The role of charge, hydrophobicity, and cooperativity in target search of SOX2 and ESRRB

Transcription factors (TFs) locate and bind specific DNA sequences within a crowded nuclear environment, yet how their biophysical properties and interactions influence their search remains unclear. Here, we modulate hydrophobicity and charge of the pluripotency TFs SOX2 and ESRRB, and combine single-molecule imaging with genomic analyses to dissect their target search dynamics. We show that increased hydrophobicity impairs specific binding while promoting confined intranuclear interactions, whereas additional negative charges decrease sampling of weak binding sites. While SOX2 retains effective target site recognition despite decreased search efficiency, impairment of ESRRB search results in a global loss of genome occupancy and redirection to SOX2-bound regions, indicating a strong reliance of ESRRB on TF-TF interactions for locating its binding sites. Single molecule imaging analysis of ESRRB search upon SOX2 depletion demonstrated that SOX2 reduces ESRRB search time by restricting its nuclear diffusion and facilitating stable DNA binding. Together, our findings highlight how TF biophysical features and cooperativity jointly shape the efficiency and specificity of target search in the nucleus.