Figure 1. RNF2 is enriched at HSATII... | Rockefeller University Press

$RNF2 is enriched at HSATII genomic regions and RNF2 protein aggregation correlates with loss of nuclear H2AUb signal in DUX4-expressing cells. (A) Combined RNA fluorescence in situ hybridization (RNA-FISH) and immunofluorescence of HSATII RNA (green) and RNF2 (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 four independent experiments conducted on separate days. (B) Percentage of individual RNF2 foci that have at least partial signal overlap with HSATII RNA foci per nucleus in +dox iDUX4 cells. N = 50 nuclei. (C) Fraction of RNF2 nuclear aggregates present within HSATII RNA foci in +dox iDUX4 cells. Fraction calculated represents the total proportion within each individual nuclei for a representative experiment and N ≥ 100 nuclei. (D) RNF2 signal intensity measured within nuclei in −dox, +dox HSATII−, or HSATII+ nuclei compared with RNF2 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 ≥ 100 nuclei per condition or N ≥ 40 ROI. (E) Fraction nuclei with RNF2 signal type: foci versus pan-nuclear (pan) staining patterns within +dox cells either with HSATII RNA (pos) or without HSAT RNA (neg). Fraction calculated represents fields taken from each independent experiment and N ≥ 100 nuclei. (F) Quantification of total RNF2 protein levels in +eto, −dox, pulse, or continuous +dox conditions. Blot shows two biological replicates for each condition. (G) Chromatin immunoprecipitation (ChIP) of RNF2 or IgG isotype control in −dox or +dox (4 h pulse and fixed/analyzed 20 h after induction) iDUX4 cells. Quantitative PCR (qPCR) was performed on isolated DNA from RNF2- or IgG-bound chromatin fractions. Primers targeting a control gene desert region (GDR) h16q21 (Maston et al., 2012), canonical PcG-target gene HOXA1, or HSATII 1q12 were used. N = 3 per IP per condition. (H) Representative immunofluorescence images of RNF2 signal (green) and H2AUb signal (magenta) 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. (I) Nuclear H2AUb MFI was calculated for nuclei that contained RNF2 pan-nuclear signal (pan) or RNF2 aggregates (foci). Nuclei are indicated for representative experiment and N ≥ 50 nuclei per condition. (J) Combined RNA-FISH and immunofluorescence of HSATII RNA (green) and H2AUb (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. (K) Nuclear H2AUb MFI was calculated 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 ≥ 50 nuclei per condition. (L) Combined RNA-FISH and immunofluorescence of HSATII RNA (green), H2AUb (cyan), and RNF2 (red) signal in +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. (M) Nuclear H2AUb MFI was calculated in +dox cells in nuclei that contained no HSATII RNA (HSATII−) or HSATII RNA foci (HSATII+) with RNF2 signal type. Nuclei are indicated for representative experiment and N ≥ 50 nuclei per condition unless stated otherwise. (C–G, I, K, and M) 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 or two-way ANOVA Sidak’s multiple comparison test between each group and a control. Source data are available for this figure: SourceData F1.$

Figure 1.

RNF2 is enriched at HSATII genomic regions and RNF2 protein aggregation correlates with loss of nuclear H2AUb signal in DUX4-expressing cells. (A) Combined RNA fluorescence in situ hybridization (RNA-FISH) and immunofluorescence of HSATII RNA (green) and RNF2 (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 four independent experiments conducted on separate days. (B) Percentage of individual RNF2 foci that have at least partial signal overlap with HSATII RNA foci per nucleus in +dox iDUX4 cells. N = 50 nuclei. (C) Fraction of RNF2 nuclear aggregates present within HSATII RNA foci in +dox iDUX4 cells. Fraction calculated represents the total proportion within each individual nuclei for a representative experiment and N ≥ 100 nuclei. (D) RNF2 signal intensity measured within nuclei in −dox, +dox HSATII−, or HSATII+ nuclei compared with RNF2 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 ≥ 100 nuclei per condition or N ≥ 40 ROI. (E) Fraction nuclei with RNF2 signal type: foci versus pan-nuclear (pan) staining patterns within +dox cells either with HSATII RNA (pos) or without HSAT RNA (neg). Fraction calculated represents fields taken from each independent experiment and N ≥ 100 nuclei. (F) Quantification of total RNF2 protein levels in +eto, −dox, pulse, or continuous +dox conditions. Blot shows two biological replicates for each condition. (G) Chromatin immunoprecipitation (ChIP) of RNF2 or IgG isotype control in −dox or +dox (4 h pulse and fixed/analyzed 20 h after induction) iDUX4 cells. Quantitative PCR (qPCR) was performed on isolated DNA from RNF2- or IgG-bound chromatin fractions. Primers targeting a control gene desert region (GDR) h16q21 (Maston et al., 2012), canonical PcG-target gene HOXA1, or HSATII 1q12 were used. N = 3 per IP per condition. (H) Representative immunofluorescence images of RNF2 signal (green) and H2AUb signal (magenta) 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. (I) Nuclear H2AUb MFI was calculated for nuclei that contained RNF2 pan-nuclear signal (pan) or RNF2 aggregates (foci). Nuclei are indicated for representative experiment and N ≥ 50 nuclei per condition. (J) Combined RNA-FISH and immunofluorescence of HSATII RNA (green) and H2AUb (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. (K) Nuclear H2AUb MFI was calculated 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 ≥ 50 nuclei per condition. (L) Combined RNA-FISH and immunofluorescence of HSATII RNA (green), H2AUb (cyan), and RNF2 (red) signal in +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. (M) Nuclear H2AUb MFI was calculated in +dox cells in nuclei that contained no HSATII RNA (HSATII−) or HSATII RNA foci (HSATII+) with RNF2 signal type. Nuclei are indicated for representative experiment and N ≥ 50 nuclei per condition unless stated otherwise. (C–G, I, K, and M) 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 or two-way ANOVA Sidak’s multiple comparison test between each group and a control. Source data are available for this figure: SourceData F1.

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