**3. Discussion**

Sox2 is a tightly regulated transcription factor; both significant increases and decreases in Sox2 dosage can be detrimental to its biological function [48,49]. Alterations in Sox2 dosage result in multiple developmental and acquired disorders [50–54]. We show that the Sox2 HMG box can induce concentration-dependent alternate DNA bends (Figure 3). Alternate promoter bends are likely to regulate genes differentially and initiate downstream cascades crucial for Sox2's diverse functions. Our results provide a mechanism for Sox2's strict dosage dependence in its function-dysfunction dichotomy [50,55–58].

**Figure 3.** Schematic representation of the Sox2 stoichiometry-dependent dsDNA bending transitions.

In summary, our smFRET experiments clearly demonstrate the role of Sox2 dosage in modulating the conformational landscape of HMG box-binding DNA motifs. Previous studies on Sox family members suggested that heterodimeric homeodomain TFs can induce sequential bending as they interact with their DNA partners [59–62]. Here, we utilize the strengths of smFRET to demonstrate that a representative sequence-specific HMG-TF alone induces concentration-dependent multistep DNA bending transitions. We envision additional layers of tunability for heteromeric HMG-TFs in respective regulatory complexes where affinities of individual transcription factors for DNAs as well as inter-TF interactions can vary dramatically.
