Extended Data Fig. 6: Combinatorial binding to signpost elements guides OSK to pluripotency enhancers during reprogramming.

a, Line plots showing O,S,K motifs enrichment on the top (red) and bottom (blue) DNA strands around the midpoints of OSK nucleosome arrays (±5 kb) in fully reprogrammed iPS cells. The average array size highlighted in yellow with dotted lines at the borders. b, Histograms showing size distribution of OSK nucleosome arrays bound in final reprogramming (purple) compared to early reprogramming (grey). c, The early reprogramming OSK nucleosome arrays are adjacent to enhancers in iPS cells. Bar plot showing the distance between OSK nucleosome arrays in early reprogramming and enhancers in iPSCs (represented by a schematic in the inset). The experimental distance (actual) was compared to random sequences. Two-sided Wilcoxon rank-sum test with continuity correction: w = 10,696,640,768 and P = 2.286 × 10⁻⁶. d, OSK nucleosome arrays in early reprogramming are silenced in iPS cells. Line plots showing that histone marks and co-factors associated with heterochromatin are enriched within the early OSK nucleosome arrays (highlighted in yellow) after the completion of reprogramming. The GEO access codes of the data used are indicated. e, Same as (d) for histone marks associated with active chromatin. f, The expression of eGFP and tdTomato were measured by flow cytometry during reprogramming, showing motif directionality in Nanog signpost element is important for gene reactivation. g, eGFP and tdTomato expression gradually increases in iPSCs after passaging. The expression of eGFP and tdTomato were measured by flow cytometry in iPSCs after different passages. h, Bar plot quantifying eGFP+ve and tdTomato+ve cells during reprogramming as measured in (g). Data are mean ± s.d. from biological replicates (n = 3). i, Fluorescence images of iPSC colony after passage four. Bright field (BF) and merged images are also shown. Representative image from n = 3 biological replicates. Scale bar = 100 µm.