Fig. 3

The impact of CTCFL on 3D chromatin organization. a IgV tracks showing principal component analysis characterizing the A/B status of compartments (red track: A compartment, PC1 > 0; blue track: B compartment, PC1 < 0) in cells harboring the CTCFL transgene under U, I, D, and ID conditions. Data from chromosome 1 is shown. b Hi-C data from Juicebox corresponding to Chr 8: 63,616,214-69,456,200 at 10 kb resolution. TADs show up as triangles on Hi-C contact maps whose intensity represents interaction strength. Heatmap of Hi-C interactions demonstrates loss of TADs following CTCF depletion (CTCFL I). CTCFL expression does not have a major impact on global TAD structure in the presence (D) or absence (ID) of CTCF. Strengthening of TADs is seen in CTCF D and rescue of TADs in CTCF ID. c Subtraction heatmaps of Hi-C data from Juicebox corresponding to CTCFL (U–I), CTCFL (U–D), and CTCF ID–CTCFL ID. CTCF binding sites (CTCF ID: FLAG ChIP) are shown on the y-axis and FLAG ChIPs of CTCFL-D and CTCFL ID on the x-axis, as indicated. d Aggregate peak analysis demonstrates the strength of the loops at sites where CTCF and CTCFL bind competitively. The transgenes and the respective treatments are indicated. The color intensity at the center of the plot is indicative of loop strength. APA scores are shown in the corners. Values > 1 indicate presence of loops. Examples of altered loops at specific loci are shown in Additional file 1: Figure S4B. e Screenshots of UCSC genome browser showing features of chromatin organization including mean boundary scores (MBS), presence of TADs, and alterations in loops as well as RNA and ChIP-seq tracks. The ChIP-seq peaks unique to CTCF and CTCFL as well as those that are overlapping are shown. The transgenes harbored by the cells and the respective treatments are indicated. Lower panel shows a representative case where alterations in loops and differential expression of genes occur at sites where CTCF and CTCFL binding overlaps. A snapshot of subtraction heatmap from Juicebox is shown with the loops highlighted in boxes. Loops appear as dots at the apex of TADs, the intensity of which defines the “loop strength.” f Co-IP experiments showing the interaction of RAD21 with transgenic CTCF and CTCFL in both D and ID conditions