TCF4 is one of the key transcription factors – that is, proteins that regulate gene expression. It plays a crucial role in the development of the nervous system, muscles, heart, and blood-forming tissues. Mutations in the Tcf4 gene (which encodes the TCF4 protein) are associated with a range of disorders, including neurodevelopmental conditions such as Pitt-Hopkins syndrome, schizophrenia, and autism. Despite its major biological significance, the properties of the full-length TCF4 molecule have remained poorly understood, as previous studies have focused only on its small, structured fragment responsible for DNA binding, known as the basic helix-loop-helix (bHLH) domain.

In our recent study, carried out in collaboration with colleagues from Wrocław University of Science and Technology and the Institute of Biochemistry and Biophysics of the Polish Academy of Sciences (Cell Communication and Signaling 23, 2025), we provided the first characterization of the full-length TCF4 protein. Our findings show that nearly the entire polypeptide chain lacks a defined spatial structure, classifying TCF4 as an intrinsically disordered protein (IDP). Only a small segment – the bHLH domain – adopts a stable structure, enabling DNA sequence recognition. This structural organization allows TCF4 to function as a “molecular anchor”: the stable bHLH domain tethers the protein to specific gene sequences, while the remaining intrinsically disordered regions, due to their flexibility, can freely interact with various molecular partners within the cell. TCF4 is one of the largest structurally disordered proteins ever studied and is the second-largest IDP described to date.

Using advanced biophysical techniques, including hydrogen-deuterium exchange mass spectrometry (HDX-MS), analytical ultracentrifugation (AUC), and circular dichroism spectroscopy (CD), the team demonstrated that TCF4 forms stable dimers. Interestingly, even when bound to DNA, the protein still retains its disordered character, with only the bHLH domain becoming stabilized. This selective stabilization suggests that DNA binding influencesthe dynamics of specific regions of the protein, which may be critical for gene regulation.

A particularly interesting part of the study involved the use of fluorescence correlation spectroscopy (FCS), a technique that allows researchers to observe single molecules diffusing in solution and track their behaviour. We measured how long fluorescently labelled DNA molecules diffuse in a microscopic volume of about one femtolitre (i.e., quadrillionth of a litre) in the presence of TCF4. Based on the diffusion times, we determined the strength of their interaction that leads to complexformation. This technique revealed that TCF4 binds DNA with high affinity (with a dissociation constant in the low submicromolar range), despite most of its chain remaining in a disordered state. This is a rare example of using FCS to study such a large and flexible protein polymer.

Our findings indicate that TCF4 functions as a flexible molecular “hub” that integrates multiple cellular signals and regulates the activity of numerous genes. This may help explain why its mutations are associated with such a broad spectrum of neurological disorders and contribute to the development of more effective therapies.