The molecular properties of the bHLH TCF4 protein as an intrinsically disordered hub transcription factor
Abstract Background Transcription factor 4 (TCF4) is a member of the basic helix-loop-helix (bHLH) family of transcription factors that guides proper embryogenesis, particularly neurogenesis, myogenesis, heart development and hematopoiesis. The interaction of TCF4 with DNA is dependent on the presen...
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BMC
2025-03-01
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| Series: | Cell Communication and Signaling |
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| Online Access: | https://doi.org/10.1186/s12964-025-02154-7 |
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| author | Nikola Sozańska Barbara P. Klepka Anna Niedzwiecka Lilia Zhukova Michał Dadlez Beata Greb-Markiewicz Andrzej Ożyhar Aneta Tarczewska |
| author_facet | Nikola Sozańska Barbara P. Klepka Anna Niedzwiecka Lilia Zhukova Michał Dadlez Beata Greb-Markiewicz Andrzej Ożyhar Aneta Tarczewska |
| author_sort | Nikola Sozańska |
| collection | DOAJ |
| description | Abstract Background Transcription factor 4 (TCF4) is a member of the basic helix-loop-helix (bHLH) family of transcription factors that guides proper embryogenesis, particularly neurogenesis, myogenesis, heart development and hematopoiesis. The interaction of TCF4 with DNA is dependent on the presence of a conserved bHLH domain, particularly the presence of a basic (b) motif. Most mutations in the Tcf4 gene are either associated with the development of serious nervous system disorders, such as Pitt-Hopkins syndrome or schizophrenia, or are lethal. Although TCF4 is essential for the proper development and function of the human body, there is a lack of fundamental knowledge about the structure of TCF4 since structural studies were previously limited exclusively to its bHLH. Methods Recombinant full-length TCF4 was expressed in bacterial cells and purified using chromatographic techniques. To compare the properties of TCF4 in its apo and holo form, we determined the dissociation constant (KD) of the TCF4:DNA complex using independent methods, including fluorescence polarization (FP), electrophoretic mobility shift assay (EMSA), and fluorescence correlation spectroscopy (FCS). Then we compared the properties of TCF4 in its apo and holo form in relation to the changes of the conformation of the polypeptide chain (hydrogen/deuterium exchange mass spectrometry; HDX-MS), hydrodynamic properties (e.g., sedimentation-velocity analytical ultracentrifugation; SV-AUC), and stability (thermal shift, circular dichroism; CD). Results We demonstrate the molecular characteristics of TCF4, the dimer of which is one of the largest intrinsically disordered proteins (IDPs) described to date. According to our findings, the structure of TCF4 is extensively disordered. Only the bHLH domain exhibits a stable fold. Strikingly, Ephrussi-box (E-box) binding via the bHLH domain has no significant effect on the disordered nature of TCF4, but it does influence the dynamic of bHLH and stability of the protein. Conclusions We suggest that bHLH plays the role of an anchor localizing TCF4 to specific gene sequences. The dual nature of the TCF4 structure and the fact that the intrinsically disordered regions (IDRs) represent most of the protein sequence, suggest that TCF4 may act as a hub transcription factor regulating the expression of specific genes through the interaction of IDRs with gene-specific partners. |
| format | Article |
| id | doaj-art-c860d78a3f0c40ea9eb07d59b4b1b67c |
| institution | DOAJ |
| issn | 1478-811X |
| language | English |
| publishDate | 2025-03-01 |
| publisher | BMC |
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| series | Cell Communication and Signaling |
| spelling | doaj-art-c860d78a3f0c40ea9eb07d59b4b1b67c2025-08-20T02:49:27ZengBMCCell Communication and Signaling1478-811X2025-03-0123112010.1186/s12964-025-02154-7The molecular properties of the bHLH TCF4 protein as an intrinsically disordered hub transcription factorNikola Sozańska0Barbara P. Klepka1Anna Niedzwiecka2Lilia Zhukova3Michał Dadlez4Beata Greb-Markiewicz5Andrzej Ożyhar6Aneta Tarczewska7Department of Biochemistry, Molecular Biology and Biotechnology, Faculty of Chemistry, Wrocław University of Science and TechnologyLaboratory of Biological Physics, Institute of Physics, Polish Academy of SciencesLaboratory of Biological Physics, Institute of Physics, Polish Academy of SciencesInstitute of Biochemistry and Biophysics, Polish Academy of SciencesInstitute of Biochemistry and Biophysics, Polish Academy of SciencesDepartment of Biochemistry, Molecular Biology and Biotechnology, Faculty of Chemistry, Wrocław University of Science and TechnologyDepartment of Biochemistry, Molecular Biology and Biotechnology, Faculty of Chemistry, Wrocław University of Science and TechnologyDepartment of Biochemistry, Molecular Biology and Biotechnology, Faculty of Chemistry, Wrocław University of Science and TechnologyAbstract Background Transcription factor 4 (TCF4) is a member of the basic helix-loop-helix (bHLH) family of transcription factors that guides proper embryogenesis, particularly neurogenesis, myogenesis, heart development and hematopoiesis. The interaction of TCF4 with DNA is dependent on the presence of a conserved bHLH domain, particularly the presence of a basic (b) motif. Most mutations in the Tcf4 gene are either associated with the development of serious nervous system disorders, such as Pitt-Hopkins syndrome or schizophrenia, or are lethal. Although TCF4 is essential for the proper development and function of the human body, there is a lack of fundamental knowledge about the structure of TCF4 since structural studies were previously limited exclusively to its bHLH. Methods Recombinant full-length TCF4 was expressed in bacterial cells and purified using chromatographic techniques. To compare the properties of TCF4 in its apo and holo form, we determined the dissociation constant (KD) of the TCF4:DNA complex using independent methods, including fluorescence polarization (FP), electrophoretic mobility shift assay (EMSA), and fluorescence correlation spectroscopy (FCS). Then we compared the properties of TCF4 in its apo and holo form in relation to the changes of the conformation of the polypeptide chain (hydrogen/deuterium exchange mass spectrometry; HDX-MS), hydrodynamic properties (e.g., sedimentation-velocity analytical ultracentrifugation; SV-AUC), and stability (thermal shift, circular dichroism; CD). Results We demonstrate the molecular characteristics of TCF4, the dimer of which is one of the largest intrinsically disordered proteins (IDPs) described to date. According to our findings, the structure of TCF4 is extensively disordered. Only the bHLH domain exhibits a stable fold. Strikingly, Ephrussi-box (E-box) binding via the bHLH domain has no significant effect on the disordered nature of TCF4, but it does influence the dynamic of bHLH and stability of the protein. Conclusions We suggest that bHLH plays the role of an anchor localizing TCF4 to specific gene sequences. The dual nature of the TCF4 structure and the fact that the intrinsically disordered regions (IDRs) represent most of the protein sequence, suggest that TCF4 may act as a hub transcription factor regulating the expression of specific genes through the interaction of IDRs with gene-specific partners.https://doi.org/10.1186/s12964-025-02154-7Transcription factor 4 (TCF4)Immunoglobulin transcription factor 2 (ITF2)SL3-3 enhancer factor 2 (SEF2)Basic helix-loop-helix (bHLH)Intrinsically disordered regions (IDRs)Intrinsically disordered proteins (IDPs) |
| spellingShingle | Nikola Sozańska Barbara P. Klepka Anna Niedzwiecka Lilia Zhukova Michał Dadlez Beata Greb-Markiewicz Andrzej Ożyhar Aneta Tarczewska The molecular properties of the bHLH TCF4 protein as an intrinsically disordered hub transcription factor Cell Communication and Signaling Transcription factor 4 (TCF4) Immunoglobulin transcription factor 2 (ITF2) SL3-3 enhancer factor 2 (SEF2) Basic helix-loop-helix (bHLH) Intrinsically disordered regions (IDRs) Intrinsically disordered proteins (IDPs) |
| title | The molecular properties of the bHLH TCF4 protein as an intrinsically disordered hub transcription factor |
| title_full | The molecular properties of the bHLH TCF4 protein as an intrinsically disordered hub transcription factor |
| title_fullStr | The molecular properties of the bHLH TCF4 protein as an intrinsically disordered hub transcription factor |
| title_full_unstemmed | The molecular properties of the bHLH TCF4 protein as an intrinsically disordered hub transcription factor |
| title_short | The molecular properties of the bHLH TCF4 protein as an intrinsically disordered hub transcription factor |
| title_sort | molecular properties of the bhlh tcf4 protein as an intrinsically disordered hub transcription factor |
| topic | Transcription factor 4 (TCF4) Immunoglobulin transcription factor 2 (ITF2) SL3-3 enhancer factor 2 (SEF2) Basic helix-loop-helix (bHLH) Intrinsically disordered regions (IDRs) Intrinsically disordered proteins (IDPs) |
| url | https://doi.org/10.1186/s12964-025-02154-7 |
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