(A) Phase contrast images of the reprogramming model system. (B) Genome-wide ES cell Hi-C data () at different bin sizes illustrating chromosome territories, A/B compartments and TADs. Images made with the Juicebox tool (http://www.aidenlab.org/juicebox/). The 4–12 kb resolution heatmaps from the present study query fine scale genome folding at the sub-Mb scale within TADs. (C) Relative contact frequency heatmaps are displayed for all biological replicates and regions queried. Color bars range from low (grey) to high (red/black) interaction frequencies. (D) Distance-corrected interaction score heatmaps for a select region around the Sox2 gene illustrating the presence of dynamic chromatin architecture among ES, NPC and iPS cells. Color bars range from low (blue) to high (red/black) interaction scores.

Principal component analysis of (A) distance-corrected interaction frequency data and (B) normalized RNAseq data for ES, NPC and iPS replicates. (A, B) Principal components 1 and 2 are scattered and the proportion of variance explained by each principal component is plotted below each scatterplot.

(A–C) Scatterplot comparison of distance-corrected interaction scores between (A) ES cells and NPCs, (B) ES and iPS cells and (C) NPCs and iPS cells. Thresholds are displayed as blue lines. For pairwise plots, cell type-specific, invariant and background interactions are represented by blue, grey and brown colored shading, respectively. (D) 3D scatterplot of distance-corrected interaction scores for cellular states in which both replicates cross the thresholds displayed in (A–C). Interaction classes are indicated by color (red, ES only; green, NPC only; orange, iPS only; gold, ES-NPC; purple, ES-iPS; blue, NPC-iPS; black, Background). Empirical false discovery rates computed from simulated data in (E–G) are reported for each classification. (E–G) Scatterplots of distance-corrected interaction scores from simulated replicates. Empirical false discovery rates were computed based on the number of interactions that cross pre-established thresholds in the simulated data versus the real data. (H) 3D scatterplot of distance-corrected interaction scores for simulated libraries that cross the thresholds displayed in (A–C, E–G). (I) Number of interactions called significant in each cell-type specific interaction class. (J) Schematic illustrating the 3D interaction behavior for each interaction class. (K–L) Zoomed-in heatmaps of distance-corrected interaction scores for specific (K) ES-iPS (purple class) and (L) ES only (red class) interactions. Classified interaction pixels are outlined in green. (M) Number of interactions called significant for each 3-D classification after clustering directly adjacent 4 kb bins. (N) Depiction of all interactions called as significant in the Sox2 region. Each interaction is outlined by the corresponding classification color.

(A) Schematic illustrating the ES-iPS (purple) interaction class. (B,D) Relative contact frequency heatmaps (top) and zoomed-in distance-corrected interaction score heatmaps (bottom) highlighting key ES-iPS interactions (purple class) between (B) Sox2 and (D) Oct4 genes and their target enhancers. Heatmaps are overlaid on ChIPseq tracks of H3K27ac and H3K4me1 in ES cells and NPCs. (C+E) Distance-corrected interaction score changes at (C) the Sox2-enhancer interaction and (E) Oct4-enhancer interaction among ES, NPC and iPS cells. Error bars represent the standard deviation across two 5C replicates. (F) Fold enrichment of cell type-specific regulatory elements in ES-iPS (purple class) interactions compared to the enrichment expected by chance across the genome. Color bar represents fold change enrichment over background (blue, depletion; red, enrichment). P-values are computed with Fisher’s Exact test and listed in each bin. (G–H) Normalized gene expression is plotted for (G) Sox2 and (H) Oct4 genes. Error bars represent standard deviation across two RNAseq replicates.

(A) Relative contact frequency heatmaps (top) and zoomed-in distance-corrected interaction score heatmaps (bottom) highlighting an NPC-iPS interaction (blue class) around the Sox2 gene. Heatmaps are overlaid on ChIPseq tracks of H3K27ac and CTCF in ES cells and NPCs. (B) Schematic illustrating the NPC-iPS (blue) interaction class. (C) Distance-corrected interaction score changes at an NPC-iPS interaction around the Sox2 gene among ES, NPC, iPS, ES+2i and iPS+2i conditions. Error bars represent standard deviation across two 5C replicates. (D) Normalized expression for the Sox2 gene. Error bars represent standard deviation across two RNAseq replicates. (E, F) Fold enrichment of cell type-specific regulatory elements in NPC-iPS (blue class) interactions compared to the enrichment expected by chance across the genome. P-values are computed with Fisher’s Exact test and listed in each bin. (E) Enrichment for any given genomic annotation at the base of NPC-iPS interactions. (F) Enrichment for any given pairwise combination of genomic annotations in the two anchoring bins at the base of NPC-iPS interactions. (G) Relative ChIP-qPCR enrichment of CTCF binding at the NPC-iPS interaction (left, denoted by blue star in (A)) and ES only interaction (right, denoted by red star in (A)).

(A) Relative contact frequency heatmaps (top) and zoomed-in distance-corrected interaction score heatmaps (bottom) highlighting an ES only (red class) interaction at ES-specific CTCF binding sites at the Zfp462 gene (indicated in green). Heatmaps are overlaid on ChIPseq tracks of H3K27ac and CTCF in ES cells and NPCs. (B) Schematic illustrating the ES only (red class) interactions. (C) Fraction of ES only (red class) interactions enriched with distinct cell type-specific regulatory elements compared to the expected enrichment in background. P-values are computed with Fisher’s Exact test and listed in each bin. (D) Bar plot displaying the fraction of each interaction class containing ES-specific CTCF binding sites compared to the expected background fraction. Fisher’s Exact test: *, P= 2.06016e-21; **, P= 0.000541696. (E) Distance-corrected interaction score changes at an ES only interaction around the Zfp462 gene among ES, NPC, iPS, ES+2i and iPS+2i conditions. Error bars represent standard deviation across two 5C replicates. (F) Zfp462 gene expression among ES, NPC, iPS, ES+2i and iPS+2i conditions. Error bars represent standard deviation across two RNAseq replicates. (G) Aggregate distance-corrected interaction score changes among ES, NPC, iPS, ES+2i and iPS+2i conditions for genes anchoring red class. (H) Relative ChIP-qPCR enrichment of CTCF binding at the ES only interaction (denoted by blue star in (A)).

Connectivity of distinct regulatory elements in ES cells, ES-derived NPCs and NPC-derived iPS cells. (A) ES-specific enhancers; (B) ES-specific genes; (C) NPC-specific enhancers; (D) NPC-specific genes; (E) Poised enhancers; (F) Invariant CTCF; (G) ES-specific CTCF; (H) NPC-specific CTCF. (I) Schematic illustrating a model of the ‘hyper-connectivity’ of certain pluripotency genes in our NPC-derived iPS clone. Key ES-specific genes (denoted by colored arrows) display the ability to reprogram their connections with ES-specific enhancers (denoted by green/blue ‘transcription factor’ binding sites) and retain remnants of their somatic connections. This intermediate architectural state correlates with inaccurate reprogramming of gene expression levels (represented by colored +/−) and can be fully restored upon culture in 2i/LIF media.