Intrinsically disordered bHLH family member TCF4 drives phase separation in a DNA-dependent manner.

Transcription factor 4 (TCF4), a class I basic helix-loop-helix (bHLH) protein, plays a critical role in neurodevelopment and is linked to disorders such as Pitt-Hopkins syndrome and schizophrenia. As an intrinsically disordered transcription factor, TCF4 interacts with various partners, serving as a hub in multicomponent transcriptional complexes. A growing body of evidence links intrinsically disordered regions of transcription factors to liquid-liquid phase separation (LLPS) in transcriptional regulation. It was demonstrated that in the nucleus, a deletion mutant of the canonical TCF4 isoform forms puncta resembling liquid-like condensates. These are reminiscent of patterns observed in disease-associated mutants, however, such observations remain limited, and nothing is known about the ability of class I bHLH proteins to form liquid condensates. Our analyses demonstrated that the brain-predominant isoform I‾(TCF4 I‾) undergoes LLPS in vitro. LLPS is promoted by increased ionic strength and molecular crowding, resulting in dynamic, liquid-like condensates that coalesce and show rapid fluorescence recovery after photobleaching. Over time, condensates mature, exhibiting increased viscosity while retaining molecular mobility. Importantly, we show that specific and non-specific DNA sequences dissolve TCF4 I‾condensates, suggesting that DNA binding interfaces with multivalent interactions necessary for phase separation. Given the existence of multiple TCF4 isoforms differing in the length and composition of their IDRs, it will be important to conduct comparative studies to fully understand the molecular determinants of TCF4's phase separation behavior and its functional consequences. This study provides a solid and strong foundation for future investigations, which may ultimately shed light on the molecular basis of TCF4-related pathologies.