Figure 6. Repo-Man depletion affects chromosome positioning.

(a) HT1080 cells containing a LacO array inserted at 13q22 and expressing GFP:LacI were fixed and stained for Nup153. The arrow indicates the integration site. (b) Position of the chr13q22 was measured using an erosion script software⁶⁹ across five concentric shells (1—most outer shell to 5—most inner shell) after RNAi with control or Repo-Man oligos or transiently transfected with the dominant-negative Repo-Man^RAXA mutant. (c) 3D FISH with probe CTC-820M16 (red signal) mapping to the subtelomeric region of chromosome 14 performed on HeLa cells. (d) Quantification of spots location described in c, using the erosion script software. (Fisher test, *P<0.05, **P<0.01, ***P<0.001 using two-three replicates). (e) Enrichment of H3K27me2/3 at the nuclear periphery after Nup153 RNAi. (f) Enrichment of H3K9me3 at the nuclear periphery after Nup153 RNAi. Enrichment was calculated as in Fig. 2e. Mann–Whitney test (*P<0.05), n is depicted in the figure. In box plots in e and f, central line represents the median, box limits are the 25th and 75th percentiles and whiskers extend to 1.5 x interquartile range. (g) Differential expression of telomeric genes bound by Repo-Man between control and Repo-Man (green) or Nup153 (blue) RNAi. Delta–delta-CT method was used and normalized for GAPDH. Error bars=s.e.m. between three replicates. t-test was used for statistical analysis (*P<0.05, **P<0.01).