R-loops Associated with Triplet Repeat Expansions Promote Gene Silencing in Friedreich Ataxia and Fragile X Syndrome
Figure 6
R-loops are formed over (CGG)n expanded repeats of FMR1 gene.
A. Diagram of FMR1 gene. Black boxes are exons, white box is 5′ UTR and lines are introns. Red triangle is (CGG)n expansion. qPCR amplicons are shown below the diagram. TSS is the transcriptional start site. Numbers indicate the distances to TSS in kilobases. B. RT-qPCR analysis of FMR1 mRNA in control and FXS cells, treated with 1 µM 5-azadC for 7 days, normalized to GAPDH. C. DIP analysis on endogenous FMR1 gene in control and FXS cells, treated with 1 µM 5-azadC for 7 days. Values are relative to ex1 region in control untreated cells. D. FMR1 R-loops are sensitive to RNase H digestion, following the treatment with 25 U of RNase H for 6 hours at 37°C prior to immuno-precipitation. Values are relative to in15 region in control untreated cells. E. R-loop kinetics on exon 1 of FMR1 gene in control and FXS cells during the process of transcriptional re-activation with 1 µM 5-azadC (7 days) followed by 5-azadC wash out with drug-free media (28 days). Values are relative to ex1 region in control untreated cells on day 7. F. RT-qPCR analysis of FMR1 mRNA in control and FXS cells, treated with 1 µM 5-azadC (7 days) followed by 5-azadC wash out with drug-free media (28 days). The level of FMR1 mRNA in control cells is taken as 1. Bars in B–D are average values +/− SEM (n>3).