KDM2 proteins recruit RNF2 to HSATII genomic loci impacting global H2AUb signal. (A) siRNA depletion of either KDM2A and KDM2B (siA/B) or control (siCTRL) sequences in −dox or +dox (4 h pulse and fixed/analyzed 20 h after induction) iDUX4 cells. Western blot was performed on whole cell lysate and probed for KDM2A, KDM2B, and RNF2. GAPDH was used as the loading control. N = 3 per condition, blot shows two replicates. (B) Paired cells from A were used for ChIP-qPCR of RNF2 or IgG isotype control in −dox or +dox with (siA/B) or without (siCTRL) KDM2A/B depletion in iDUX4 cells. Primers targeting control GDR h16q21 (Maston et al., 2012) or HSATII 1q12 were used. N = 3 per IP per condition. (C) Combined RNA-FISH and immunofluorescence of HSATII RNA (green) and H2AUb (red) signal in +dox (4 h pulse and fixed/analyzed 20 h after induction) iDUX4 cells with (siKDM2A/B) or without (siCTRL) KDM2A/B depletion, (scale bar = 10 μm). Images are representative of two independent experiments conducted on separate days. (D) Quantification of nuclear H2AUb MFI was calculated in +dox cells with (siKDM2A/B) or without (siCTRL) KDM2A/B depletion in nuclei that contained no HSATII RNA (HSATII−) or HSATII RNA foci (HSATII+). Nuclei are indicated for each independent experiment and N ≥ 100 nuclei per condition. (E) Combined RNA-FISH and immunofluorescence of HSATII RNA (green) and H2AUb (red) signal in +dox (4-h pulse and fixed/analyzed 20 h after induction) iDUX4 cells with (siKDM2B) or without (siCTRL) KDM2B depletion, (scale bar = 10 μm). Images are representative of two independent experiments conducted on separate days. (F) Quantification of nuclear H2AUb MFI was calculated in +dox cells with (siKDM2B) or without (siCTRL) KDM2B depletion in nuclei that contained no HSATII RNA (HSATII−) or HSATII RNA foci (HSATII+). Nuclei are indicated for each independent experiment and N ≥ 100 nuclei per condition. (A, B, D, and F) Data represent means ± SD. Statistical differences between groups were analyzed with one-way ANOVA Tukey’s multiple comparison test, Kruskal–Wallis test, or two-way ANOVA Sidak’s multiple comparison test between each group and a control. Source data are available for this figure: SourceData F4.
Figure 4.

KDM2 proteins recruit RNF2 to HSATII genomic loci impacting global H2AUb signal. (A) siRNA depletion of either KDM2A and KDM2B (siA/B) or control (siCTRL) sequences in −dox or +dox (4 h pulse and fixed/analyzed 20 h after induction) iDUX4 cells. Western blot was performed on whole cell lysate and probed for KDM2A, KDM2B, and RNF2. GAPDH was used as the loading control. N = 3 per condition, blot shows two replicates. (B) Paired cells from A were used for ChIP-qPCR of RNF2 or IgG isotype control in −dox or +dox with (siA/B) or without (siCTRL) KDM2A/B depletion in iDUX4 cells. Primers targeting control GDR h16q21 (Maston et al., 2012) or HSATII 1q12 were used. N = 3 per IP per condition. (C) Combined RNA-FISH and immunofluorescence of HSATII RNA (green) and H2AUb (red) signal in +dox (4 h pulse and fixed/analyzed 20 h after induction) iDUX4 cells with (siKDM2A/B) or without (siCTRL) KDM2A/B depletion, (scale bar = 10 μm). Images are representative of two independent experiments conducted on separate days. (D) Quantification of nuclear H2AUb MFI was calculated in +dox cells with (siKDM2A/B) or without (siCTRL) KDM2A/B depletion in nuclei that contained no HSATII RNA (HSATII−) or HSATII RNA foci (HSATII+). Nuclei are indicated for each independent experiment and N ≥ 100 nuclei per condition. (E) Combined RNA-FISH and immunofluorescence of HSATII RNA (green) and H2AUb (red) signal in +dox (4-h pulse and fixed/analyzed 20 h after induction) iDUX4 cells with (siKDM2B) or without (siCTRL) KDM2B depletion, (scale bar = 10 μm). Images are representative of two independent experiments conducted on separate days. (F) Quantification of nuclear H2AUb MFI was calculated in +dox cells with (siKDM2B) or without (siCTRL) KDM2B depletion in nuclei that contained no HSATII RNA (HSATII−) or HSATII RNA foci (HSATII+). Nuclei are indicated for each independent experiment and N ≥ 100 nuclei per condition. (A, B, D, and F) Data represent means ± SD. Statistical differences between groups were analyzed with one-way ANOVA Tukey’s multiple comparison test, Kruskal–Wallis test, or two-way ANOVA Sidak’s multiple comparison test between each group and a control. Source data are available for this figure: SourceData F4.

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