Unveiling the Formation and Stability of the Droplet Phase of the Thyroid Transcription Factor TTF-1HD/dsDNA Complex under Cell-like Crowding Conditions

Abstract Liquid−liquid phase separation (LLPS) of protein−nucleic acid systems plays a key role in gene expression and regulation, yet the molecular mechanisms governing transcription factor condensation remain poorly understood. Using complementary biophysical techniques, we investigated the ability of TTF1-Helix III, a peptide derived from the DNA-binding homeodomain of thyroid transcription factor 1, to form biomolecular condensates with its cognate 14 bp dsDNA under cell-like crowding conditions. By varying concentration, pressure, and temperature, we identified the key molecular determinants driving phase separation. TTF1-Helix III first forms a high-affinity, reversible complex with dsDNA, followed by droplet formation through weak multivalent interactions between peptide/dsDNA complexes when DNA negative charges are balanced by peptide positive charges. The resulting condensates remain stable up to the DNA melting temperature and resist pressures up to 1.4 kbar. These findings demonstrate that the DNA-binding domain of a transcription factor is sufficient to drive condensate formation with its target DNA.