Skip to main content
. 2023 Apr 3;12:e79386. doi: 10.7554/eLife.79386

Figure 1. SA interacts with CTCF in the absence of cohesin.

(a) Representative confocal images of SA1 and CTCF IF in RAD21mAC cells treated with ethanol (EtOH) as a control or Auxin (IAA) for 4 hr. Nuclei were counterstained with DAPI. (b) Imaris quantification of the relative mean fluorescence intensity (MFI) of mClover, CTCF, SA1 and SA2 in EtOH and IAA-treated RAD21mAC cells. Whiskers and boxes indicate all and 50% of values, respectively. Central line represents the median. Asterisks indicate a statistically significant difference as assessed using two-tailed t-test. **** p<0.0001. n>50 cells/condition from three biological replicates. Chromatin coIP of (c) SA1, SA2, and IgG with RAD21 and CTCF or (d) CTCF and IgG with RAD21, SA1, and SA2 in RAD21mAC cells treated with EtOH or IAA for 4 hr. Input represents (c) 2.5% and (d) 1.25% of the material used for immunoprecipitation. (e) Dual-color STORM images of SA1 (green) and CTCF (magenta) in EtOH and IAA-treated RAD21mAC cells. Representative full nuclei and zoomed nuclear areas are shown. Line denotes 2 microns and 200 nm for full nuclei and zoomed areas respectively. See figure supplements for SA2 STORM images. (f) Mean CTCF, SA1 and SA2 localization densities (localizations normalized with nuclear area) in EtOH and IAA-treated RAD21mAC cells (n = >30, >17, and>15 nuclei for CTCF, SA1, and SA2, respectively). Mean and SD are plotted, Mann Whitney test. ** p<0.005, *** p<0.0005, **** p<0.0001. (g) Mean Nearest Neighbor Distance (NND) of CTCF, SA1, and SA2 clusters in nanometers in EtOH and IAA-treated cells (n = >38, >14. and>23 nuclei for CTCF, SA1, and SA2, respectively). Mean and SD are plotted, Mann Whitney test. **** p<0.0001. (h) NND distribution plot of the distance between CTCF and SA1 (left panel) or SA2 (right panel) clusters in EtOH and IAA-treated cells. Experimental data are shown as continuous lines, random simulated data are displayed as dotted lines. (i) ChIP–seq deepTools heat map of CTCF, SA1, SA2, Rad21 and SMC3 binding profiles in control (EtOH) and IAA-treated RAD21mAC cells. Selected regions are bound by CTCF in control conditions. (j) Analysis of contact frequency hotspots from Hi-C libraries generated from EtOH-treated (top row) and IAA-treated (bottom row) RAD21mAC cells. Contact frequencies were calculated in two distance ranges of 100 kb – 1 Mb and 1–5 Mb. The last column includes contact frequencies specifically at SA-CTCFΔCoh binding sites.

Figure 1—source data 1. Original, unedited western blots corresponding to Figure 1.

Figure 1.

Figure 1—figure supplement 1. SA interacts with CTCF in the absence of cohesin.

Figure 1—figure supplement 1.

(a) Immunoblot analysis of RAD21 levels in whole cell lysates from in RAD21mAC cells with and without CMV-OsTIR1 integration in the genome. Cells were untreated (-), treated with ethanol (EtOH) or with Auxin (IAA) for 4 hr. The IAA affect was also assessed with antibody to the AID tag. Tubulin and H3 serve as loading controls. (b) Representative confocal images of SA2 and CTCF IF in RAD21mAC cells treated with ethanol (EtOH) or Auxin (IAA) for 4 hr. Nuclei were counterstained with DAPI. (c) Chromatin coIP of SA1, SA2, and CTCF, together with Mock IgG controls in HeLa cells treated with siRNA to SMC3 for 48 hr. (d) GFP-TRAP in RAD21mAC cells immunoblotted for RAD21, SA1, and SA2. TRAP was performed with two concentrations of beads. Shown also is the % residual protein in the flow-through material (post-TRAP) relative to IP and as determined by ImageJ densitometry. (e) Dual-color STORM images of SA2 (green) and CTCF (magenta) in EtOH and IAA-treated RAD21mAC cells. Representative full nuclei and zoomed nuclear areas are shown. Line denotes 2 microns and 200 nm for full nuclei and zoomed areas respectively. (f) Nearest Neighbor Distance (NND) distribution plot of the distance difference between the experimental and random simulated data for SA1 (left) or SA2 (right) at CTCF localizations in EtOH (black) and IAA (red)-treated cells. (g) NND distribution plot of the distance between CTCF and SA1 (top left) or SA2 (bottom left) clusters in EtOH and IAA-treated cells. Experimental data are shown as continuous lines, random simulated data are displayed as dotted lines. Shown also are the NND distribution plots of the distance difference between the experimental and random simulated data for CTCF at SA1 (top right) or CTCF at SA2 (bottom right) localizations in EtOH (black) and IAA (red)-treated cells. (h) Pairwise comparisons of global CTCF ChIP-seq data (from merged biological replicates) compared to RAD21, SA1 and SA2 ChIP-seq in EtOH and IAA-treated RAD21mAC cells. (i) ChromHMM analysis of our ChIP-seq data as well as publicly available ChIP data in HCT116 cells as shown (Methods). Marks enriched within a given state (left) and enriched genomic features (right) are shown. We note that state 6 includes enrichments for ChIP data from RAD21 and SMC3 controls as well as SA1, SA2, CTCF in both control and IAA. This state is also enriched for active marks such as Polr2a, H3K4me3, and H3K27ac.
Figure 1—figure supplement 1—source data 1. Original, unedited western blots for Figure 1—figure supplement 1.