the induction of proliferation in primary hematopoietic cells. J. Biol. Chem. 279, 55362–55371 19 Zhang, J. et al. (2004) E protein silencing by the leukemogenic AML1– ETO fusion protein. Science 305, 1286–1289 20 Voronova, A. and Baltimore, D. (1990) Mutations that disrupt DNA binding and dimer formation in the E47 helix–loop–helix protein map to distinct domains. Proc. Natl. Acad. Sci. U.S.A. 87, 4722–4726 21 Ellenberger, T. et al. (1994) Crystal structure of transcription factor E47: E-box recognition by a basic region helix–loop–helix dimer. Genes Dev. 8, 970–980 22 Longo, A. et al. (2008) Crystal structure of E47–NeuroD1/b2 bHLH domain–DNA complex: heterodimer selectivity and DNA recognition. Biochemistry 47, 218–229 23 Goldfarb, A.N. et al. (1998) Identification of a highly conserved module in E proteins required for in vivo helix–loop–helix dimerization. J. Biol. Chem. 273, 2866–2873 24 Blake, D.J. et al. (2010) TCF4, schizophrenia, and Pitt–Hopkins syndrome. Schizophr. Bull. 36, 443–447

TRMOME-936; No. of Pages 10

Review 25 Teachenor, R. et al. (2012) Biochemical and phosphoproteomic analysis of the helix–loop–helix protein E47. Mol. Cell. Biol. 32, 1671–1682 26 Corneliussen, B. et al. (1994) Calcium/calmodulin inhibition of basichelix–loop–helix transcription factor domains. Nature 368, 760–764 27 Onions, J. et al. (1997) Basic helix–loop–helix protein sequences determining differential inhibition by calmodulin and S-100 proteins. J. Biol. Chem. 272, 23930–23937 28 Larsson, G. et al. (2001) A novel target recognition revealed by calmodulin in complex with the basic helix–loop–helix transcription factor SEF2-1/E2-2. Protein Sci. 10, 169–186 29 Larsson, G. et al. (2005) Backbone dynamics of a symmetric calmodulin dimer in complex with the calmodulin-binding domain of the basic-helix–loop–helix transcription factor SEF2-1/E2-2: a highly dynamic complex. Biophys. J. 89, 1214–1226 30 Hauser, J. et al. (2008) B-cell receptor activation inhibits AID expression through calmodulin inhibition of E-proteins. Proc. Natl. Acad. Sci. U.S.A. 105, 1267–1272 31 Hauser, J. et al. (2008) Calcium regulation of myogenesis by differential calmodulin inhibition of basic helix–loop–helix transcription factors. Mol. Biol. Cell 19, 2509–2519 32 Kee, B.L. (2009) E and ID proteins branch out. Nat. Rev. Immunol. 9, 175–184 33 Murre, C. (2005) Helix–loop–helix proteins and lymphocyte development. Nat. Immunol. 6, 1079–1086 34 Zhuang, Y. et al. (1996) B-lymphocyte development is regulated by the combined dosage of three basic helix–loop–helix genes, E2A, E2-2, and HEB. Mol. Cell. Biol. 16, 2898–2905 35 Bergqvist, I. et al. (2000) The basic helix–loop–helix transcription factor E2-2 is involved in T lymphocyte development. Eur. J. Immunol. 30, 2857–2863 36 Reizis, B. (2010) Regulation of plasmacytoid dendritic cell development. Curr. Opin. Immunol. 22, 206–211 37 Ghosh, H.S. et al. (2010) Continuous expression of the transcription factor e2-2 maintains the cell fate of mature plasmacytoid dendritic cells. Immunity 33, 905–916 38 Cisse, B. et al. (2008) Transcription factor E2-2 is an essential and specific regulator of plasmacytoid dendritic cell development. Cell 135, 37–48 39 Hacker, C. et al. (2003) Transcriptional profiling identifies Id2 function in dendritic cell development. Nat. Immunol. 4, 380–386 40 Li, H.S. et al. (2012) The signal transducers STAT5 and STAT3 control expression of Id2 and E2-2 during dendritic cell development. Blood 120, 4363–4373 41 Banchereau, J. and Pascual, V. (2006) Type I interferon in systemic lupus erythematosus and other autoimmune diseases. Immunity 25, 383–392 42 Cano, A. and Portillo, F. (2010) An emerging role for class I bHLH E22 proteins in EMT regulation and tumor progression. Cell Adh. Migr. 4, 56–60 43 Lim, J. and Thiery, J.P. (2012) Epithelial–mesenchymal transitions: insights from development. Development 139, 3471–3486 44 Kalluri, R. and Weinberg, R.A. (2009) The basics of epithelial– mesenchymal transition. J. Clin. Invest. 119, 1420–1428 45 Perez-Moreno, M.A. et al. (2001) A new role for E12/E47 in the repression of E-cadherin expression and epithelial–mesenchymal transitions. J. Biol. Chem. 276, 27424–27431 46 Bolos, V. et al. (2003) The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors. J. Cell Sci. 116, 499–511 47 Cano, A. et al. (2000) The transcription factor snail controls epithelial–mesenchymal transitions by repressing E-cadherin expression. Nat. Cell Biol. 2, 76–83 48 Moreno-Bueno, G. et al. (2006) Genetic profiling of epithelial cells expressing E-cadherin repressors reveals a distinct role for Snail, Slug, and E47 factors in epithelial–mesenchymal transition. Cancer Res. 66, 9543–9556 49 Forrest, M.P. et al. (2013) Knockdown of human TCF4 affects multiple signaling pathways involved in cell survival, epithelial to mesenchymal transition and neuronal differentiation. PLoS ONE 8, e73169 50 Nieto, M.A. (2011) The ins and outs of the epithelial to mesenchymal transition in health and disease. Annu. Rev. Cell Dev. Biol. 27, 347–376

Trends in Molecular Medicine xxx xxxx, Vol. xxx, No. x

51 Bertrand, N. et al. (2002) Proneural genes and the specification of neural cell types. Nat. Rev. Neurosci. 3, 517–530 52 Powell, L.M. and Jarman, A.P. (2008) Context dependence of proneural bHLH proteins. Curr. Opin. Genet. Dev. 18, 411–417 53 Flora, A. et al. (2007) The E-protein Tcf4 interacts with Math1 to regulate differentiation of a specific subset of neuronal progenitors. Proc. Natl. Acad. Sci. U.S.A. 104, 15382–15387 54 Amiel, J. et al. (2007) Mutations in TCF4, encoding a class I basic helix–loop–helix transcription factor, are responsible for Pitt– Hopkins syndrome, a severe epileptic encephalopathy associated with autonomic dysfunction. Am. J. Hum. Genet. 80, 988–993 55 Pitt, D. and Hopkins, I. (1978) A syndrome of mental retardation, wide mouth and intermittent overbreathing. Aust. Paediatr. J. 14, 182–184 56 Zweier, C. et al. (2007) Haploinsufficiency of TCF4 causes syndromal mental retardation with intermittent hyperventilation (Pitt–Hopkins syndrome). Am. J. Hum. Genet. 80, 994–1001 57 Sepp, M. et al. (2012) Pitt–Hopkins syndrome-associated mutations in TCF4 lead to variable impairment of the transcription factor function ranging from hypomorphic to dominant-negative effects. Hum. Mol. Genet. 21, 2873–2888 58 Brockschmidt, A. et al. (2007) Severe mental retardation with breathing abnormalities (Pitt–Hopkins syndrome) is caused by haploinsufficiency of the neuronal bHLH transcription factor TCF4. Hum. Mol. Genet. 16, 1488–1494 59 Zweier, C. et al. (2009) CNTNAP2 and NRXN1 are mutated in autosomal-recessive Pitt–Hopkins-like mental retardation and determine the level of a common synaptic protein in Drosophila. Am. J. Hum. Genet. 85, 655–666 60 Alarcon, M. et al. (2008) Linkage, association, and gene-expression analyses identify CNTNAP2 as an autism-susceptibility gene. Am. J. Hum. Genet. 82, 150–159 61 Kirov, G. et al. (2009) Neurexin 1 (NRXN1) deletions in schizophrenia. Schizophr. Bull. 35, 851–854 62 Talkowski, M.E. et al. (2012) Sequencing chromosomal abnormalities reveals neurodevelopmental loci that confer risk across diagnostic boundaries. Cell 149, 525–537 63 Rauch, A. et al. (2012) Range of genetic mutations associated with severe non-syndromic sporadic intellectual disability: an exome sequencing study. Lancet 380, 1674–1682 64 Hamdan, F.F. et al. (2013) Parent–child exome sequencing identifies a de novo truncating mutation in TCF4 in non-syndromic intellectual disability. Clin. Genet. 83, 198–200 65 Sullivan, P.F. et al. (2012) Genetic architectures of psychiatric disorders: the emerging picture and its implications. Nat. Rev. Genet. 13, 537–551 66 Ripke, S. et al. (2011) Genome-wide association study identifies five new schizophrenia loci. Nat. Genet. 43, 969–976 67 Lewis, D.A. and Levitt, P. (2002) Schizophrenia as a disorder of neurodevelopment. Annu. Rev. Neurosci. 25, 409–432 68 Insel, T.R. (2010) Rethinking schizophrenia. Nature 468, 187–193 69 Wright, C. et al. (2013) Potential impact of miR-137 and its targets in schizophrenia. Front. Genet. 4, 58 70 Williams, H.J. et al. (2011) Association between TCF4 and schizophrenia does not exert its effect by common nonsynonymous variation or by influencing cis-acting regulation of mRNA expression in adult human brain. Am. J. Med. Genet. B: Neuropsychiatr. Genet. 156B, 781–784 71 Hu, X. et al. (2013) A survey of rare coding variants in candidate genes in schizophrenia by deep sequencing. Mol. Psychiatry http:// dx.doi.org/10.1038/mp.2013.131 72 Pastinen, T. (2010) Genome-wide allele-specific analysis: insights into regulatory variation. Nat. Rev. Genet. 11, 533–538 73 Buonocore, F. et al. (2010) Effects of cis-regulatory variation differ across regions of the adult human brain. Hum. Mol. Genet. 19, 4490– 4496 74 Hill, M.J. and Bray, N.J. (2012) Evidence that schizophrenia risk variation in the ZNF804A gene exerts its effects during fetal brain development. Am. J. Psychiatry 169, 1301–1308 75 Quednow, B.B. et al. (2014) Transcription factor 4 (TCF4) and schizophrenia: integrating the animal and the human perspective. Cell. Mol. Life Sci. http://dx.doi.org/10.1007/s00018-013-1553-4

9

TRMOME-936; No. of Pages 10

Review 76 Toulopoulouand, T. and Murray, R.M. (2004) Verbal memory deficit in patients with schizophrenia: an important future target for treatment. Expert Rev. Neurother. 4, 43–52 77 Lennertz, L. et al. (2011) Novel schizophrenia risk gene TCF4 influences verbal learning and memory functioning in schizophrenia patients. Neuropsychobiology 63, 131–136 78 Wirgenes, K.V. et al. (2012) TCF4 sequence variants and mRNA levels are associated with neurodevelopmental characteristics in psychotic disorders. Transl. Psychiatry 2, e112 79 Quednow, B.B. et al. (2011) The schizophrenia risk allele C of the TCF4 rs9960767 polymorphism disrupts sensorimotor gating in schizophrenia spectrum and healthy volunteers. J. Neurosci. 31, 6684–6691 80 Braff, D.L. and Geyer, M.A. (1990) Sensorimotor gating and schizophrenia. Human and animal model studies. Arch. Gen. Psychiatry 47, 181–188 81 Brzozka, M.M. et al. (2010) Cognitive and sensorimotor gating impairments in transgenic mice overexpressing the schizophrenia susceptibility gene Tcf4 in the brain. Biol. Psychiatry 68, 33–40 82 Quednow, B.B. et al. (2012) Schizophrenia risk polymorphisms in the TCF4 gene interact with smoking in the modulation of auditory sensory gating. Proc. Natl. Acad. Sci. U.S.A. 109, 6271–6276 83 Lijffijt, M. et al. (2009) P50, N100, and P200 sensory gating: relationships with behavioral inhibition, attention, and working memory. Psychophysiology 46, 1059–1068 84 Ellinghaus, D. et al. (2013) Genome-wide association analysis in Primary sclerosing cholangitis and ulcerative colitis identifies risk loci at GPR35 and TCF4. Hepatology 58, 1074–1083 85 Aron, J.H. and Bowlus, C.L. (2009) The immunobiology of primary sclerosing cholangitis. Semin. Immunopathol. 31, 383–397 86 Liu, J.Z. et al. (2013) Dense genotyping of immune-related disease regions identifies nine new risk loci for primary sclerosing cholangitis. Nat. Genet. 45, 670–675 87 Baratz, K.H. et al. (2010) E2-2 protein and Fuchs’s corneal dystrophy. N. Engl. J. Med. 363, 1016–1024 88 Jurkunas, U.V. et al. (2008) Increased clusterin expression in Fuchs’ endothelial dystrophy. Invest. Ophthalmol. Vis. Sci. 49, 2946–2955 89 Igo, R.P., Jr et al. (2012) Differing roles for TCF4 and COL8A2 in central corneal thickness and fuchs endothelial corneal dystrophy. PLoS One 7, e46742 PMID: 23110055 90 Wieben, E.D. et al. (2012) A common trinucleotide repeat expansion within the transcription factor 4 (TCF4, E2-2) gene predicts Fuchs corneal dystrophy. PLoS ONE 7, e49083 91 Mootha, V.V. et al. (2014) Association and familial segregation of CTG18.1 trinucleotide repeat expansion of TCF4 gene in Fuchs’ endothelial corneal dystrophy. Invest. Ophthalmol. Vis. Sci. 55, 33–42 92 Schmedt, T. et al. (2012) Molecular bases of corneal endothelial dystrophies. Exp. Eye Res. 95, 24–34 93 Riazuddin, S.A. et al. (2013) Mutations in AGBL1 cause dominant late-onset Fuchs corneal dystrophy and alter protein–protein interaction with TCF4. Am. J. Hum. Genet. 93, 758–764 94 Massari, M.E. and Murre, C. (2000) Helix–loop–helix proteins: regulators of transcription in eucaryotic organisms. Mol. Cell. Biol. 20, 429–440

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95 Bain, G. and Murre, C. (1998) The role of E-proteins in B- and Tlymphocyte development. Semin. Immunol. 10, 143–153 96 Murre, C. et al. (1989) Interactions between heterologous helix–loop– helix proteins generate complexes that bind specifically to a common DNA sequence. Cell 58, 537–544 97 Benezra, R. et al. (1990) The protein Id: a negative regulator of helix– loop–helix DNA binding proteins. Cell 61, 49–59 98 Sun, X.H. et al. (1991) Id proteins Id1 and Id2 selectively inhibit DNA binding by one class of helix–loop–helix proteins. Mol. Cell. Biol. 11, 5603–5611 99 Iavarone, A. and Lasorella, A. (2006) ID proteins as targets in cancer and tools in neurobiology. Trends Mol. Med. 12, 588–594 100 Akazawa, C. et al. (1992) Molecular characterization of a rat negative regulator with a basic helix–loop–helix structure predominantly expressed in the developing nervous system. J. Biol. Chem. 267, 21879–21885 101 Ohsako, S. et al. (1994) Hairy function as a DNA-binding helix–loop– helix repressor of Drosophila sensory organ formation. Genes Dev. 8, 2743–2755 102 Sasai, Y. et al. (1992) Two mammalian helix–loop–helix factors structurally related to Drosophila hairy and Enhancer of split. Genes Dev. 6, 2620–2634 103 Kageyama, R. et al. (2007) The Hes gene family: repressors and oscillators that orchestrate embryogenesis. Development 134, 1243–1251 104 Acloque, H. et al. (2009) Epithelial–mesenchymal transitions: the importance of changing cell state in development and disease. J. Clin. Invest. 119, 1438–1449 105 Gotz, M. and Huttner, W.B. (2005) The cell biology of neurogenesis. Nat. Rev. Mol. Cell Biol. 6, 777–788 106 Pacary, E. et al. (2012) Crucial first steps: the transcriptional control of neuron delamination. Neuron 74, 209–211 107 Castro, D.S. and Guillemot, F. (2011) Old and new functions of proneural factors revealed by the genome-wide characterization of their transcriptional targets. Cell Cycle 10, 4026–4031 108 Farah, M.H. et al. (2000) Generation of neurons by transient expression of neural bHLH proteins in mammalian cells. Development 127, 693–702 109 Heng, J.I. et al. (2008) Neurogenin 2 controls cortical neuron migration through regulation of Rnd2. Nature 455, 114–118 110 Ge, W. et al. (2006) Coupling of cell migration with neurogenesis by proneural bHLH factors. Proc. Natl. Acad. Sci. U.S.A. 103, 1319– 1324 111 Schwab, M.H. et al. (2000) Neuronal basic helix–loop–helix proteins (NEX and BETA2/Neuro D) regulate terminal granule cell differentiation in the hippocampus. J. Neurosci. 20, 3714–3724 112 Steinberg, S. et al. (2011) Common variants at VRK2 and TCF4 conferring risk of schizophrenia. Hum. Mol. Genet. 20, 4076–4081 113 Aberg, K.A. et al. (2013) A comprehensive family-based replication study of schizophrenia genes. JAMA Psychiatry 70, 573–581 114 International Schizophrenia Consortium, Purcell, S.M. et al. (2009) Common polygenic variation contributes to risk of schizophrenia and bipolar disorder. Nature 460, 748–752 115 Wojciechowska, M. and Krzyzosiak, W.J. (2011) Cellular toxicity of expanded RNA repeats: focus on RNA foci. Hum. Mol. Genet. 20, 3811–3821