The molecular properties of the bHLH TCF4 protein as an intrinsically disordered hub transcription factor

Transcription factor 4 (TCF4) is a member of the basic helix-loop-helix (bHLH) family of transcription factors and guides proper embryogenesis, particularly neurogenesis, myogenesis, heart development and hematopoiesis. bHLH proteins exhibit variations in length, expression patterns and the presence of defined motifs. 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, apart from the isolated bHLH domain, there is a lack of fundamental knowledge about the structure of TCF4.

Recombinant full-length TCF4 was expressed in E. coli cells and purified using chromatographic techniques. To determine the conformation of the polypeptide chain of TCF4, we performed in vitro analysis using hydrogen/deuterium exchange mass spectrometry (HDX-MS). We then 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). Finally, we compared the properties of TCF4 in its apo and holo form in relation to the changes of the conformation of the polypeptide chain (HDX-MS), hydrodynamic properties (e.g., sedimentation-velocity analytical ultracentrifugation; SV-AUC), and stability (thermal shift, circular dichroism; CD).

In this study, we demonstrate the molecular characteristics of full-length 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, which comprises 10% of the polypeptide chain, 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 stability of TCF4.

We suggest that bHLH plays the role of an anchor localizing TCF4 to specific gene sequences. The dual nature of the TCF4 structure, as well as the fact that the intrinsically disordered regions (IDRs) represents most of the protein sequence, suggests that TCF4 may act as a hub transcription factor regulating the expression of specific genes through the interaction of IDRs with gene-specific partners. Competing Interest Statement The authors have declared no competing interest. List of abbreviations - ASCL1: - Achaete-scute homolog 1 - ATOH1: - Atonal bHLH transcription factor 1 - bHLH: - Basic helix-loop-helix - CaM - Calmodulin - CD: - Circular dichroism - cv: - Column volumes - DOL: - Degree of labeling - dsDNA: - Double-stranded DNA - dTCF4: - Dimeric TCF4 - E-box: - Ephrussi box - EMSA: - Electrophoretic mobility shift assay - FAM: - 6-carboxy-fluorescein - FCS: - Fluorescence correlation spectroscopy - FP: - Fluorescence polarization - HDX-MS: - Hydrogen/deuterium exchange mass spectrometry - HES: - Hairy and enhancer of split - ID: - Intellectual disability - ID-2: - DNA-binding protein inhibitor ID-2 - IDPs: - Intrinsically disordered proteins - IDRs: - Intrinsically disordered regions - IMAC: - Immobilized metal affinity chromatography - ITF2: - Immunoglobulin transcription factor 2 - KD: - Dissociation constant - MW: - Molecular weight - PAS: - Per-ARNT-Sim - PTHS: - Pitt-Hopkins syndrome - Rh: - Hydrodynamic radius - SEC: - Size exclusion chromatography - SEF2: - SL3-3 enhancer factor 2 - SV-AUC: - Sedimentation-velocity analytical ultracentrifugation - TCF4: - Transcription factor 4 - TF: - Transcription factor