Article Title: Network analysis of promoter interactions reveals the hierarchical differences in genome organisation between human pluripotent states
Figure Lengend Snippet: Dynamics of enhancer activity and interactivity between human pluripotent states. (A) Plot showing the number of ROSE-called SEs in naïve and primed PSCs. As illustrated in the diagram, two values are given for shared SEs because a SE in one cell type may overlap with two individually-called SEs in the other cell type. (B) Diagram showing the number of genes that are contacted by SEs in the two pluripotent cell types. Shared genes (orange) are genes that are contacted by SE elements in both naïve and primed PSCs. Naïve-specific genes (blue) and primed-specific genes (red) are contacted by SEs in either naïve or primed PSCs, respectively. (C) Plots showing the log2 FPKM expression of genes that interact with SEs in each cell type (naïve, n=648; primed, n=286; shared, n=174). P-values are derived from a Mann Whitney U test. (D) Plot showing the distribution of ROSE-called enhancers in naïve and primed PSCs. (E) Diagram showing the number of genes that are contacted by enhancers in the two pluripotent cell types. Genes that are also in contact with a SE have been removed from this list of enhancer-interacting genes. (F) Plots showing the log2 FPKM expression of genes that interact with enhancer elements in each cell type (naïve, n=2077; primed, n=1210; shared, n=3158). P-values are derived from a Mann Whitney U test. (G) Genome browser view of the DPPA5 promoter interactomes in naïve (upper) and primed (lower) PSCs. Significant interactions are shown as blue arcs that connect the baited HindIII fragment containing the DPPA5 promoter (shaded in red) with promoter-interacting regions (shaded in green). ChIP-seq (H3K4me1, H3K4me3 and H3K27ac) and RNA-seq tracks are shown. Chromatin states include active chromatin, light green; H3K4me1-only chromatin, dark green; bivalent chromatin, purple; background, grey. ROSE tracks show the location of enhancers (green) and super-enhancers (red), and OSN tracks show the position of shared (orange) and naïve-specific (blue) regions of OSN occupancy. (H) Network graph showing the locations and cell-type-origin of enhancer and SE elements. Colours depict naïve-specific (blue), primed-specific (red) and shared (orange) enhancer and SE elements. Node size represents SE (large nodes) and enhancers (small nodes). Lines represent interactions and are coloured according to the colour of the node of origin. (I-J) Plots show the (I) percent DNA methylation or (J) histone modification levels in naïve and primed PSCs at shared (n=18,735) and cell type-specific active enhancers (naïve, n=26,955; primed, n=34,805). Regions that are in the background chromatin state in both cell types are shown to indicate genome-wide levels (n=467,772). See also Figure S6 .
Article Snippet: Using biotin-14-dATP (Life Technologies), dCTP, dGTP and dTTP (Life Technologies; all at a final concentration of 30 μM), the HindIII restriction sites were then filled in with Klenow (NEB) for 75 minutes at 37°C, followed by ligation for 4 hours at 16°C (50 units T4 DNA ligase (Life Technologies) per 7 million cells starting material) in a total volume of 5.5 mL ligation buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, 100 μg/mL BSA) per 7 million cells starting material.
Techniques: Activity Assay, Expressing, Derivative Assay, MANN-WHITNEY, Chromatin Immunoprecipitation, RNA Sequencing Assay, DNA Methylation Assay, Modification, Genome Wide