Fig. 6: NANOG PrD assembly mediates distant DNA–DNA contacts by Hi-C 3.0 analyses.
a, P(s) curves showing the probability of contact in relation to genomic distance in HEK 293T cells expressing either NANOG WT (red) or W8A (green). b, Contact heatmaps showing normalized interaction frequencies (20-kb bin) in one example region (chr7: 15–30 Mb). c, Magnified view of a region (chr7: 26–27.5 Mb) that hosts TAD structures. d, Diagram explaining the strategies in calculating pairwise DNA contacts between NANOG binding sites (Methods; adopted from a previous method, paired-end spatial chromatin analysis (PE-SCAn)). Black ovals indicate NANOG, and a and b indicate two distant genomic bins (25 kb) harbouring NANOG binding sites. Sliding windows of 25 kb were used to scan each site of a or b for 250 kb, and the interactions between each sliding window bin next to a or b were calculated as the background interactions surrounding specific a–b interactions. For any NANOG binding sites (a1–an versus b1–bn), the aggregated interactions between each pair of sites and nearby background are shown below. e, DNA contact strength under two conditions (WT or W8A) between WT only sites (n = 1,979), shared sites (n = 1,152) or W8A only sites (n = 2,577) (high-density NANOG binding cluster with ≥2 peaks in 25 kb). (i) Data plots based on the PE-SCAn method. (ii) Box plots showing quantitative counts of the central peaks shown in (i). The boxplot centre lines represent medians, the box limits indicate the 25th and 75th percentiles, and the whiskers extend 1.5 times the interquartile range (IQR) from the 25th and 75th percentiles. P values are based on two-sided paired Student’s t-tests. (iii) Cartoon diagrams based on DNA contacts. f, Example of Hi-C 3.0 DNA contacts between NANOG binding sites. (i)–(vi) Representative genomic loci showing DNA–DNA contacts formed between NANOG binding sites in Hi-C 3.0 (arc plots). From the top to the bottom, the data shown are (i,ii) NANOG ChIP-seq (either WT or W8A), (iii,iv) DNA interaction arc plots (either WT in purple or W8A in blue), (v) log2 fold changes of DNA interactions (with green–red coloured scales) and (vi) genomic annotations and coordinates. The purple and blue arcs in panels (iii) and (iv) indicate chromatin interactions between two NANOG sites in cells expressing either WT (purple) or W8A (blue), respectively. The arc thickness represents the interaction strength in Hi-C 3.0. For the fold changes shown in (v), they were plotted on the colour scale shown to the right, which were calculated using Hi-C 3.0 normalized contacts between the sites; green and red indicate their reduction or increase, respectively. Most of these contacts were weaker in the W8A condition than in the WT condition. The shown Hi-C 3.0 data were derived using two biologically independent replicates. g, Model of how NANOG can reshape the pluripotent genome. Through prion-like assembly, NANOG can initiate or stabilize intragenomic (promoter-enhancer) contacts, as well as connect distant intergenomic loci to form superenhancer clusters with other TFs and coactivators (green/yellow).
