VGLL4-driven TEAD4 multimerization orchestrates DNA binding and YAP recruitment

The Hippo pathway, a highly conserved kinase signaling cascade, is central to regulating cell growth and proliferation, tissue homeostasis, and organ development. As downstream effectors of this pathway, TEAD1-4 proteins serve as sequence-specific transcription factors in mammals. They collaborate with cofactors, such as VGLL and YAP/TAZ, to modulate gene expression, thereby controlling diverse cellular processes. Here, employing fluorescence-combined optical tweezers, we demonstrate that monomeric TEAD4 binds to consensus motifs with association rates significantly higher than nonspecific DNA, while the dissociation rates are fast and comparable. Yet, TEAD4, through multimerization, gains multiple DNA binding sites, supporting elongated DNA residence time and YAP recruitment. Moreover, both YAP and VGLL4 can promote TEAD4 multimerization and strengthen its DNA binding and sequence specificity. Unexpectedly, the presence of two Tondu domains in VGLL4 elicits a stoichiometry-dependent effect on YAP recruitment to DNA-bound TEAD4: A low VGLL4:TEAD4 molar ratio enhances this process, whereas a high ratio inhibits it. These findings offer a dynamic understanding of how a eukaryotic TF interacts with DNA and underscore a distinct molecular mechanism by which VGLL4 modulates TEAD4-mediated YAP recruitment in the Hippo pathway.