Fig. 3: Signpost elements guide OSK binding to pluripotency enhancers during reprogramming.

a, Genome browser screenshot of the Nanog locus bound by OSK (ChIP–seq) in early (top) and final reprogramming (bottom). A schematic of the Nanog promoter (pro.) and enhancer (enh.) separated by a signpost element, with the directionality of OSK motifs indicated by chevrons, is shown below. b, Schematic of the PiggyBac (PB) construct containing the dual eGFP/tdTomato reporter cassettes. eGFP is driven by the WT Nanog promoter–signpost–enhancer shown in a, and tdTomato is driven by the same promoter–enhancer but separated by a flipped signpost element. ITR, inverted terminal repeat. c, Experimental flow chart illustrating PB construct integration into ES cells, which contributed to chimeric embryos from which MEFs were derived; these cells were then used for iPS reprogramming to examine the reactivation of the dual reporters. d, The expression of eGFP and tdTomato in ES cells targeted by the PB construct was measured using flow cytometry and the percentages of eGFP⁺ and tdTomato⁺ cells are indicated. FL8, fluorescence channel 8 (non-specific channel). e, Expression of eGFP and tdTomato in the male gonad isolated from chimeric embryos at E13.5, reflecting Nanog expression. Representative image from n = 3 biological replicates. Scale bar, 100 µm. f, Motif directionality in the signpost element leads to more efficient eGFP activation during reprogramming. Quantification of eGFP⁺ and tdTomato⁺ cells during reprogramming is shown, as measured using flow cytometry. Statistical significance was determined using two-sided paired t-tests; *P = 0.03, **P = 0.01, ***P < 0.001. Data are mean ± s.d. from three biological replicates (n = 3). g, eGFP expression precedes tdTomato in reprogramming. Fluorescence images of an iPS cell colony showing expression of eGFP and tdTomato at day 15 followed by 4 days without doxycycline (dox.). Bright-field (BF) and merged images are also shown. Representative image from n = 3 biological replicates. Scale bar, 100 µm.