The establishment of mesoderm and neuroectoderm in the early Drosophila embryo relies on interactions between the Dorsal morphogen and basic-helix-loop-helix (bHLH) activators. Here we show that Dorsal and the bHLH activator Twist synergistically activate transcription in cell culture and in vitro from a promoter containing binding sites for both factors. Somewhat surprisingly, a region of Twist outside the conserved bHLH domain is required for the synergy. In Dorsal, the rel homology domain appears to be sufficient for synergy. Protein-protein interaction assays show that Twist and Dorsal bind to one another in vitro. However, this interaction does not appear to be of sufficient strength to yield cooperative binding to DNA. Nonetheless, the regions of Twist and Dorsal required for the binding interaction are also required for synergistic transcriptional activation.
TWIST is a basic helix-loop-helix (bHLH) transcription factor that regulates mesodermal development, promotes tumor cell metastasis, and, in response to cytotoxic stress, enhances cell survival. Our screen for bHLH gene expression in rat C6 glioma revealed TWIST. To delineate a possible oncogenic role for TWIST in the human central nervous system (CNS), we analyzed TWIST message and protein expression in gliomas and normal brain. TWIST was detected in the large majority of human glioma-derived cell lines and human gliomas examined. Increased TWIST mRNA levels were associated with the highest grade gliomas, and increased TWIST expression accompanied transition from low grade to high grade in vivo, suggesting a role for TWIST in promoting malignant progression. In accord, elevated TWIST mRNA abundance preceded the spontaneous malignant transformation of cultured mouse astrocytes hemizygous for p53. Overexpression of TWIST protein in a human glioma cell line significantly enhanced tumor cell invasion, a hallmark of high-grade gliomas. These findings support roles for TWIST both in early glial tumorigenesis and subsequent malignant progression. TWIST was also expressed in embryonic and fetal human brain, and in neurons, but not glia, of mature brain, indicating that, in gliomas, TWIST may promote the functions also critical for CNS development or normal neuronal physiology.
cancer; brain tumor; neuron; oncogene; invasion
Twist is a basic helix-loop-helix (bHLH) transcriptional factor that has been identified to play an important role in epithelial-mesenchymal transition (EMT)-mediated metastasis through the regulation of E-cadherin expression. However, few authors have examined the expression of Twist and E-cadherin and their prognostic value in patients with esophageal squamous cell carcinoma (ESCC). The purpose of this study is to evaluate the clinical significance of Twist and E-cadherin expression in ESCC.
We immunohistochemically investigated the relationship between their expression and clinicopathological factors including prognosis in surgical specimens of primary tumors in 166 patients with ESCC.
The expression rate of high Twist was 42.0% and that of preserved E-cadherin was 40.4%. The expression of high Twist and reduced E-cadherin was significantly associated with depth of tumor invasion, lymph node metastasis, distant nodal metastasis, stage and lymphatic invasion, and poor prognosis. High Twist expression significantly correlated with reduced E-cadherin expression. In the preserved E-cadherin group, the 5-year survival rate was better for patients who were low for Twist expression than for those who were high for Twist expression. Multivariate analysis indicated that the combination of low Twist and preserved E-cadherin expression was an independent prognostic factor along with tumor depth, distant nodal metastasis and E-cadherin expression.
Evaluation of Twist and E-cadherin expressions should be useful for determining tumor properties, including prognosis, in patients with ESCC.
Parathyroid hormone (PTH) is an essential regulator of endochondral bone formation and an important anabolic agent for the reversal of bone loss. PTH mediates its functions in part by regulating binding of the bone-related activating transcription factor 4 (ATF4) to the osteoblast-specific gene, osteocalcin. The basic helix-loop-helix (bHLH) factors Twist1 and Twist2 also regulate osteocalcin transcription in part through the interaction of the C-terminal “box” domain in these factors and Runx2. In this study, we discovered a novel function of PTH: its ability to dramatically decrease Twist1 transcription. Since ATF4 is a major regulator of the PTH response in osteoblasts, we assessed the mutual regulation between these factors and determined that Twist proteins and ATF4 physically interact in a manner that affects ATF4 DNA binding function. We mapped the interaction domain of Twist proteins to the C-terminal “box” domain and of ATF4, to the N-terminus. Furthermore, we demonstrate that Twist1 overexpression in osteoblasts attenuates ATF4 binding to the osteocalcin promoter in response to PTH. This study thus identifies Twist proteins as novel inhibitory binding partners of ATF4 and explores the functional significance of this interaction.
Twist1; ATF4; PTH; osteoblasts; osteocalcin
The muscle-specific basic helix-loop-helix (bHLH) protein myogenin activates muscle transcription by binding to target sequences in muscle-specific promoters and enhancers as a heterodimer with ubiquitous bHLH proteins, such as the E2A gene products E12 and E47. We show that dimerization with E2A products potentiates phosphorylation of myogenin at sites within its amino- and carboxyl-terminal transcription activation domains. Phosphorylation of myogenin at these sites was mediated by the bHLH region of E2A products and was dependent on dimerization but not on DNA binding. Mutations of the dimerization-dependent phosphorylation sites resulted in enhanced transcriptional activity of myogenin, suggesting that their phosphorylation diminishes myogenin's transcriptional activity. The ability of E2A products to potentiate myogenin phosphorylation suggests that dimerization induces a conformational change in myogenin that unmasks otherwise cryptic phosphorylation sites or that E2A proteins recruit a kinase for which myogenin is a substrate. That phosphorylation of these dimerization-dependent sites diminished myogenin's transcriptional activity suggests that these sites are targets for a kinase that interferes with muscle-specific gene expression.
Hairy-related proteins include the Drosophila Hairy and Enhancer of Split proteins and mammalian Hes proteins. These proteins are basic helix-loop-helix (bHLH) transcriptional repressors that control cell fate decisions such as neurogenesis or myogenesis in both Drosophila melanogaster and mammals. Hairy-related proteins are site-specific DNA-binding proteins defined by the presence of both a repressor-specific bHLH DNA binding domain and a carboxyl-terminal WRPW (Trp-Arg-Pro-Trp) motif. These proteins act as repressors by binding to DNA sites in target gene promoters and not by interfering with activator proteins, indicating that these proteins are active repressors which should therefore have specific repression domains. Here we show the WRPW motif to be a functional transcriptional repression domain sufficient to confer active repression to Hairy-related proteins or a heterologous DNA-binding protein, Ga14. This motif was previously shown to be necessary for interactions with Groucho, a genetically defined corepressor for Drosophila Hairy-related proteins. Here we show that the WRPW motif is sufficient to recruit Groucho or the TLE mammalian homologs to target gene promoters. We also show that Groucho and TLE proteins actively repress transcription when directly bound to a target gene promoter and identify a novel, highly conserved transcriptional repression domain in these proteins. These results directly demonstrate that Groucho family proteins are active transcriptional corepressors for Hairy-related proteins and are recruited by the 4-amino acid protein-protein interaction domain, WRPW.
Proper metazoan mesoderm development requires the function of a basic helix-loop-helix (bHLH) transcription factor, Twist. Twist-containing dimers regulate the expression of target genes by binding to E box promoter elements containing the site CANNTG. In Caenorhabditis elegans, CeTwist functions in a subset of mesodermal cells. Our study focuses on how CeTwist controls the expression of its target gene, arg-1. We find that a 385 bp promoter region of arg-1, which contains three different E box elements, is sufficient for maintaining the full CeTwist-dependent expression pattern. Interestingly, the expression of arg-1 in different tissues is regulated distinctly, and each of the three E boxes plays a unique role in the regulation. The first and the third E boxes (E1 and E3) are required for expression in a distinct subset of the mesodermal tissues where arg-1 is normally expressed, and the second E box (E2) is required for expression in the full set of those tissues. The essential role of E2 in arg-1 regulation is correlated with the finding that E2 binds with greater affinity than E1 or E3 to CeTwist dimers. A potential role for additional transcription factors in mesodermal gene regulation is suggested by the discovery of a novel site that is also required for arg-1 expression in a subset of the tissues but is not bound in vitro by CeTwist. On the basis of these results, we propose a model of CeTwist gene regulation in which expression is controlled by tissue-specific binding of distinct sets of E boxes.
C. elegans; Twist; bHLH; E box; hlh-2; hlh-8; arg-1; E/DA; mesoderm; DSL homolog
Neural crest cells are multipotent progenitor cells that can generate both ectodermal cell types, such as neurons, and mesodermal cell types, such as smooth muscle. The mechanisms controlling this cell fate choice are not known. The basic Helix-loop-Helix (bHLH) transcription factor Twist1 is expressed throughout the migratory and post-migratory cardiac neural crest. Twist1 ablation or mutation of the Twist-box causes differentiation of ectopic neuronal cells, which molecularly resemble sympathetic ganglia, in the cardiac outflow tract. Twist1 interacts with the pro-neural factor Sox10 via its Twist-box domain and binds to the Phox2b promoter to repress transcriptional activity. Mesodermal cardiac neural crest trans-differentiation into ectodermal sympathetic ganglia-like neurons is dependent upon Phox2b function. Ectopic Twist1 expression in neural crest precursors disrupts sympathetic neurogenesis. These data demonstrate that Twist1 functions in post-migratory neural crest cells to repress pro-neural factors and thereby regulate cell fate determination between ectodermal and mesodermal lineages.
During vertebrate development, a unique population of cells, termed neural crest cells, migrates throughout the developing embryo, generating various cell types, for example, the smooth muscle that divides the aorta and pulmonary artery where they connect to the heart, and the autonomic neurons, which coordinate organ function. The distinctions between neural crest cells that will form smooth muscle and those that will become neurons are thought to occur prior to migration. Here, we show that, in mice with mutations of the transcription factor Twist1, a subpopulation of presumptive smooth muscle cells, following migration to the heart, instead mis-specify to resemble autonomic neurons. Twist1 represses transcription of the pro-neural factor Phox2b both through antagonism of its upstream effector, Sox10, and through direct binding to its promoter. Phox2b is absolutely required for autonomic neuron development, and indeed, the aberrant neurons in Twist1 mutants disappear when Phox2b is also mutated. Ectopic Twist1 expression within all neural crest cells disrupts the specification of normal autonomic neurons. Collectively, these data reveal that neural crest cells can alter their cell fate from mesoderm to ectoderm after they have migrated and that Twist1 functions to maintain neural crest cell potency during embryonic development.
The Saethre-Chotzen syndrome is characterized by premature fusion of cranial sutures resulting from mutations in Twist, a basic helix-loop-helix (bHLH) transcription factor. We have identified Twist target genes using human mutant calvaria osteoblastic cells from a child with Saethre-Chotzen syndrome with a Twist mutation that introduces a stop codon upstream of the bHLH domain. We observed that Twist mRNA and protein levels were reduced in mutant cells and that the Twist mutation increased cell growth in mutant osteoblasts compared with control cells. The mutation also caused increased alkaline phosphatase and type I collagen expression independently of cell growth. During in vitro osteogenesis, Twist mutant cells showed increased ability to form alkaline phosphatase-positive bone-like nodular structures associated with increased type I collagen expression. Mutant cells also showed increased collagen synthesis and matrix production when cultured in aggregates, as well as an increased capacity to form a collagenous matrix in vivo when transplanted into nude mice. In contrast, Twist mutant osteoblasts displayed a cell-autonomous reduction of osteocalcin mRNA expression in basal conditions and during osteogenesis. The data show that genetic deletion of Twist causing reduced Twist dosage increases cell growth, collagen expression, and osteogenic capability, but inhibits osteocalcin gene expression. This provides one mechanism that may contribute to the premature cranial ossification induced by deletion of the bHLH Twist domain in Saethre-Chotzen syndrome.
Twist1, a bHLH transcription factor, promotes breast tumor cell epithelial-mesenchymal transition (EMT), invasiveness and metastasis. However, the mechanisms responsible for regulating Twist1 stability are unknown in these cells. We identified the serine 68 (S68) as a major phosphorylation site of Twist1 by mass spectrometry and with specific antibodies. This S68 is phosphorylated by p38, JNK and ERK1/2 in vitro, and its phosphorylation levels positively correlate with Twist1 protein levels in HEK293 and breast cancer cells. Prevention of S68 phosphorylation by an alanine (A) mutation (S68A) dramatically accelerates Twist1 ubiquitination and degradation. Furthermore, activation of MAPKs by an active Ras protein or TGF-β treatment significantly increases S68 phosphorylation and Twist1 protein levels without altering Twist1 mRNA expression, while blocking of MAPK activities by either specific inhibitors or dominant negative inhibitory mutants effectively reduces the levels of both induced and un-induced S68 phosphorylation and Twist protein. Accordingly, the mammary epithelial cells expressing Twist1 exhibit much higher degrees of EMT and invasiveness upon stimulation with TGF-β or the active Ras as well as taxol resistance compared to same cells expressing the S68A-Twist1 mutant. Importantly, the levels of S68 phosphorylation in the invasive human breast ductal carcinomas positively correlate with the levels of Twist1 protein and JNK activity and are significantly higher in progesterone receptor-negative and HER2-positive breast cancers. These findings suggest that activation of MAPKs by tyrosine kinase receptors and Ras signaling pathways may substantially promote breast tumor cell EMT and metastasis via phoshorylation and stabilization of Twist1.
Twist1 is a basic helix-loop-helix (bHLH) factor that plays an important role in limb development. Haploinsufficiency of Twist1 results in polydactyly via the inability of Twist1 to antagonistically regulate the related factor Hand2. The mechanism modulating Twist1-Hand2 antagonism is via phosphoregulation of conserved threonine and serine residues in helix I of the bHLH domain. Phosphoregulation alters the dimerization affinities for both proteins. Here we show that the expression of Twist1 and Twist1 phosphoregulation mutants result in distinct limb phenotypes in mice. In addition to dimer regulation, Twist1 phosphoregulation affects the DNA-binding affinities of Twist1 in a partner dependent and cis-element dependent manner. In order to gain a better understanding of the specific Twist1 transcriptional complexes that function during limb morphogensis, we employ a series of Twist1-tethered dimers that include the known Twist1 partners, E12 and Hand2, as well as a tethered Twist1 homodimer. We show that these dimers behave in a manner similar to monomerically expressed bHLH factors and result in distinct limb phenotypes that correlate well with those observed from the limb expression of Twist1 and Twist1 phosphoregulation mutants. Taken together, this study shows that the Twist1 dimer affinity for a given partner can modulate the DNA binding affinity and that Twist1 dimer choice determines phenotypic outcome during limb development.
bHLH factors; Twist1; Hand2; limb development; transcription; and dimerization
In vertebrates, the basic helix-loop-helix (bHLH) protein Twist may be involved in the negative regulation of cellular determination and in the differentiation of several lineages, including myogenesis, osteogenesis, and neurogenesis. Although it has been shown that mouse twist (M-Twist) (i) sequesters E proteins, thus preventing formation of myogenic E protein-MyoD complexes and (ii) inhibits the MEF2 transcription factor, a cofactor of myogenic bHLH proteins, overexpression of E proteins and MEF2 failed to rescue the inhibitory effects of M-Twist on MyoD. We report here that M-Twist physically interacts with the myogenic bHLH proteins in vitro and in vivo and that this interaction is required for the inhibition of MyoD by M-Twist. In contrast to the conventional HLH-HLH domain interaction formed in the MyoD/E12 heterodimer, this novel type of interaction uses the basic domains of the two proteins. While the MyoD HLH domain without the basic domain failed to interact with M-Twist, a MyoD peptide containing only the basic and helix 1 regions was sufficient to interact with M-Twist, suggesting that the basic domain contacts M-Twist. The replacement of three arginine residues by alanines in the M-Twist basic domain was sufficient to abolish both the binding and inhibition of MyoD by M-Twist, while the domain retained other M-Twist functions such as heterodimerization with an E protein and inhibition of MEF2 transactivation. These findings demonstrate that M-Twist interacts with MyoD through the basic domains, thereby inhibiting MyoD.
The Twist1-family basic helix-loop-helix (bHLH) transcription factors including Twist1, Hand1 and Hand2, play an essential role in heart development and are implicated in pathological heart remodeling. Previously, it was reported that these bHLH transcription factors can be regulated by phosphorylation within the basic-helix I domain, which is involved in developmental processes such as limb formation and trophoblast differentiation. However, how phosphorylation of Twist1 family functions in post-natal heart is elusive.
Here, we generated transgenic mice with over-expression of Hand1 and Twist1 mutants (to mimic or to abolish phosphorylation) in cardiomyocytes and found pathological cardiac remodeling leading to heart failure and sudden death. Gene expression profile analysis revealed up-regulation of growth-promoting genes and down-regulation of metabolic genes. It is well known that aberrant activation of Akt signaling causes pathological cardiac remodeling and results in heart failure. The basic-helix I domain of Twist1 family members contain Akt substrate consensus motif and may be downstream targets of Akt signaling. Using biochemical analysis, we demonstrated that Hand1 and Twist1 were phosphorylated by Akt in the basic-helix I domain. Phosphorylation of Hand1 regulated its transcriptional activation of luciferase reporter genes and DNA binding ability.
This study provides novel insights into the regulation of Twist1 family in cardiac remodeling and suggests that the Twist1 family can be regulated by Akt signaling.
Basic helix-loop-helix (bHLH) transcription factors play critical roles in lymphoid and erythroid development; however, little is known about their role in myeloid lineage development. In this study, we identify the bHLH transcription factor Twist-2 as a key negative regulator of myeloid lineage development, as manifested by marked increases in mature myeloid populations of macrophages, neutrophils, and basophils in Twist-2–deficient mice. Mechanistic studies demonstrate that Twist-2 inhibits the proliferation as well as differentiation of granulocyte macrophage progenitors (GMP) by interacting with and inhibiting the transcription factors Runx1 and C/EBPα. Moreover, Twist-2 was found to have a contrasting effect on cytokine production: inhibiting the production of proinflammatory cytokines such as interleukin-12 (IL-12) and interferon-γ (IFNγ) while promoting the regulatory cytokine IL-10 by myeloid cells. The data from further analyses suggest that Twist-2 activates the transcription factor c-Maf, leading to IL-10 expression. In addition, Twist-2 was found to be essential for endotoxin tolerance. Thus, this study reveals the critical role of Twist-2 in regulating the development of myeloid lineages, as well as the function and inflammatory responses of mature myeloid cells.
Hematopoiesis is coordinated by transcription factors that regulate proliferation, differentiation, and cell fate determinations. Myelopoiesis refers to the development of all white blood cells, excluding lymphocytes (B and T cells); however, the molecular regulation of this developmental process is still incompletely understood. In this study using mice that lack expression of Twist-2, we establish a novel role for this basic helix-loop-helix transcription factor as regulator of myeloid progenitors and fully differentiated myeloid cells. Specifically, Twist-2 acts to inhibit proliferation as well as differentiation of progenitors that give rise to macrophages, neutrophils, and basophils by inhibiting the important transcription factors Runx1 and C/EBPα. In mature myeloid cells, Twist-2 negatively regulates the production of proinflammatory cytokines while positively promoting the production of regulatory cytokine IL-10 by these cells. These findings provide significant insight into regulation of myeloid lineage development and function.
The transcription factor Twist-2 is a new regulator that inhibits the proliferation and differentiation of granulocyte macrophage progenitors. Twist-2 also inhibits proinflammatory cytokine production, while stimulating IL-10 by myeloid cells.
The heart is a complex organ that is composed of numerous cell types, which must integrate their programs for proper specification, differentiation and cardiac morphogenesis. During cardiogenesis members of the Twist-family of basic helix-loop-helix (bHLH) transcription factors play distinct roles within cardiac lineages such as the endocardium and extra-cardiac lineages such as the cardiac neural crest (cNCC) and epicardium. While the study of these cell populations is often eclipsed by that of cardiomyocytes, the contributions of non-cardiomyocytes to development and disease are increasingly being appreciated as both dynamic and essential. This review summarizes what is known regarding Twist-family bHLH function in extra-cardiac cell populations and the endocardium, with a focus on regulatory mechanisms, downstream targets, and expression profiles. Improving our understanding of the molecular pathways that Twist-family bHLH factors mediate in these lineages will be necessary to ascertain how their dysfunction leads to congenital disease and adult pathologies such as myocardial infarctions and cardiac fibroblast induced fibrosis. Indeed, this knowledge will prove to be critical to clinicians seeking to improve current treatments.
Tumor cell invasion into adjacent normal brain is a mesenchymal feature of GBM and a major factor contributing to their dismal outcomes. Therefore, better understandings of mechanisms that promote mesenchymal change in GBM are of great clinical importance to address invasion. We previously showed that the bHLH transcription factor TWIST1 which orchestrates carcinoma metastasis through an epithelial mesenchymal transition (EMT) is upregulated in GBM and promotes invasion of the SF767 GBM cell line in vitro.
To further define TWIST1 functions in GBM we tested the impact of TWIST1 over-expression on invasion in vivo and its impact on gene expression. We found that TWIST1 significantly increased SNB19 and T98G cell line invasion in orthotopic xenotransplants and increased expression of genes in functional categories associated with adhesion, extracellular matrix proteins, cell motility and locomotion, cell migration and actin cytoskeleton organization. Consistent with this TWIST1 reduced cell aggregation, promoted actin cytoskeletal re-organization and enhanced migration and adhesion to fibronectin substrates. Individual genes upregulated by TWIST1 known to promote EMT and/or GBM invasion included SNAI2, MMP2, HGF, FAP and FN1. Distinct from carcinoma EMT, TWIST1 did not generate an E- to N-cadherin "switch" in GBM cell lines. The clinical relevance of putative TWIST target genes SNAI2 and fibroblast activation protein alpha (FAP) identified in vitro was confirmed by their highly correlated expression with TWIST1 in 39 human tumors. The potential therapeutic importance of inhibiting TWIST1 was also shown through a decrease in cell invasion in vitro and growth of GBM stem cells.
Together these studies demonstrated that TWIST1 enhances GBM invasion in concert with mesenchymal change not involving the canonical cadherin switch of carcinoma EMT. Given the recent recognition that mesenchymal change in GBMs is associated with increased malignancy, these findings support the potential therapeutic importance of strategies to subvert TWIST1-mediated mesenchymal change.
The basic helix-loop-helix (bHLH) transcription factor TWIST1 is essential to embryonic development, and hijacking of its function contributes to the development of numerous cancer types. It forms either a homodimer or a heterodimeric complex with an E2A or HAND partner. These functionally distinct complexes display sometimes antagonistic functions during development, so that alterations in the balance between them lead to pronounced morphological alterations, as observed in mice and in Saethre–Chotzen syndrome patients. We, here, describe the structures of TWIST1 bHLH–DNA complexes produced in silico through molecular dynamics simulations. We highlight the determinant role of the interhelical loops in maintaining the TWIST1–DNA complex structures and provide a structural explanation for the loss of function associated with several TWIST1 mutations/insertions observed in Saethre–Chotzen syndrome patients.
An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:27
TWIST; dimerization; DNA interaction; homology models; 3D-models; molecular dynamics; embryonic transcription factors; bHLH
Basic Helix-loop-Helix (bHLH) factors play a significant role in both development and disease. bHLH factors function as protein dimers where two bHLH factors compose an active transcriptional complex. In various species, the bHLH factor Twist has been shown to play critical roles in diverse developmental systems such as mesoderm formation, neurogenesis, myogenesis, and neural crest cell migration and differentiation. Pathologically, Twist1 is a master regulator of epithelial-to-mesenchymal transition (EMT) and is causative of the autosomal-dominant human disease Saethre Chotzen Syndrome (SCS). Given the wide spectrum of Twist1 expression in the developing embryo and the diverse roles it plays within these forming tissues, the question of how Twist1 fills some of these specific roles has been largely unanswered. Recent work has shown that Twist’s biological function can be regulated by its partner choice within a given cell. Our work has identified a phosphoregulatory circuit where phosphorylation of key residues within the bHLH domain alters partner affinities for Twist1; and more recently, we show that the DNA binding affinity of the complexes that do form is affected in a cis-element dependent manner. Such perturbations are complex as they not only affect direct transcriptional programs of Twist1, but they indirectly affect the transcriptional outcomes of any bHLH factor that can dimerize with Twist1. Thus, the resulting lineage-restricted cell fate defects are a combination of loss-of-function and gain-of-function events. Relating the observed phenotypes of defective Twist function with this complex regulatory mechanism will add insight into our understanding of the critical functions of this complex transcription factor.
Twist1; bHLH; transcription; dimerization; DNA binding; Saethre Chotzen Syndrome; limb development; phosphorylation.
Basic Helix-loop-Helix (bHLH) factors play a significant role in both development and disease. bHLH factors function as protein dimers where two bHLH factors compose an active transcriptional complex. In various species, the bHLH factor Twist has been shown to play critical roles in diverse developmental systems such as mesoderm formation, neurogenesis, myogenesis, and neural crest cell migration and differentiation. Pathologically Twist1 is a master regulator of epithelial-to-mesenchymal transition (EMT) and is causative of the autosomal-dominant human disease Saethre Chotzen Syndrome (SCS). Given the wide spectrum of Twist1 expression in the developing embryo and the diverse roles it plays within these forming tissues, the question of how Twist1 fills some of these specific roles has been largely unanswered. Recent work has shown that Twist’s biological function can be regulated by its partner choice within a given cell. Our work has identified a phosphoregulatory circuit where phosphorylation of key residues within the bHLH domain alters partner affinities for Twist1; and more recently, we show that the DNA binding affinity of the complexes that do form is affected in a cis-element dependent manner. Such perturbations are complex as they not only affect direct transcriptional programs of Twist1, but they indirectly affect the transcriptional outcomes of any bHLH factor that can dimerize with Twist1. Thus the resulting lineage-restricted cell fate defects are a combination of loss-of-function and gain-of-function events. Relating the observed phenotypes of defective Twist function with this complex regulatory mechanism will add insight into our understanding of the critical functions of this complex transcription factor.
Twist1; bHLH; transcription; dimerization; DNA binding; Saethre Chotzen Syndrome; limb development; phosphorylation
Some higher vertebrates can display unique muscle regenerative abilities through dedifferentiation. Research evidence suggests that induced dedifferentiation can be achieved in mammalian cells. TWIST is a bHLH (basic helix-loop-helix) transcription factor that is expressed during embryonic development and plays critical roles in diverse developmental systems including myogenesis. Several experiments demonstrated its role in inhibition of muscle cell differentiation. We have previously shown that overexpression of TWIST can reverse muscle cell differentiation in the presence of growth factors. Here we show that TWIST reverses muscle cell differentiation through binding and down-regulation of myogenin. Moreover, it can reverse cellular morphology in the absence of growth factors.
dedifferentiation; myogenesis; myogenin; myotubes; Twist; AdC, control adenoviral vector; AdMyoD, MyoD-overexpressing adenoviral vector; AdT, TWIST-overexpressing adenoviral vector; Ara-C, cytosine β-D-arabinofuranoside; bHLH, basic helix-loop-helix; ChIP, chromatin immunoprecipitation; DM, differentiation medium; EdU, 5-ethynyl-2′-deoxyuridine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GM, growth medium; MEF, myocyte enhancer factor; MRF, myogenic regulatory factor
Twist1 is a bHLH transcription factor that regulates cell proliferation, migration, and differentiation in embryonic progenitor cell populations and transformed tumor cells. While much is known about Twist1’s function in a variety of mesenchymal cell types, the role of Twist1 in endocardial cushion cells is unknown. Twist1 gain and loss of function experiments were performed in primary chicken endocardial cushion cells in order to elucidate its role in endocardial cushion development. These studies indicate that Twist1 can induce endocardial cushion cell proliferation as well as promote endocardial cushion cell migration. Furthermore, Twist1 is subject to BMP regulation and can induce expression of cell migration marker genes including Periostin, Cadherin 11, and Mmp2 while repressing markers of valve cell differentiation including Aggrecan. Previously, Tbx20 has been implicated in endocardial cushion cell proliferation and differentiation, and in the current study, Tbx20 also promotes cushion cell migration. Twist1 can induce Tbx20 expression, while Tbx20 does not affect Twist1 expression. Taken together, these data indicate a role for Twist1 upstream of Tbx20 in promoting cell proliferation and migration and repressing differentiation in endocardial cushion cells during embryonic development.
Twist1; Tbx20; endocardial cushion development; Cadherin 11; Periostin; Mmp2; Aggrecan; Versican; cell proliferation; cell migration; siRNA; chicken
Autosomal dominant mutations in the bHLH transcription factor TWIST1 are associated with limb and craniofacial defects in humans with Saethre-Chotzen syndrome (SCS). The molecular mechanism underlying these phenotypes is poorly understood. We show that the ectopic expression of the related bHLH factor Hand2 phenocopies Twist1 loss-of-function phenotypes in the limb, and that they display a gene dosage-dependent antagonistic interaction. Twist1 and Hand2 dimerization partner choice can be modulated by PKA and protein phosphatase 2A-regulated phosphorylation of conserved helix I residues. Interestingly, multiple TWIST1 mutations associated with SCS alter PKA-mediated Twist1 phosphorylation, suggesting that misregulation of Twist1 dimerization via either stoichiometric or posttranslational mechanisms underlies SCS phenotypes.
Human TWIST1 is a highly conserved member of the regulatory basic helix-loop-helix (bHLH) transcription factors. TWIST1 forms homo- or heterodimers with E-box proteins, such as E2A (isoforms E12 and E47), MYOD and HAND2. Haploinsufficiency germ-line mutations of the twist1 gene in humans are the main cause of Saethre-Chotzen syndrome (SCS), which is characterized by limb abnormalities and premature fusion of cranial sutures. Because of the importance of TWIST1 in the regulation of embryonic development and its relationship with SCS, along with the lack of an experimentally solved 3D structure, we performed comparative modeling for the TWIST1 bHLH region arranged into wild-type homodimers and heterodimers with E47. In addition, three mutations that promote DNA binding failure (R118C, S144R and K145E) were studied on the TWIST1 monomer. We also explored the behavior of the mutant forms in aqueous solution using molecular dynamics (MD) simulations, focusing on the structural changes of the wild-type versus mutant dimers.
The solvent-accessible surface area of the homodimers was smaller on wild-type dimers, which indicates that the cleft between the monomers remained more open on the mutant homodimers. RMSD and RMSF analyses indicated that mutated dimers presented values that were higher than those for the wild-type dimers. For a more careful investigation, the monomer was subdivided into four regions: basic, helix I, loop and helix II. The basic domain presented a higher flexibility in all of the parameters that were analyzed, and the mutant dimer basic domains presented values that were higher than the wild-type dimers. The essential dynamic analysis also indicated a higher collective motion for the basic domain.
Our results suggest the mutations studied turned the dimers into more unstable structures with a wider cleft, which may be a reason for the loss of DNA binding capacity observed for in vitro circumstances.
Twist1; Transcription factor; bHLH; Comparative modeling; Molecular dynamics simulation; Collective motions
The basic Helix-Loop-Helix (bHLH) transcription factor Twist1 fulfills an essential function in neural crest cell formation, migration and survival and is associated with the craniosynostic Saethre-Chotzen syndrome in humans. However, its functions during mandibular development, when it may interact with other bHLH transcription factors like Hand2, are unknown since mice homozygous for the Twist1 null mutation die in early embryogenesis. To determine the role of Twist1 during mandibular development, we used the Hand2-Cre transgene to conditionally inactivate the gene in the neural crest cells populating the mandibular pharyngeal arch.
The mutant mice exhibited a spectrum of craniofacial anomalies, including mandibular hypoplasia, altered middle ear development, and cleft palate. It appears that Twist1 is essential for the survival of the neural crest cells involved in the development of the mandibular ramal elements. Twist1 plays a role in molar development and cusp formation by participating in the reciprocal signaling needed for the formation of the enamel knot. This gene is also needed to control the ossification of the mandible, a redundant role shared with Hand2.
Twist1, along with Hand2, is essential for the proximo-distal patterning and development of the mandible and ossification.
mandible; neural crest cells; Twist1; Hand2; palate; Meckel’s cartilage; mineralization; basic Helix-Loop-Helix transcription factor
Twist1, a basic helix-loop-helix transcription factor, is expressed in mesenchymal precursor populations during embryogenesis and in metastatic cancer cells. In the developing heart, Twist1 is highly expressed in endocardial cushion (ECC) valve mesenchymal cells and is down regulated during valve differentiation and remodeling. Previous studies demonstrated that Twist1 promotes cell proliferation, migration, and expression of primitive extracellular matrix (ECM) molecules in ECC mesenchymal cells. Furthermore, Twist1 expression is induced in human pediatric and adult diseased heart valves. However, the Twist1 downstream target genes that mediate increased cell proliferation and migration during early heart valve development remain largely unknown. Candidate gene and global gene profiling approaches were used to identify transcriptional targets of Twist1 during heart valve development. Candidate target genes were analyzed for evolutionarily conserved regions (ECRs) containing E-box consensus sequences that are potential Twist1 binding sites. ECRs containing conserved E-box sequences were identified for Twist1 responsive genes Tbx20, Cdh11, Sema3C, Rab39b, and Gadd45a. Twist1 binding to these sequences in vivo was determined by chromatin immunoprecipitation (ChIP) assays, and binding was detected in ECCs but not late stage remodeling valves. In addition identified Twist1 target genes are highly expressed in ECCs and have reduced expression during heart valve remodeling in vivo, which is consistent with the expression pattern of Twist1. Together these analyses identify multiple new genes involved in cell proliferation and migration that are differentially expressed in the developing heart valves, are responsive to Twist1 transcriptional function, and contain Twist1-responsive regulatory sequences.