Aberrant FUS condensates limit the binding of CCCTC-binding factor with chromatin. (A) Heatmaps showing the CCCTC-binding factor (CTCF) peaks around the CTCF motif in FUS^low and FUS^high LSK cells (±1 kb). Freshly isolated 5 × 10⁴ LSK from young (2-4 months) Fus-gfp mice are subjected to a CTCF CUT&Tag assay. (B) Representative example showing the merged TAD, insulation score, and CTCF peaks at chr3: 30-31.5 Mb of FUS^low and FUS^high HSCs. (C) Representative western blot showing the interaction of FUS and CTCF. HEK293T cells were cotransfected with plasmids encoding SFB-tagged FUS and Myc-tagged CTCF. Lysates were subjected to immunoprecipitation by using anti-S-protein agarose beads for 3 hours. Input and copurified proteins were blotted by probing with corresponding antibodies. (D) Representative images showing the immunofluorescence staining of CTCF (red) in FUS^low and FUS^high LSK cells from a 2-month-old Fus-gfp mouse. DAPI (blue) was used for nuclei counterstaining. Scale bar represents 2 μm. (E) These histograms depict the CTCF intensity, overlap count, and overlap area between FUS and CTCF in FUS^low and FUS^high LSK cells. Data are shown as mean ± SEM, N = 32-40 cells per group. (F) Representative images showing immunofluorescence staining of CTCF (red) in CSK buffer–preextracted FUS^low and FUS^high LSK cells from young (2-4 months old) Fus-gfp mice. DAPI (blue) was used for nuclei counterstaining. Scale bar represents 2 μm. (G) These histograms depict the intensity, count, and area of CTCF condensates in FUS^low and FUS^high LSK cells preextracted with CSK buffer. Data are shown as mean ± SEM, N = 33-34 cells per group. (H) Representative western blot showing the detection of CTCF binding to the biotinylated M1 motifs in the presence FUS^FL or variants by DNA pull-down assay. HEK293T cells were cotransfected with plasmids encoding SFB-tagged FUS^FL or FUS variants and Myc-tagged CTCF. A total of 2 μg biotinylated M1 motifs were added to lysates and subjected to immunoprecipitation by using streptavidin agarose beads for 3 hours. Input and copurified proteins were blotted by probing with corresponding antibodies. (I-K) These pie charts show the distribution of DEG (I), upregulated genes (J), and downregulated genes (K) in FUS^high HSCs compared with FUS^low HSCs. The green background represents the genes that originated from merged TADs, wherein the red lines indicate the genes that have been reported to modulate HSC function.