KDM2B is enriched at HSATII genomic regions and has increased association with RNF2 in DUX4-expressing cells. (A) Combined RNA-FISH and immunofluorescence of HSATII RNA (green) and KDM2B (magenta) signal in −dox or +dox (4-h pulse and fixed/analyzed 20 h after induction) iDUX4 cells, (scale bar = 20 μm). Images are representative of two independent experiments conducted on separate days. (B) Nuclear KDM2B MFI in −dox cells or +dox cells in nuclei that contained no HSATII RNA (HSATII−) or HSATII RNA foci (HSATII+). Nuclei are indicated for representative experiment and N = 20–50 nuclei per condition. (C) ChIP-qPCR of KDM2B or IgG isotype control in −dox or +dox iDUX4 cells. Primers targeting control GDR h16q21 (Maston et al., 2012) or HSATII 1q12 were used. Three biological replicates were performed per IP per condition. (D) Immunofluorescence of RNF2 (green) and KDM2B (magenta) signal in −dox or +dox (4 h pulse and fixed/analyzed 20 h after induction) iDUX4 cells, (scale bar = 20 μm). Images are representative of two independent experiments conducted on separate days. (E) Quantification of nuclear KDM2B MFI of nuclei that contained RNF2 pan-nuclear signal (pan) or RNF2 aggregates (foci). Nuclei are indicated for representative experiment and N = 15–50 nuclei per condition. (F) Combined RNA-FISH and immunofluorescence of PLA of KDM2B and RNF2 (magenta) and HSATII RNA (green) in −dox or +dox (4-h pulse and fixed/analyzed 20 h after induction) iDUX4 cells (scale bar = 20 μm). Images are representative of two independent experiments conducted on separate days. N = 50–100 nuclei imaged. (G) KDM2B–RNF2 PLA signal intensity measured within nuclei in −dox, +dox HSATII− or HSATII+ nuclei, compared with KDM2B–RNF2 PLA signal intensity measured within HSATII RNA foci or randomly drawn ROI within the nucleoplasm in +dox HSATII+ nuclei. Each dot represents either individual nuclei or individual foci, respectively. Nuclei are indicated for representative experiment and N ≥ 50 nuclei per condition or N ≥ 140 ROI. (B, C, E, and G) Data represent means ± SD. Statistical differences between groups were analyzed employing nonparametric Mann–Whitney test in the absence of normal distribution, with one-way ANOVA Dunnett’s or Tukey’s multiple comparison test, or two-way ANOVA Sidak’s multiple comparison test between each group and a control.
Figure 3.

KDM2B is enriched at HSATII genomic regions and has increased association with RNF2 in DUX4-expressing cells. (A) Combined RNA-FISH and immunofluorescence of HSATII RNA (green) and KDM2B (magenta) signal in −dox or +dox (4-h pulse and fixed/analyzed 20 h after induction) iDUX4 cells, (scale bar = 20 μm). Images are representative of two independent experiments conducted on separate days. (B) Nuclear KDM2B MFI in −dox cells or +dox cells in nuclei that contained no HSATII RNA (HSATII−) or HSATII RNA foci (HSATII+). Nuclei are indicated for representative experiment and N = 20–50 nuclei per condition. (C) ChIP-qPCR of KDM2B or IgG isotype control in −dox or +dox iDUX4 cells. Primers targeting control GDR h16q21 (Maston et al., 2012) or HSATII 1q12 were used. Three biological replicates were performed per IP per condition. (D) Immunofluorescence of RNF2 (green) and KDM2B (magenta) signal in −dox or +dox (4 h pulse and fixed/analyzed 20 h after induction) iDUX4 cells, (scale bar = 20 μm). Images are representative of two independent experiments conducted on separate days. (E) Quantification of nuclear KDM2B MFI of nuclei that contained RNF2 pan-nuclear signal (pan) or RNF2 aggregates (foci). Nuclei are indicated for representative experiment and N = 15–50 nuclei per condition. (F) Combined RNA-FISH and immunofluorescence of PLA of KDM2B and RNF2 (magenta) and HSATII RNA (green) in −dox or +dox (4-h pulse and fixed/analyzed 20 h after induction) iDUX4 cells (scale bar = 20 μm). Images are representative of two independent experiments conducted on separate days. N = 50–100 nuclei imaged. (G) KDM2B–RNF2 PLA signal intensity measured within nuclei in −dox, +dox HSATII− or HSATII+ nuclei, compared with KDM2B–RNF2 PLA signal intensity measured within HSATII RNA foci or randomly drawn ROI within the nucleoplasm in +dox HSATII+ nuclei. Each dot represents either individual nuclei or individual foci, respectively. Nuclei are indicated for representative experiment and N ≥ 50 nuclei per condition or N ≥ 140 ROI. (B, C, E, and G) Data represent means ± SD. Statistical differences between groups were analyzed employing nonparametric Mann–Whitney test in the absence of normal distribution, with one-way ANOVA Dunnett’s or Tukey’s multiple comparison test, or two-way ANOVA Sidak’s multiple comparison test between each group and a control.

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