Intrinsically disordered bHLH family member TCF4 drives LLPS in a DNA-dependent manner

Transcription factor 4 (TCF4), a basic helix-loop-helix (bHLH) protein, plays a critical role in neurodevelopment and is genetically linked to disorders such as Pitt-Hopkins syndrome (PTHS) and schizophrenia. While TCF4 is known to function through sequence-specific DNA binding, the influence of its intrinsically disordered regions (IDRs) on its molecular behavior remains scattered. The TCF4 deletion mutant, lacking a fragment of the bHLH domain, forms puncta in the nucleus, which are reminiscent of patterns observed in disease-associated TCF4 mutants. Our in vitro analyses have demonstrated that the shortest human isoform of TCF4 (TCF4-I-), which is predominant in the brain and contains extensive IDRs, undergoes liquid-liquid phase separation (LLPS) in vitro. LLPS is promoted by increased ionic strength and molecular crowding, resulting in dynamic, liquid-like condensates that coalesce and display rapid fluorescence recovery after photobleaching (FRAP). Over time, these condensates mature, exhibiting increased viscosity while retaining molecular mobility. Importantly, we show that both specific and non-specific DNA sequences dissolve TCF4 condensates. This suggests that DNA binding competitively inhibits the multivalent interactions necessary for phase separation. These findings indicate that LLPS driven by TCF4 is a reversible process, likely involved in transcriptional activation bursts. We propose that mutations that disrupt DNA binding or localization may trap TCF4 in aberrant condensates, contributing to PTHS pathogenesis through dysregulated phase behavior. Our findings offer a mechanistic link between TCF4 structural features, phase separation, and its transcriptional function, providing novel insight into how mutations in this TF may drive neurological disease through aberrant biomolecular condensation.