Sozańska Nikola, Klepka Barbara P, Niedzwiecka Anna, Zhukova Lilia, Dadlez Michał, Greb-Markiewicz Beata, Ożyhar Andrzej, Tarczewska Aneta
Department of Biochemistry, Molecular Biology and Biotechnology, Faculty of Chemistry, Wrocław University of Science and Technology, Wybrzeże Wyspiańskiego 27, Wrocław, 50-370, Poland.
Laboratory of Biological Physics, Institute of Physics, Polish Academy of Sciences, Aleja Lotnikow 32/46, Warsaw, PL-02668, Poland.
Cell Commun Signal. 2025 Mar 27;23(1):154. doi: 10.1186/s12964-025-02154-7.
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.
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 (K) 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).
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.
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.
转录因子4（TCF4）是转录因子基本螺旋-环-螺旋（bHLH）家族的成员，其指导正常胚胎发育，尤其是神经发生、肌肉生成、心脏发育和造血作用。TCF4与DNA的相互作用依赖于保守的bHLH结构域的存在，特别是碱性（b）基序的存在。Tcf4基因中的大多数突变要么与严重神经系统疾病的发展相关，如皮特-霍普金斯综合征或精神分裂症，要么是致死性的。尽管TCF4对人体的正常发育和功能至关重要，但由于之前的结构研究仅局限于其bHLH结构域，因此缺乏关于TCF4结构的基础知识。
重组全长TCF4在细菌细胞中表达，并使用色谱技术进行纯化。为了比较TCF4的无辅基形式和全蛋白形式的性质，我们使用包括荧光偏振（FP）、电泳迁移率变动分析（EMSA）和荧光相关光谱（FCS）在内的独立方法测定了TCF4:DNA复合物的解离常数（K）。然后，我们比较了TCF4的无辅基形式和全蛋白形式在多肽链构象变化（氢/氘交换质谱法；HDX-MS）、流体动力学性质（如沉降速度分析超离心法；SV-AUC）和稳定性（热位移、圆二色性；CD）方面的性质。
我们展示了TCF4的分子特征，其作为二聚体是迄今为止所描述的最大的内在无序蛋白（IDP）之一。根据我们的发现，TCF4的结构广泛无序。只有bHLH结构域表现出稳定的折叠。引人注目的是，通过bHLH结构域与Ephrussi盒（E-box）结合对TCF4的无序性质没有显著影响，但确实影响bHLH的动力学和蛋白质的稳定性。
我们认为bHLH起到将TCF4定位到特定基因序列的锚定作用。TCF4结构的双重性质以及内在无序区域（IDR）代表了大部分蛋白质序列这一事实，表明TCF4可能作为一个中枢转录因子，通过IDR与基因特异性伴侣的相互作用来调节特定基因的表达。
