R-loops Associated with Triplet Repeat Expansions Promote Gene Silencing in Friedreich Ataxia and Fragile X Syndrome
Figure 5
R-loops trigger transcriptional repression of FXN gene.
A. DIP analysis on FXN gene in control and FRDA cells, treated with 10 µM camptothecin (CPT) for 6 hours. B. H3K9me2 ChIP on FXN gene in control and FRDA cells, treated with 10 µM camptothecin (CPT) for 6 hours. H3K9me2 levels were normalized to the total H3 levels. C. RT-qPCR analysis of FXN nascent RNA in control and FRDA cells, treated with 10 µM camptothecin for 6 hours. Values are relative to untreated control cells and normalized to γ-actin nascent RNA. D. G9a ChIP on FXN gene in control and FRDA cells. G9a levels are normalised relative to amplicon B in control cells. E. Western blot analysis of 20 and 40 µg of protein extracts obtained from FXN-Luc and FXN-GAA-Luc cells, treated with control and Top1 siRNAs. Western blot was probed with anti-Top1 and anti-actin antibody. F. DIP analysis on FXN gene in FXN-Luc and FXN-GAA-Luc HEK293 cells, treated with control and Top1 siRNAs. Bars in A–D and F are average values +/− SEM (n>3).