Extended Data Fig. 5: OSK motif readout on nucleosome arrays decipher combinatorial binding in early reprogramming.

a, Histograms showing size distribution of OSK nucleosome arrays (grey) compared to arrays targeted by O,S,K individually (blue, red, magenta, respectively). b, Read density heatmaps showing OCT4 ChIP-seq around OSK nucleosome arrays (left) or OCT4 lone-bound sites (right) in OSKM-48h and O-48h, spanning ±5 kb around the array midpoints. The nucleosome arrays were ranked ordered based on size and grouped into open (top panels) and closed (bottom panels) according to ATAC-seq in MEFs (not shown). The number of nucleosome arrays (n) are indicated. c, OCT4 and SOX2 bind specifically together to Fgf4 enhancer containing OCT/SOX composite motif. Super-shift EMSA showing three retarded bands when OSKM-48h nuclear lysates were incubated with Cy5-labelled oligonucleotide from Fgf4 enhancer. The bands correspond to OCT4-DNA, SOX2-DNA and OCT4/SOX2-DNA complexes. All three bands were diminished when excessive amounts of the specific (Fgf4) but not the non-specific competitor (P19) were added. Specific bands were also diminished when the corresponding antibodies were added. Representative image from (n = 3) biological replicates. Uncropped gels are shown in Supplementary Fig. 1. d, EMSA showing OCT4/SOX2-DNA complexes were formed only when OSKM-48h nuclear lysates were incubated with Fgf4 enhancer but not O-48h lysates. Representative image from (n = 2) biological replicates. Uncropped gels are shown in Supplementary Fig. 1. e, Histograms showing OSK motif frequency distribution within OSK nucleosome arrays. f, Profile plots showing SOX2 motif distribution around OSK nucleosome array borders, ranging in motif density from ≥ 4-to-7 motif/kb. Motif enrichment is measured in both strands separately, showing motif unidirectional orientation. The number (n) of nucleosome arrays with the different SOX2 motif densities are indicated. g, Venn diagram showing the overlap between OSK nucleosome arrays containing SOX2 motifs on the top and bottom strands. Only arrays with motif density ≥ 7 motif/kb and 0.8-2.8 kb in size are shown here. h, Heatmaps showing the OSK motif distribution patterns (logos on top) around nucleosome array midpoint (±5 kb) bound by OSK in early reprogramming within closed chromatin and contain ≥ 7 SOX2 motif/kb on the top strand. Motifs are scored on the top (red) and bottom (blue) strands as indicated by the colour gradient scale at the bottom. MNase heatmaps (purple) within the same OSK nucleosome arrays are shown on the left. The arrays were rank ordered based on size and those within 0.8-2.8 kb are indicated by arrowheads and dashed lines. The number (n) of the OSK nucleosome arrays is indicated.