Extended Data Fig. 10: Competitive interaction between EOMES and ESRRB for TFAP2C/MYC on nucleosome arrays expands GETMR reprogramming.

a, Bar plot of GETM peaks identified by ChIP-seq in GETM-48h and GETMR-48h cells showing a significant loss of TFAP2C and MYC sites in the presence of ESRRB. b, ChIP-seq read density heatmaps (blue) of GETM in GETM-48h cells and GETMR in GETMR-48h cells spanning ±1 kb from TFAP2C summits of retained (top) versus lost sites (bottom) when comparing GETM-48h to GETMR-48h cells. Sites are sorted based on the central enrichment of TFAP2C in GETM-48h cells. The average enrichment of the corresponding TFs in retained (solid line) versus lost (dotted line) TFAP2C sites are shown above. The number (n) of sites are indicated in the left. Colour scale indicated indicate normalized ChIP-seq enrichment (RPGC). c, Genome browser tracks of representative loci containing a TFAP2C retained or lost site, showing GETM and GETMR enrichment (ChIP-seq) in GETM-48h and GETMR-48h cells, respectively, and chromatin accessibility (ATAC-seq) in MEFs, GETM-72h and GETMR-72h cells. d, Bar plots showing the percentage of TFAP2C sites bound individually or co-bound with other TFs. e, Profile plots of TFAP2C and ESRRB motif distribution around the dyad of nucleosomes bound by ESRRB overlapping with or away from TFAP2C retained sites (left and right panels, respectively) in GETMR-48h cells. f, Micro-C pile-up heatmaps around nucleosome arrays containing retained (left) or lost (right) TFAP2C sites after adding ESRRB to GETM during early reprogramming. Maps plotted at 100 bp resolution. Yellow arrowheads indicate local cross-interactions. Schematic on top showing the co-binding of ESRRB and TFAP2C mediated by inter-nucleosome interactions. g, Immunoprecipitation of EOMES or ESRRB and western blot for TFAP2C in the presence of constant EOMES and increasing ESRRB (left) or constant ESRRB and increasing EOMES (right). The amount of each transfected plasmid encoding the corresponding TF is shown below. The bands representing TFAP2C, and antibody heavy chain (IgG-HC) are indicated. Representative image from n = 2 biological replicates. Molecular weight marker (KDa) is indicated. Raw blots are shown in Supplementary Fig. 1. h, Bar plot showing the reprogramming efficiency of MEFs to iPS cells using OSKM and TMR. Mean values of biological replicates (n = 4) with error bars representing ± s.d. i, Immunofluorescence of the indicated pluripotency markers (green fluorescence) in stable TMR iPS clonal lines. Bright field images showing typical morphology of the corresponding TMR-iPS cells. Nuclear DAPI staining (Blue) images are also shown. Representative image from n = 6 independent iPS clones shown in (j). Scale-bar = 100 µm. j, Gene expression of pluripotency markers by q-PCR from six independent TMR-iPS clones (n = 6) as compared to MEFs (negative control) and ESCs (positive control). Mean values of technical replicates (n = 2) with error bars representing ± s.d. k, Two stable TMR-iPS clonal lines carrying a tdTomato reporter in Rosa26 locus were used for chimera assays. Both lines display significant chimeric contribution as measured by tdTomato fluorescence across the whole embryo, which was equivalent the ES cells counterparts. Non-chimeric embryos were used as negative control. l, Sketch illustrating guided search model of TF combinatorial binding to signpost elements. In a standard random sampling, TFs transiently interact with low-affinity sites (dotted arrows), searching for gene regulatory targets (red bullseye). In guided search model, chromatin loops displaying oriented OSKM motifs (blue arrows) or loop junctions condensing GET/R motifs act as signposts that direct TF binding to their target enhancers.