Extended Data Fig. 6: EMSAs using SOX2, and wild-type or mutant SOX11 in complex with DNA or nucleosomes.
From: Nucleosome-bound SOX2 and SOX11 structures elucidate pioneer factor function
Related to Figs. 1–3. EMSAs reveal the formation of SOX-factor complexes with DNA or nucleosomes. All experiments were repeated at least twice with similar results. For gel source data, see Supplementary Fig. 1. a, EMSA of DNA-1 or nucleosome-1 with wild-type SOX2. b, EMSA of DNA-1 or nucleosome-1 with wild-type SOX11. c, EMSA of DNA-1 or nucleosome-1 with SOX11(H75A/S80A). d, EMSA of DNA-1 or nucleosome-1 with SOX11(Y118A). Relevant bands are labelled. To observe a wider range of the binding curve for the mutants, a larger amount of glycerol (12% final concentration) was used here (as compared to the EMSAs shown in Extended Data Fig. 3, which used 5% glycerol)—thus, the apparent affinity is higher. DNA or nucleosome concentration is 1.1 nM. e, EMSAs reveal formation of SOX–DNA or SOX–nucleosome complexes at different concentrations of salt. There is virtually no difference in binding at 10 or 30 mM NaCl, whereas binding is weaker at 150 mM NaCl. DNA or nucleosome concentration is 1.1 nM. f, EMSAs reveal formation of SOX complexes with DNA-1a or nucleosome-1a (only one canonical motif present) as compared to DNA-1b and nucleosome-1b (in which all canonical SOX motifs were mutated). SOX concentrations ranged from 0 to 200 nM, DNA concentration was 1.1 nM. g, Superimposition of two nucleosome–SOX11 cryo-EM maps obtained in the presence of 1 mM MgCl2 (grey density) or in the absence of MgCl2 (coloured). Magnesium does not influence the structure of the SOX–nucleosome complex. Cross-correlation between the two unmasked maps is 0.94 (Chimera).
