Human Molecular Genetics, 2000, Vol. 9, No. 5 813-819
© 2000 Oxford University Press
Saethre–Chotzen mutations cause TWIST protein degradation or impaired nuclear location
Unité de Recherches sur les Handicaps Génétiques de l’Enfant, INSERM U393, Institut Necker, 149 rue de Sèvres, 75743 Paris cedex 15, France and 1INSERM U248, Institut Curie, Section de Recherche, 26 rue d’Ulm, Paris, France
Received 8 December 1999; Revised and Accepted 18 January 2000.
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ABSTRACT |
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H-TWIST belongs to the family of basic helix–loop–helix (bHLH) transcription factors known to exert their activity through dimer formation. We have demonstrated recently that mutations in H-TWIST account for Saethre–Chotzen syndrome (SCS), an autosomal dominant craniosynostosis syndrome characterized by premature fusion of coronal sutures and limb abnormalities of variable severity. Although insertions, deletions, nonsense and missense mutations have been identified, no genotype–phenotype correlation could be found, suggesting that the gene alterations lead to a loss of protein function irrespective of the mutation. To assess this hypothesis, we studied stability, dimerization capacities and subcellular distribution of three types of TWIST mutant. Here, we show that: (i) nonsense mutations resulted in truncated protein instability; (ii) missense mutations involving the helical domains led to a complete loss of H-TWIST heterodimerization with the E12 bHLH protein in the two-hybrid system and dramatically altered the ability of the TWIST protein to localize in the nucleus of COS-transfected cells; and (iii) in-frame insertion or missense mutations within the loop significantly altered dimer formation but not the nuclear location of the protein. We conclude that at least two distinct mechanisms account for loss of TWIST protein function in SCS patients, namely protein degradation and subcellular mislocalization.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Saethre–Chotzen syndrome (SCS) is an autosomal dominant craniosynostosis characterized by premature fusion of the coronal sutures leading to skull deformation, distal limb abnormalities and dysmorphic facial features of variable severity. This disorder has been ascribed to mutations in the H-Twist gene encoding a basic helix–loop–helix (bHLH) transcription factor (1,2). Twist was first identified in Drosophila as a zygotic developmental gene involved in early mesoderm patterning (3,4). In contrast, its murine homologue M-Twist is not critical for mesoderm formation as homozygous Twist-null embryos die at embryonic day 11.5 after the mesoderm has formed (5). The specific expression of M-Twist transcripts in cephalic and branchial mesectoderm and in subsets of mesodermal cells (6,7) indicates that TWIST belongs to the class B bHLH proteins. Most of these transcription factors are known to form stable heterodimers with ubiquitous class A bHLH proteins including members of the E family (8,9) although variations among species have been reported. Indeed, Ce-TWIST, the Caenorhabditis elegans homologue of H-TWIST, heterodimerizes with the E/daughterless homologue to activate target genes (10). In contrast the Drosophila D-TWIST protein is able to form homodimers and does not appear to require heterodimerization for DNA binding (11). In the mouse model, M-TWIST has been shown to form either homo- or heterodimers in vitro (12,13). Whether H-TWIST forms homo- or heterodimers in human remains to be elucidated.
The identification of single base mutations or deletions producing a premature termination codon in the H-Twist gene and the SCS-like phenotype of Twist-null heterozygous mice strongly suggest that haploinsufficiency is the pathogenic mechanism underlying SCS (1,2,14,15). That gene alterations could result in a loss of protein function irrespective of the mutation is further supported by the absence of genotype–phenotype correlation among patients carrying nonsense, missense or in-frame insertions. However, the hypothesis has been raised that some missense mutations could confer a dominant-negative function to the protein (16). In an attempt to understand how mutations affected the TWIST protein, cDNAs carrying different types of mutation identified in SCS patients were generated and used for in vitro and in vivo experiments. Here we show by transfection assay and immunoblotting that nonsense mutations result in the synthesis of truncated proteins that are rapidly degraded, whereas other mutant proteins are stable. Using a two-hybrid assay, we demonstrate that the human E12 protein is a partner for heterodimerization with H-TWIST. Dimer formation is abolished by missense mutations in the TWIST helical domains but not by in-frame insertion or missense mutations in the loop. This helix–loop correlation is further supported by immunostaining experiments showing that, unlike mutations in the loop, helix mutations dramatically reduce the number of TWIST-positive cells in the nucleus.
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RESULTS |
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Nonsense mutations induce degradation of truncated TWIST proteins
To determine how H-Twist mutations alter protein function in SCS, human Twist cDNAs carrying different types of mutation including three nonsense mutations (Y103X, E126X, Q161X), three missense mutations affecting either the helices (A129P, helix I; L159F, helix II) or the loop (K145E) of the HLH motif and a 21 bp in-frame insertion (P139ins7) were generated (Fig. 1a). Twist mutants cloned in an expression vector were in vitro-translated in a reticulocyte lysate system and resulted in molecules of the expected size (Fig. 1b). RNA and protein stability was then tested by ectopically expressing wild-type and mutant constructs in transfected COS7 cells. Northern blot analysis of total RNAs detected similar amounts of Twist transcripts irrespective of the mutation, indicating that RNA stability was unaffected (data not shown). Immunoblot analyses of cell lysates revealed that truncated proteins due to nonsense mutations were unstable, as their expression level was reduced at 24 h post-transfection compared with the wild-type (Fig. 1c) and was undetectable after 48 h (Fig. 1d). In contrast, the amount of missense and insertion mutant gene products was similar to control (Fig. 1c and d). The variable stability of mutant proteins suggested that markedly different mechanisms cause the SCS phenotype.
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Direct interaction between H-TWIST and E12 in vitro and in vivo
Since bHLH transcription factors require either homo- or heterodimerization for DNA binding, we tested by using a two-hybrid assay whether H-TWIST could form homo- or hetero- dimers. The entire coding sequence of the gene was expressed in yeast as fusions with either the LexA DNA-binding domain or the GAL4 activation domain (GAD) and both constructs were co-transformed in yeast. Co-expression of wild-type LexA–TWIST and GAD–TWIST fusion proteins failed to transactivate HIS3, as evidenced by their inability to grow on medium lacking histidine (Fig. 2a). In contrast, co-expression of wild-type LexA–TWIST and GAD–E12 fusion proteins led to rapid yeast growth on medium lacking histidine with the same efficiency as LexA–rap2A/GAD ral-GDS (17), which was used as a positive control (Fig. 2a). Similar results were obtained with a ß-galactosidase reporter gene (data not shown). We conclude that H-TWIST does not homodimerize but can efficiently interact with the human E12 protein in the yeast two-hybrid system. Additional evidence for this interaction was derived from binding assays of in vitro-translated wild-type and mutant proteins to the glutathione S-transferase–E12 fusion protein (GST–E12). Indeed, H-TWIST directly interacted with E12 fused to GST and deletion of the bHLH domain from either partners hampered their interaction, thus indicating that these domains are required for hetero- dimerization (Fig. 2d). Finally, H-TWIST and E12 also interacted in mammalian cells co-transfected with expression constructs, as evidenced by co-immunoprecipitation of the two partners (Fig. 2e).
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Analysis of TWIST mutant proteins by the two-hybrid assay
Next we investigated whether the TWIST–E12 interaction could be altered by TWIST mutations. Mutant constructs fused to the LexA DNA-binding domain were co-expressed in yeast with a GAD–E12 fusion protein and their capacity to interact was assessed as described above. Interestingly, two distinct patterns of yeast growth were observed (Fig. 2b). First, nonsense mutations consistently led to a complete loss of interaction with E12, irrespective of the extent of protein truncation. Similarly, the two missense mutations affecting either helix of the HLH motif failed to transactivate the reporter gene when co-expressed with E12. The absence of transactivation of the HIS3 gene could not be ascribed to the lack of expression of the mutant protein, as evidenced by immunoblotting with an anti-TWIST antibody (data not shown). Second, missense mutations and the in-frame insertion within the loop were still able to lead to histidine auxotrophy (Fig. 2b) albeit quantitation of ß-galactosidase activity revealed a 10- to 50-fold decreased interaction compared with the wild-type heterodimer (Fig. 2c). Hence, protein truncation and mutations of the helix domains hampered the ability of TWIST to interact with its partner whereas proteins carrying loop mutations were still able to form dimers with E12.
Subcellular localization of the TWIST mutant proteins
The M-TWIST protein has been detected in the cell nuclei of normal mouse embryos (18). Considering that the subcellular localization of transcription factors in either the cytoplasm or the nucleus regulates their biological activity, we wondered whether an abnormal subcellular distribution of the mutant H-TWIST protein could account for the disease. To address this issue, TWIST mutant proteins carrying either the in-frame insertion or missense mutations were co-expressed with E12 in COS7 cells and the localization of both proteins was examined by immunofluorescence microscopy. Cells transfected with wild-type constructs exhibited a strong nuclear staining with either anti-TWIST or anti-E12 antibodies (Fig. 3a–c) whereas no signal was detected in untransfected cells (Fig. 3p–r). Three patterns of immunofluorescence were observed using the transfected mutant constructs: (i) mutations altering helices caused a marked staining of the cytoplasm using anti-TWIST antibodies but these mutations had no effect on the localization of E12 which remained in the nucleus (Fig. 3d–i); (ii) missense mutations within the loop did not alter the nuclear localization of the TWIST mutant protein (Fig. 3m–o); and (iii) the in-frame insertion gave an intermediate figure, as shown by the presence of cytoplasm-positive and nuclear-positive cells (Fig. 3j–l).
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These observations were further documented by quantifying the proportion of cells showing either a nuclear or a cytoplasmic localization of TWIST. Although >90% of cells expressing the wild-type protein or the loop mutant exhibited a normal nuclear staining, this percentage fell to 25–40% for mutations altering TWIST helices (Fig. 4a). These data were further confirmed by fractionating transfected cells into nuclear and non-nuclear fractions. Immunoblotting revealed that the wild-type protein and the loop mutant were only detectable in the nuclear fraction. Helix mutants were mostly present in the cytoplasm whereas mutant proteins carrying the insertion were detected in both fractions (Fig. 4b). However, in both assays the L159F helix mutant was apparently more cytoplasmic than the A129P helix mutant.
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DISCUSSION |
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Although compelling evidence now exists that different types of mutation in the Twist gene account for common clinical features including craniofacial and limb anomalies, the phenotypic variability seen in SCS patients and the absence of genotype–phenotype correlation is far from understood (14,19). The present study was thus undertaken to determine how mutations in the H-Twist gene affected the protein function. To this purpose, a series of recombinant plasmids carrying mutations identified in SCS patients were produced. Expression analyses first demonstrated that nonsense mutations resulted in the synthesis of truncated proteins that were rapidly degraded, thereby leading to haploinsufficiency. Along these lines, it is worth keeping in mind that individuals carrying monosomy 7p21 and thus hemizygous for the Twist gene had a SCS phenotype (20). Similarly, the Twist-null heterozygous mouse model mimicked the human phenotype (1,15).
Since missense mutations or in-frame insertions had no detectable effect on the stability of the translation product in transfected cells, we speculated that these mutations altered the TWIST function through a different mechanism. Class B bHLH proteins to which H-TWIST belongs are known to form stable heterodimers with members of class A bHLH transcription factors including gene products of E2A (E12 and E47) (8,9), E2-2 and E2-5 (ITF-2 and ITF-1, respectively) (21) or HEB/HTF-4 (22). However, D-TWIST homodimerization has been reported in Drosophila (11), and homodimerization of the mouse M-TWIST protein has been observed in an in vitro binding assay (13). The yeast two-hybrid assay thus appeared as a relevant model to test the possible H-TWIST homo- or heterodimerization with a member of the E protein family (23). We found no homodimerization of the H-TWIST protein in this in vivo system, further emphasizing the striking differences between in vitro and in vivo HLH dimerization (24). In contrast, E12 strongly interacted with H-TWIST in yeast. This finding was consistent with the previous obser- vation that E12 was able to heterodimerize with M-TWIST in an in vitro assay (12). In addition, DERMO-1, a bHLH transcription factor closely related to M-TWIST, has emerged from a two-hybrid screen for factors interacting with E12 (25). The capacity of H-TWIST to form stable heterodimers with E12 was further supported by in vitro binding experiments and co-immunoprecipitation studies. Hence, E12 appears as a putative partner in vivo for regulating the H-TWIST transcriptional activity. Interestingly, Twist and E2A transcripts have been detected in mouse osteoblastic cells (26) and were found to be co-expressed in the developing mandible and limb buds of human embryos (unpublished data). Based on the recent demonstration that H-TWIST has an inhibitory effect on osteogenic differentiation (27), the TWIST–E12 heterodimer might be regarded as a negative regulator of transcription in human osteoblastic cells.
Heterodimerization proved to be mediated through the bHLH motif as deletion of the E12 bHLH domain or nonsense mutations truncating the TWIST bHLH domain hampered heterodimer formation. This result is in accordance with previous in vitro data obtained with MyoD (28), E47 (29) and Lyl-1 (30) but does not rule out the possible influence of additional adjacent HLH sequences for in vivo dimerization (24). Within the HLH motif, the two helices seem to play a more critical role than the loop as their alteration results in both a markedly impaired heterodimerization and an abnormal cellular location of the protein. Likewise, the helix 2 mutation affecting a highly conserved leucine residue at position 159 had a more dramatic effect on the protein mislocalization than the A129P substitution in helix 1. This observation is consistent with the critical role of leucine residues in the stability of 
-helices (8). Interestingly, significant increase of the loop length due to in-frame insertion of seven amino acids had little effect on dimer formation and nuclear location. Similarly, random mutations within the loop region of E47 do not alter protein dimerization (29) and replacement of five residues of the loop in M-TWIST fail to abolish its interaction with MyoD or E12 (13).
Mutations in transcription factors may affect their nuclear localization and thus account for their inability to bind DNA (31). Accordingly, the subcellular localization of mutant TWIST proteins was investigated in COS cells following co-transfection with E12. Since heterodimerization of HLH proteins has been proposed to take place in the cytoplasm prior to nuclear import (32), the expected nuclear location of the wild-type TWIST protein appears to be a direct reflection of the in vivo interaction with E12. Consequently, the cyto- plasmic location of TWIST proteins carrying single amino acid changes in the helices might result from the loss of dimer formation. Alternatively, a rapid nuclear export of the mutant protein could account for its abnormal subcellular distribution (33). Nevertheless, the normal nuclear location of loop mutants suggests that loss of TWIST function in these cases occurs at still another step. Alteration of the DNA-binding properties of the TWIST/E12 complex is the most likely explanation, but this will require identification of the human target DNA sequences involved.
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MATERIALS AND METHODS |
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cDNAs constructs and expression vectors
For eukaryotic expression in COS7 cells and in vitro expression of TWIST proteins, genomic DNA from control and SCS patients was PCR-amplified, and amplification products were directly cloned into the pCR3 unidirectional plasmid (Invitrogen, San Diego, CA). Primers used for PCR allowed generation of a 5' BamHI and a 3' SalI restriction site used for subsequent subcloning. Normal and mutant inserts were sequenced to ensure integrity of the cloned allele. For E12 expression, a human full-length cDNA in a pSP65 vector was subcloned into the pCMV5 vector. For expression of the LexA fusion protein in yeast, we used the pVJL10 plasmid (34). Wild-type and mutant Twist cDNAs were subcloned from pCR3uni into pVJL10 using the BamHI and SalI restriction sites. The fusion between the activation domain of GAL4 and TWIST was generated by subcloning the wild-type Twist cDNA from pCR3uni into pGAD1318 using BamHI as the 5' restriction site and SalI–XhoI half-sites at the 3' end. The GAL4 activation domain–E12 fusion was obtained by subcloning the E12 cDNA into the pGAD-GE plasmid using the EcoRI site. To produce GST–E12 fusion proteins, the full-length and a bHLH-deleted form of E12 were subcloned into the pGEX4T-1 vector (Amersham Pharmacia Biotech, Uppsala, Sweden) between the EcoRI and XhoI sites.
Cell cultures, transfection and immunofluorescence
COS7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and antibiotics. Six-well tissue culture plates and eight-well chamber slides were seeded with 1.5 x 105 and 1 x 104 cells/well, respectively, and cultured to 70% confluency. Transfections were performed with Fugene-6 (Roche Molecular Biochemicals, Uppsala, Sweden) and 2 or 0.1 µg DNA/well in FCS supplemented DMEM according to the manufacturer’s instructions. Twenty-four or 48 h later, cells were used for immunofluorescence studies. Transfected cells in culture chamber slides were fixed with 4% paraformaldehyde for 30 min. After permeabilization with 0.1% Triton X-100 for 15 min, cells were incubated for 1 h at room temperature with the above-mentioned primary antibodies at the respective dilutions of 1:100 and 1:1000. Cells were then incubated with either a fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat antibody (Santa Cruz Biotechnology, Santa Cruz, CA) to visualize TWIST, or a Cy3-conjugated goat anti-mouse antibody (Jackson Immuno Research, West Grove, PA) to visualize E12. Cells were examined with a Zeiss LSM 510 confocal microscope.
Immunoblotting and co-immunoprecipitation
Cell lysates were denatured by boiling in sample buffer, separated by 7 or 12% SDS–PAGE and electroblotted on PVDF membranes (Immobilon; Millipore, Bedford, MA). The membranes were pre-incubated for 1 h at 4°C in TBS (20 mM Tris–HCl pH 7.4, 150 mM NaCl) containing 5% skim milk. The blots were then incubated overnight at 4°C with either goat polyclonal anti-TWIST or mouse monoclonal anti-E12 antibodies (Santa Cruz Biotechnology) at respective dilutions of 1:200 and 1:100. After membrane washing, a second antibody coupled to peroxidase was added. Proteins were visualized with the ECL detection kit (Amersham Pharmacia Biotech, Uppsala, Sweden). Co-immunoprecipitation was achieved by adding 5 µg of anti-E12 antibody to 50 µl of cell lysate followed by incubation at 4°C for 2 h. Protein A–Sepharose beads were added and the mixture was rotated at 4°C for 1 h. Beads were washed twice with 0.1 M Tris–HCl pH 7.6 buffer containing 0.25 M NaCl, and twice with 10 mM Tris–HCl pH 7.6, 0.1 M NaCl, 1 mM EDTA then resuspended in 40 µl of SDS–PAGE sample buffer and boiled for 5 min. Cytoplasmic and nuclear extracts were prepared as described (35). Both fractions were submitted to immunoblot analysis as described above.
Yeast two-hybrid assay
The genotype of the Saccharomyces cerevisiae reporter strain L40 was MATa trp1 leu 2 his3 ade2, LYS2::lexA-HIS3 URA3::lexA-lacZ. Yeast strains were grown at 30°C in a rich medium (1% yeast extract, 2% Bacto-peptone, 2% glucose) or in a synthetic minimal medium containing appropriate supplements. The yeast reporter strain L40, which contains the reporter genes LacZ and HIS3 downstream of the binding sequences for LexA, was co-transformed with pVJL10–TWIST (wild-type or mutants) and either pGAD1318–TWIST (wild-type) or pGAD-GE–E12 plasmids using the lithium acetate method. Double transformants were plated to synthetic medium lacking leucine and tryptophan. The plates were incubated at 30°C for 2 days. Leu+Trp+ colonies were patched on selective plates lacking histidine and assayed for ß-galactosidase activity by a filter assay (36). Quantification of ß-galactosidase activity was performed as described (37).
In vitro protein–protein interaction assay
Wild-type and mutant human Twist cDNAs were transcribed and translated in vitro from pCR3uni constructs using the TNT T7 Quick transcription/translation system (Promega, Madison, WI) in the presence of [35S]methionine (1000 Ci/mmol; Amersham Pharmacia Biotech). Synthesis of radiolabelled proteins was checked by SDS–PAGE in a 12% polyacrylamide gel, followed by fluorography. BL21 Escherichia coli cells, transformed with pGEX4T-1/E12wt or E12
bHLH were incubated for 3 h at 30°C in the presence of 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) to induce the production of wild-type GST–E12 and bHLH-deleted GST–E12 fusion proteins, respectively. Recombinant GST fusion proteins were purified on glutathione–Sepharose 4B beads (Amersham Pharmacia Biotech). Purity and yield of the GST fusion proteins were assessed by SDS–PAGE in 7% polyacrylamide gels stained with Coomassie blue.
In vitro interaction assays were performed by incubating 10 µl of GST–E12 beads with 5 µl of [35S]TWIST at 4°C for 1 h in a 50 mM Tris–HCl pH 7.5, 100 mM NaCl, 5 mM MgCl2 buffer containing a mixture of protease inhibitors (Roche Molecular Biochemicals) and 0.1% Triton X-100. Beads were washed six times for 2 min in the same buffer containing 125 mM NaCl and 1% Triton X-100, boiled in SDS–PAGE sample buffer and analysed on 12% polyacrylamide gels.
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ACKNOWLEDGEMENTS |
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We thank Dr Michael L. Cleary (Stanford University School of Medicine, San Diego, CA) for kindly providing human E12 cDNA, Yann Goureau for technical assistance with the confocal microscope and Yves Deris for artwork. This work was supported by the Association Française contre les Myopathies (AFM).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +33 1 44 49 40 00 ext. 97833; Fax: +33 1 47 34 85 14; Email: bonavent@necker.fr
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REFERENCES |
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1 El Ghouzzi, V., Le Merrer, M., Perrin-Schmitt, F., Lajeunie, E., Benit, P., Renier, D., Bourgeois, P., Bolcato-Bellemin, A.L., Munnich, A. and Bonaventure, J. (1997) Mutations of the TWIST gene in the Saethre–Chotzen syndrome. Nature Genet., 15, 42–46.[Web of Science][Medline]
2 Howard, T.D., Paznekas, W.A., Green, E.D., Chiang, L., Ma, N., Ortiz De Luna, R.I., Garcia Delgado, C., Gonzalez-Ramos, M., Kline, A.D. and Jabs, E.W. (1997) Mutations in TWIST, a basic helix–loop–helix transcription factor in Saethre–Chotzen syndrome. Nature Genet., 15, 36–41.[Web of Science][Medline]
3 Thisse, B., Stoetzel, C., El Messal, M. and Perrin-Schmitt, F. (1987) The twist gene: isolation of a Drosophila zygotic gene necessary for the establishment of dorso-ventral pattern. Nucleic Acids Res., 15, 3439–3453.
4 Leptin, M. and Grunewald, B. (1990) Cell shape changes during gastrulation in Drosophila. Development, 110, 73–84.[Abstract]
5 Chen, Z.F. and Behringer, R.R. (1995) Twist is required in head mesenchyme for cranial neural tube morphogenesis. Genes Dev., 9, 686–699.
6 Wolf, C., Thisse, C., Stoetzel, C., Thisse, B., Gerlinger, P. and Perrin-Schmitt, F. (1991) The M-Twist gene of mus is expressed in subsets of mesodermal cells and is closely related to the Xenopus X-twi and Drosophila twist genes. Dev. Biol., 143, 363–373.[Web of Science][Medline]
7 Füchtbauer, E.M. (1995) Expression of M-twist during postimplantation development of the mouse. Dev. Dyn., 204, 316–322.[Web of Science][Medline]
8 Murre, C., Schonleber McCaw, P. and Baltimore, D. (1989) A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell, 56, 777–783.[Web of Science][Medline]
9 Murre, C., Schonleber McCaw, P., Vaessin, H., Caudy, M., Jan, L.Y., Jan, Y.N., Cabrera, C.V., Buskin, J.N., Hauschka, D., Lassar, A.B. et al. (1989) Interactions between heterologous helix–loop–helix proteins generate complexes that bind specifically to a common DNA sequence. Cell, 58, 537–544.[Web of Science][Medline]
10 Harfe, B.D., Vaz Gomes, A., Kenyon, C., Liu, J., Krause, M. and Fire, A. (1998) Analysis of a Caenorhabditis elegans Twist homologue identifies conserved and divergent aspects of mesodermal patterning. Genes Dev., 12, 2623–2635.
11 Lee, Y.M., Park, T., Schulz, R.A. and Kim, Y. (1997) Twist-mediated activation of the NK-4 homeobox gene in the visceral mesoderm of Drosophila requires two distinct clusters of E-box regulatory elements. J. Biol. Chem., 272, 17531–17541.
12 Spicer, D.B., Rhee, J., Cheung, W.L. and Lassar, A.B. (1996) Inhibition of myogenic bHLH and MEF2 transcription factors by the bHLH protein Twist. Science, 272, 1476–1480.[Abstract]
13 Hamamori, Y., Wu, H.Y., Sartorelli, V. and Kedes, L. (1997) The basic domain of myogenic basic helix–loop–helix (bHLH) proteins is the novel target for direct inhibition by another bHLH protein, Twist. Mol. Cell. Biol., 17, 6563–6573.[Abstract]
14 El Ghouzzi, V., Lajeunie, E., Le Merrer, M., Cormier-Daire, V., Renier, D., Munnich, A. and Bonaventure, J. (1999) Mutations within or upstream of the basic helix–loop–helix domain of the TWIST gene are specific to Saethre–Chotzen syndrome. Eur. J. Hum. Genet., 7, 27–33.[Web of Science][Medline]
15 Bourgeois, P., Bolcato-Bellemin, A.L., Danse, J.M., Bloch-Zupan, A., Yoshiba, K., Stoetzel, C. and Perrin-Schmitt, F. (1998) The variable expressively and incomplete penetrance of the twi-null heterozygous mouse phenotype resemble those of human Saethre–Chotzen syndrome. Hum. Mol. Genet., 7, 945–957.
16 Rose, C.S.P. and Malcolm, S. (1997) A TWIST in development. Trends Genet., 13, 384–387.[Web of Science][Medline]
17 Nancy, V., Wolthuis, R.M.F., de Tand, M.F., Janoueix-Lerosey, I., Bos, J.L. and de Gunzburg, J. (1999) Identification and characterization of potential effectors molecules of the Ras-related GTPase Rap2. J. Biol.Chem., 274, 8737–8745.
18 Gitelman, I. (1997) Twist protein in mouse embryogenesis. Dev. Biol., 189, 205–214.[Web of Science][Medline]
19 Paznekas, W.A., Cunningham, M.L., Howard, T.D., Korf, B.R., Lipson, M.H., Grix, A.W., Feingold, M., Goldberg, R., Borochowitz, Z., Aleck, K. et al. (1998) Genetic heterogeneity of Saethre–Chotzen syndrome, due to TWIST and FGFR mutations. Am. J. Hum. Genet., 62, 1370–1380.[Web of Science][Medline]
20 Johnson, D., Horsley, S.W., Moloney, D.M., Oldridge, M., Twigg, R.F., Walsh, S., Barrow, M., Njolstad, P.R., Kunz, J., Ashworth, J. et al. (1998) A comprehensive screen for TWIST mutations in patients with craniosynostosis identifies a new microdeletion syndrome of chromosome band 7p21.1. Am. J. Hum. Genet., 63, 1282–1293.[Web of Science][Medline]
21 Henthorn, P., Kiledjian, M. and Kadesch, T. (1990) Two distinct transcription factors that bind the immunoglobulin enhancer µE5/kE2 motif. Science, 247, 467–470.
22 Hu, J.S., Olson, E.N. and Kingston, R.E. (1992) HEB, a helix–loop–helix protein related to E2A and ITF2 that can modulate the DNA-binding ability of myogenic regulatory factors. Mol. Cell. Biol., 12, 1031–1042.
23 Staudinger, J., Perry, M., Elledge, S.J. and Olson, E.N. (1993) Interactions among vertebrate helix–loop–helix proteins in yeast using the two-hybrid system. J. Biol. Chem., 268, 4608–4611.
24 Goldfarb, A.N., Lewandowska, K. and Pennell, C.A. (1998) Identification of a highly conserved module in E proteins required for in vivo helix–loop–helix dimerization. J. Biol. Chem., 273, 2866–2873.
25 Li, L., Cserjesi, P. and Olson, E.N. (1995) Dermo-1: a novel Twist-related bHLH protein expressed in the developing dermis. Dev. Biol., 172, 280–292.[Web of Science][Medline]
26 Murray, S.S., Glackin, C.A., Winters, K.A., Gazit, D., Karn, A.J. and Murray, E.J. (1992) Expression of helix–loop–helix regulatory genes during differentiation of mouse osteoblastic cells. J. Bone Mineral Res., 10, 1131–1138.
27 Lee, M.S., Lowe, G.N., Strong, D.D., Wergedal, J.E. and Glackin, C.A. (1999) TWIST, a basic helix–loop–helix transcription factor can regulate the human osteogenic lineage. J. Cell. Biochem., 75, 566–577.[Web of Science][Medline]
28 Tapscott, S.J., Davis, R.L., Thayer, M.J., Cheng, P.F., Weintraub, H. and Lassar A.B. (1988) MyoD 1: a nuclear phosphoprotein requiring a myc homology region to convert fibroblasts to myoblasts. Science, 242, 405–411.
29 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. USA, 87, 4722–4726.
30 Miyamoto, A., Cui, X., Naumovski, L. and Cleary, M.L. (1996) Helix-loop-helix proteins LYL1 and E2a form heterodimeric complexes with distinctive DNA-binding properties in hematolymphoid cells. Mol. Cell. Biol., 16, 2394–2401.[Abstract]
31 Takebayashi, K., Chida, K., Tsukamoto, I., Morii, E., Munakata, H., Arnheiter, H., Kuroki, T., Kitamura, Y. and Nomura, S. (1996) The recessive phenotype displayed by a dominant negative Microphthalmia-associated transcription factor mutant is a result of impaired nuclear location potential. Mol. Cell. Biol., 16, 1203–1211.[Abstract]
32 Goldfarb, A.N. and Lewandowska, K. (1994) Nuclear redirection of a cytoplasmic helix–loop–helix protein via heterodimerization with a nuclear localizing partner. Exp. Cell Res., 214, 481–485.[Web of Science][Medline]
33 Gorlich, D. and Mattaj, I.W. (1996) Nucleocytoplasmic transport. Science, 271, 1513–1518.[Abstract]
34 Romero, F., Dargemont, C., Pozo, F., Reeves, W.N., Camonis, J., Gisselbrecht, S. and Fisher, S. (1996) p95vav associates with the nuclear protein Ku-70. Mol. Cell. Biol., 16, 37–44.[Abstract]
35 Schreiber, E., Matthias, P., Muller, M.M. and Schaffner, W. (1989) Rapid detection of octamer binding proteins with mini extracts prepared from a small number of cells. Nucleic Acids Res., 17, 6419.
36 Janoueix-Lerosey, I., Jollivet, F., Camonis, J., Marche, P.N. and Goud, B. (1995) Two-hybrid system screen with the small GTP-binding protein Rab6. Identification of a novel mouse GDP dissociation inhibitor isoform and two other potential partners of Rab6. J. Biol. Chem., 270, 14801–14808.
37 Rose, M.D., Winston, F. and Hieter, P. (1990) Methods in Yeast Genetics: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
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