Interplay between minor and major groove-binding transcription factors Sox2 and Oct1 in translocation on DNA studied by paramagnetic and diamagnetic NMR

The pathways whereby Sox2 scans DNA to locate its specific binding site are investigated by NMR in specific and nonspecific Sox2·DNA complexes and in a specific ternary complex with Oct1 on the Hoxb1 regulatory element. Direct transfer of Sox2 between nonspecific sites on different DNA molecules occurs without dissociation into free solution at a rate of ～10(6) M(-1) s(-1), whereas one-dimensional sliding proceeds with a diffusion constant of ≥0.1 μm(2)·s(-1). Translocation of Sox2 from one specific DNA site to another occurs via jumping, involving complete dissociation into free solution (k(d) ～5-6 s(-1)) followed by reassociation (k(a) ～5 × 10(8) M(-1) s(-1)). In the presence of Oct1 bound to an adjacent specific site, k(d) is reduced by more than 10-fold. Paramagnetic relaxation measurements, however, demonstrate that sparsely populated (<1%), transient states involving nonspecifically bound Sox2 in rapid exchange with specifically bound Sox2 are sampled in both binary Sox2·DNA- and ternary Oct1·Sox2·Hoxb1-DNA-specific complexes. Moreover, Sox2 modulates the mechanism of translocation of Oct1. Both Sox2 and the Oct1 POU(HD) domain are transiently released from the specific ternary complex by sliding to an adjacent nonspecific site, followed by direct transfer to another DNA molecule, whereas the Oct1 POU(S) domain is fixed to its specific site through direct interactions with Sox2. Intermolecular translocation of POU(HD) results in the formation of a bridged intermediate spanning two DNA molecules, enhancing the probability of complete intermolecular translocation of Oct1. By way of contrast, in the specific Oct1·DNA binary complex, POU(S) undergoes direct intermolecular translocation, whereas POU(HD) scans the DNA by sliding.     

Tethered-Hopping Model for Protein-DNA Binding and Unbinding Based on Sox2-Oct1-Hoxb1 Ternary Complex Simulations

The sliding and hopping models encapsulate the essential protein-DNA binding process for binary complex formation and dissociation. However, the effects of a cofactor protein on the protein-DNA binding process that leads to the formation of a ternary complex remain largely unknown. Here we investigate the effect of the cofactor Sox2 on the binding and unbinding of Oct1 with the Hoxb1 control element. We simulate the association of Oct1 with Sox2-Hoxb1 using molecular dynamics simulations, and the dissociation of Oct1 from Sox2-Hoxb1 using steered molecular dynamics simulations, in analogy to a hopping event of Oct1. We compare the kinetic and thermodynamic properties of three model complexes (the wild-type and two mutants) in which the Oct1-DNA base-specific interactions or the Sox2-Oct1 protein-protein interactions are largely abolished. We find that Oct1-DNA base-specific interactions contribute significantly to the total interaction energy of the ternary complex, and that nonspecific Oct1-DNA interactions are sufficient for driving the formation of the protein-DNA interface. The Sox2-Oct1 protein-protein binding interface is largely hydrophobic, with remarkable shape complementarity. This interface promotes the formation of the ternary complex and slows the dissociation of Oct1 from its DNA-binding site. We propose a simple two-step reaction model of protein-DNA binding, called the tethered-hopping model, that explains the importance of the cofactor Sox2 and may apply to similar ternary protein-DNA complexes.     

Impact of Protein/Protein Interactions on Global Intermolecular Translocation Rates of the Transcription Factors Sox2 and Oct1 between DNA Cognate Sites Analyzed by z-Exchange NMR Spectroscopy

Oct1 and Sox2 synergistically regulate developmental genes by binding to adjacent sites within promoters. We have investigated the kinetics of global intermolecular translocation of Sox2 and Oct1 between cognate sites located on different DNA molecules by z-exchange NMR spectroscopy. In the Hoxb1 promoter, the Sox2 and Oct1 sites are immediately adjacent to one another, and the intermolecular translocation rates are too slow to be measured by z-exchange spectroscopy. By introducing a 3-bp insertion between the Sox2 and Oct1 sites to mimic the spacing in the FGF4 enhancer, the interprotein contact surface is reduced, and the translocation rates are increased. Interaction between Sox2 and the POU-specific domain (POU(S)) of Oct1 does not affect the translocation mechanism but modulates the rates. Translocation involves only jumping (dissociation and reassociation) for Sox2, but both jumping and direct intersegment transfer (no dissociation into free solution) for Oct1. The dissociation (k(off) ～1.5 s(-1)) and association (k(on) ～5.1 × 10(9) m(-1)s(-1)) rate constants for Sox2 are reduced 4-fold and increased 5-fold, respectively, in the presence of Oct1. k(off) (～3.5 s(-1)) for Oct1 is unaffected by Sox2, whereas k(on) (～1.3 × 10(9) m(-1)s(-1)) is increased ～13-fold. The direct intermolecular translocation rate (k(inter) ～1.8 × 10(4) m(-1)s(-1)) for the POU(S) domain of Oct1 is reduced 2-fold by Sox2, whereas that for the POU homeodomain (POU(HD)) of Oct1 (k(inter) ～ 1.7 × 10(4) m(-1)s(-1)) remains unaltered, consistent with the absence of contacts between Sox2 and POU(HD). The data suggest a model for the sequence of binding events involved in synergistic gene regulation by Sox2 and Oct1.