Extended Data Fig. 7. Histone modifications modulate OCT4 and SOX2 cooperativity.

a) Left, representative native gel electrophoresis showing OCT4 binding to the LIN28B or H3K27me3 LIN28B nucleosomes. Colored asterisks indicate the number of OCT4 molecules bound to the nucleosome: red, 1 OCT4; blue, 2 OCT4; and green, 3 OCT4. Right, quantification of OCT4 binding to H3K27me3 LIN28B relative to unmodified LIN28B, data shown as s.e.m. of 4 independent experiments. Bands marked with * were used for quantification. b) Left, quantification of Mnl I digestion of free and OCT4-bound nucleosomes (unmodified, H3K27ac and H3K27me3). The y-axis shows intensity of nucleosome bands after enzyme digestion, normalized to the input (without enzyme). Data are mean and s.e.m. of 4 independent experiments. Representative gels are shown in Extended Data Fig. 6d. Right, comparison of Mnl I digestion between unmodified and H3K27me3 OCT4 bound nucleosomes. c) Quantification of sequencing of Mnase I digested OCT4-bound nucleosomes (unmodified or H3K27me3). The y-axis shows fraction of nucleosome size reads starting at defined position, the x-axis shows position of the first base pair relative to the most abundant position (0 as observed in the structure). Data are mean and spread of 2 independent experiments. d) Model of OCT4 bound to the LIN28B nucleosome with SOX2 binding site shown in blue. OCT4 bound to OBS1 is in solid green; OCT4 structure was superimposed on OBS2 and on OBS3. SOX2 binding site and SOX2 model are shown in blue. e) A representative native gel from >6 independent experiments stained for DNA showing SOX2 and OCT4 binding to nucleosome. f) Left, native gel stained for DNA showing SOX2 and OCT4 binding to nucleosome. Center, western blot with anti-H3 showing presence of histones in the complexes. Right, western blot with anti-Sox2 showing presence of Sox2 in these complexes. Each experiment has been replicated >3 times. g) Left, representative native gel electrophoresis showing OCT4 and SOX2 binding to the LIN28B nucleosome. OCT4 and SOX2 were mixed and added to nucleosomes as indicated. SOX2 binding to nucleosome was validated by western blot analyses (Extended Data Fig. 7f). Colored asterisks indicate the molecules bound to the nucleosome: black, nucleosome alone; red, 1 OCT4; gray, 1 SOX2; orange, 1 OCT4 and 1 SOX2. Right, quantification of SOX2 binding, with data shown as mean and s.e.m. of 5 independent experiments; ** p = 0.003, one-sided Student’s t-test. To assess SOX2 binding to OCT4-bound nucleosome, we used 1-OCT4-SOX2 (orange asterisk) and 1-OCT4 (red asterisk) bands; to assess SOX2 binding to nucleosome, we used SOX2-bound nucleosome (gray asterisk) and input nucleosome (black asterisk) bands. See Methods and quantification data in Supplementary Table 3.