*2.1. Multiple Sox2HMG Domains Cooperatively Interact with dsDNANANOG*

In our initial ensemble experiments, we observe that Sox2HMG cooperatively binds to the *NANOG* composite promoter (DNANANOG; Figure 1). We utilized fluorescence anisotropy to detect Sox2HMG binding to dsDNANANOG (Supplementary Methods). Anisotropy reports on fluorophore rotational properties, dependent on both probe local and global environment perturbations; fluorescence anisotropy of labeled macromolecule usually increases upon ligand binding. To characterize Sox2HMG-DNA binding, we singly-labeled dsDNANANOG with Alexa Fluor 647 (Supplementary Methods) and monitored changes in DNA fluorescence anisotropy with increasing Sox2HMG concentrations (Figure 1a). Nonlinear least squares (NLS) fitting of the anisotropy data to a Hill equation yields an apparent dissociation constant (*K*D) of 15.1 (±2.0) nM and Hill coefficient of 1.5 (±0.3). The estimated *K*<sup>D</sup> is similar to that previously reported for specific DNA-Sox2 interactions [18]. A Hill coefficient greater than 1 indicates that multiple Sox2 HMG boxes bind to the DNA in a TF concentration-dependent fashion [24]. Anisotropy measurements also indicate that Sox2HMG alone (i.e., the DNA-binding domain in the absence of dsDNANANOG) fails to dimerize/oligomerize (Figure S1). To verify the binding of multiple Sox2 molecules to DNANANOG, we carried out fluorescence electrophoretic mobility shift assay (fEMSA) of DNA with increasing [Sox2HMG]. The fEMSA micrograph shows concentration-dependent appearance of multiple electrophoretic species (Figure 1b). This suggests a multistep Sox2HMG interaction with the *NANOG* proximal promoter. The non-equilibrium nature of mobility shift assays, however, precludes precise estimation of binding affinities of individual Sox2-DNA assemblies on the basis the fEMSA micrograph [25].

**Figure 1.** Sox2 cooperatively binds to the *NANOG* upstream promoter (DNANANOG). (**a**) DNA binding of Sox2HMG was probed by monitoring changes in fluorescence anisotropy of Alexa Fluor 647-labeled dsDNA with increasing [Sox2HMG]. The solid line represents nonlinear least squares (NLS) fit of the data to a Hill equation. NLS-derived parameters: *K*<sup>D</sup> = 15.1 (±2.0) nM, Hill coefficient = 1.5 (±0.3). (**b**) Fluorescence electrophoretic mobility assay (fEMSA) of Sox2HMG-DNANANOG binding suggests a multistep Sox2HMG complex formation with dsDNANANOG involving multiple protein molecules that are able to bind the DNA partner. (See also Figure S2.)
