Figure 5. FIREs are conserved across evolution, and mediated by Cohesin.
a) Venn diagrams showing the significant number of conserved FIRE bins when lifting over mouse FIREs onto the human genome (left column), or lifting over human FIREs onto the mouse genome (right column) in either embryonic stem cells (top row, p value < 5.0e-16), neural progenitor cells (middle row, p value < 2.2e-16), and cortex tissue (bottom row, p value < 2.2e-16). Significance evaluated using a Fisher's exact test (see Supplemental Methods).
b) Normalized Hi-C contact matrix in human cortex (left) and mouse cortex (right) for a 2Mb syntenic region (human chr3:78,000,000-80,000,000; mouse chr16:71,520,000-73,520,000) showing a conserved FIRE (connected black lines) within the same tissue type but across species. Below is a UCSC gene track, and to right of the contact matrix is the continuous FIRE score across the locus. For the human data, the Hi-C contact matrix, gene track, and FIRE score plot have been inverted to show synteny with the mouse data.
c) Normalized Hi-C contact matrices (red and white) or delta matrix (green and blue) for 1.96Mb locus (chr1:55,400,000-57,360,000) illustrating the change of interaction frequency between TEV and HRV. Directly below the delta matrix are binding profiles of CTCF and the Cohesin subunit SMC3 in wild type HEK cells (Zuin et al, 2014), as well as TAD boundary annotations. To the right of the Hi-C delta matrices shows the continuous FIRE Z-score difference between TEV and HRV. Below is a delta matrix at a zoomed in 800kb region (chr1:55,560,000-56,360,000) for TEV-HRV showing the greatest reduction of FIRE score occurs at the bin with co-binding of CTCF and SMC3. FIRE Z-score difference is plotted to the right of the subtraction matrices.
d) Box plots showing the change in Z-score at FIREs overlapping bins bound by CTCF but not SMC3 “CTCF-only” (left plot), all CTCF peaks (middle plot), and CTCF and SMC3 co-binding (right plot) for the comparison of TEV and HRV. The red boxes show distributions of FIRE score change at FIRE bins called in wild type cells minus the mutant cells, while the blue boxes are distributions for FIRE score change at FIRE bins called in wild type cells but between biological replicates of wild type cells. These comparisons show the significant reduction of FIRE score at all CTCF peaks, and especially at CTCF SMC3 co-bound peaks overlapping FIRE bins (*p=1.0e-4, **p=4.04e-5; two sample t-test).
e) Similar to Panel d, except analysis of Z-score change were done considering FIREs overlapping the Cohesin subunit Rad21 peaks using previously published Hi-C data and Rad21 ChIP-seq data in mouse neural stem cells (left plot) and mouse post-mitotic astrocytes (middle plot) (Sofueva et al., 2013). Comparison of Z-score change upon deletion of Rad21 shows significant decrease compared to changes observed between biological replicates (*p<0.01; **p< 2.2e-16; two sample t-test).
f) Similar to Panel e, except analysis of Z-score change was conducted on previously published Hi-C data and Rad21 ChIP-seq data in mouse thymocytes (Seitan et al., 2013). Comparing the distributions of Z-score changes at FIRE bins bound by Rad21 shows a significant reduction in Z-score between the wild type and Rad21 knockout cells compared to changes between wild type biological replicates (**p< 2.2e-16; two sample t-test).