KDM2A is enriched at HSATII genomic regions and associates with RNF2 at HSATII-expressing loci. (A) Combined RNA-FISH and immunofluorescence of HSATII RNA (green) and KDM2A (magenta) signal in −dox or +dox (4 h pulse and fixed/analyzed 20 h after induction) iDUX4 cells, (scale bar = 20 μm). Asterisk in +dox merged image is the drawn distance measured in plot-profile. Images are representative of two independent experiments conducted on separate days. N ≥ 100 nuclei imaged. (B) Plot-profile colocalization of HSATII RNA or KDM2A. Y-axis represents fluorescent intensity (gray value) for HSATII (green line) or KDM2A (magenta line) signal; x-axis represents drawn distances in microns. Pearson R value = 0.64 (HSATII RNA foci and KDM2A foci in +dox cells). (C) KDM2A signal intensity measured within nuclei in −dox, +dox HSATII−, or HSATII+ nuclei, compared with KDM2A 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 ≥ 75 nuclei per condition or N ≥ 60 ROI. (D) ChIP-qPCR of KDM2A or IgG isotype control in −dox or +dox (4-h pulse and fixed/analyzed 20 h after induction) iDUX4 cells. Primers targeting control GAPDH or HSATII 1q12 were used. N = 3 per IP per condition. (E) Representative immunofluorescence images of RNF2 signal (green) and KDM2A signal (magenta) in −dox or +dox (4-h pulse and fixed/analyzed 20-h post-induction) iDUX4 cells (scale bar = 20 μm). Asterisk in +dox merged image is the drawn distance measured in the plot-profile. Images are representative of two independent experiments conducted on separate days. N ≥ 100 nuclei imaged. (F) Plot-profile colocalization of RNF2 and KDM2A. Y-axis represents fluorescent intensity (gray value) for RNF2 (green line) or KDM2A (magenta line) signal; x-axis represents drawn distances in microns. Pearson R value = 0.71 (KDM2A and RNF2 foci in +dox cells). (G) KDM2A signal intensity measured within nuclei in −dox, +dox cells with RNF2 pan-nuclear signal (pan) or RNF2 foci signal, compared to KDM2A signal intensity measured within RNF2 foci or randomly drawn ROI within the nucleoplasm in +dox RNF2 foci+ nuclei. Each dot represents either individual nuclei or individual foci, respectively. Nuclei are indicated for representative experiment and N = 10–50 nuclei per condition or N ≥ 40 ROI. (H) Combined RNA-FISH and immunofluorescence of proximity ligation assays (PLA) of KDM2A 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. (I) Quantification of number of PLA foci per nucleus in −dox or +dox cells. Each dot represents individual nuclei. N ≥ 80 nuclei per condition. (J) Quantification of overall nuclear PLA signal intensity within −dox or +dox cells. Each dot represents individual nuclei. N ≥ 85 nuclei per condition. (K) PLA signal intensity measured within HSATII RNA foci or random ROI within +dox HSATII+ cells only. Each dot represents individual foci. N ≥ 50 foci per condition. (C, D, G, I–K) Data represent means ± SD. Statistical differences between groups were analyzed employing either nonparametric Mann–Whitney test in the absence of normal distribution, two-tailed paired t test, or were assessed with one-way ANOVA Tukey’s multiple comparisons test between each group and a control.
Figure 2.

KDM2A is enriched at HSATII genomic regions and associates with RNF2 at HSATII-expressing loci. (A) Combined RNA-FISH and immunofluorescence of HSATII RNA (green) and KDM2A (magenta) signal in −dox or +dox (4 h pulse and fixed/analyzed 20 h after induction) iDUX4 cells, (scale bar = 20 μm). Asterisk in +dox merged image is the drawn distance measured in plot-profile. Images are representative of two independent experiments conducted on separate days. N ≥ 100 nuclei imaged. (B) Plot-profile colocalization of HSATII RNA or KDM2A. Y-axis represents fluorescent intensity (gray value) for HSATII (green line) or KDM2A (magenta line) signal; x-axis represents drawn distances in microns. Pearson R value = 0.64 (HSATII RNA foci and KDM2A foci in +dox cells). (C) KDM2A signal intensity measured within nuclei in −dox, +dox HSATII−, or HSATII+ nuclei, compared with KDM2A 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 ≥ 75 nuclei per condition or N ≥ 60 ROI. (D) ChIP-qPCR of KDM2A or IgG isotype control in −dox or +dox (4-h pulse and fixed/analyzed 20 h after induction) iDUX4 cells. Primers targeting control GAPDH or HSATII 1q12 were used. N = 3 per IP per condition. (E) Representative immunofluorescence images of RNF2 signal (green) and KDM2A signal (magenta) in −dox or +dox (4-h pulse and fixed/analyzed 20-h post-induction) iDUX4 cells (scale bar = 20 μm). Asterisk in +dox merged image is the drawn distance measured in the plot-profile. Images are representative of two independent experiments conducted on separate days. N ≥ 100 nuclei imaged. (F) Plot-profile colocalization of RNF2 and KDM2A. Y-axis represents fluorescent intensity (gray value) for RNF2 (green line) or KDM2A (magenta line) signal; x-axis represents drawn distances in microns. Pearson R value = 0.71 (KDM2A and RNF2 foci in +dox cells). (G) KDM2A signal intensity measured within nuclei in −dox, +dox cells with RNF2 pan-nuclear signal (pan) or RNF2 foci signal, compared to KDM2A signal intensity measured within RNF2 foci or randomly drawn ROI within the nucleoplasm in +dox RNF2 foci+ nuclei. Each dot represents either individual nuclei or individual foci, respectively. Nuclei are indicated for representative experiment and N = 10–50 nuclei per condition or N ≥ 40 ROI. (H) Combined RNA-FISH and immunofluorescence of proximity ligation assays (PLA) of KDM2A 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. (I) Quantification of number of PLA foci per nucleus in −dox or +dox cells. Each dot represents individual nuclei. N ≥ 80 nuclei per condition. (J) Quantification of overall nuclear PLA signal intensity within −dox or +dox cells. Each dot represents individual nuclei. N ≥ 85 nuclei per condition. (K) PLA signal intensity measured within HSATII RNA foci or random ROI within +dox HSATII+ cells only. Each dot represents individual foci. N ≥ 50 foci per condition. (C, D, G, I–K) Data represent means ± SD. Statistical differences between groups were analyzed employing either nonparametric Mann–Whitney test in the absence of normal distribution, two-tailed paired t test, or were assessed with one-way ANOVA Tukey’s multiple comparisons test between each group and a control.

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