Fig. 7
Increased DOT1L levels are responsible for DNA repeat repression at peri-nucleolar heterochromatin. A Individual data points plot showing the fold change expression (FC OE/CTR) of the indicated DNA repeats after RT-qPCR upon 3 days of DOT1L OE in N2a cells. Gapdh was used as reference gene for normalization. Abbreviations are: gSAT (gamma satellites), mSAT (major satellites). Statistical analysis was performed on n = 7 biological replicates using an unpaired two tailed t-test. *p < 0.05, **p < 0.01. Outliers were identified using a Grubbs method. Error bars represent S.D. B Individual data points plot showing the fold change enrichment over control of H3K79me2 after ChIP-qPCR upon 3 days of DOT1L OE (OE/CTR). Abbreviations as in A. Statistical analysis was performed on n = 3 biological replicates using an unpaired two tailed Student's t-test. *p < 0.05, **p < 0.01. Error bars represent S.D. C Individual data points plot showing the fold change enrichment over control of H3K27me3 at the indicated DNA repeats after ChIP-qPCR upon 3 days of DOT1L OE (OE/CTR). Abbreviations as in A. Statistical analysis was performed on n = 4 biological replicates using an unpaired two tailed t-test. *p < 0.05, **p < 0.01. Error bars represent S.D. D Individual data points plot showing the fold change expression (FC OE/CTR) of the indicated genes after RT-qPCR upon 3 days of DOT1L OE in N2a cells. Gapdh was used as reference gene for normalization. Statistical analysis was performed on n = 7 biological replicates using an unpaired one tailed (Dot1l) and two tailed t-test (all other genes). *p < 0.05, **p < 0.01. Outliers were identified using a Grubbs method. Error bars represent S.D. E Individual data points plot showing the fold change enrichment over control of H3K79me2 at the indicated genes, calculated by ChIP-qPCR upon 3 days of DOT1L OE (OE/CTR). C3T2.1 is used as negative control gene. Statistical analysis was performed on n = 3 biological replicates using an unpaired two tailed Student's t-test. *p < 0.05, **p < 0.01. Error bars represent S.D. F Confocal immunofluorescence images of immune-FISH performed on N2a cells after 3 days of DOT1L OE using cy3-labeled probes specific for mSat (red). GFP antibody (green) was used to highlight cells with DOT1L OE. Scale bars 10μm. G Quantification of the average number of mSat foci in control (CTR) and after 3 days of DOT1L OE. Statistical analysis was performed on n = 3 biological replicates and on n = 68 (CTR) and n = 68 (OE) cells using a two-way ANOVA with Sidak’s post-hoc test. *p < 0.05, **p < 0.01. Error bars represent S.D. H Quantification of the mean area of mSat foci per cell in control (CTR) and after 3 days of DOT1L OE. Statistical analysis was performed on n = 3 biological replicates and n = 20 nuclei per condition using a two-way ANOVA with Sidak’s post-hoc test. *p < 0.05, **p < 0.01. Error bars represent S.D. I Nuclei were divided in 3 categories according to the average size of mSat foci per nuclei: small (0.2–1 μm2), medium (1–2 μm2) and large (> 2 μm2). Statistical analysis was done between classes using a two-way ANOVA with Sidak’s post-hoc test. *p < 0.05, **p < 0.01