R-loops Associated with Triplet Repeat Expansions Promote Gene Silencing in Friedreich Ataxia and Fragile X Syndrome (original) (raw)
Figure 1
R-loops are formed over expanded repeats of FXN gene in FRDA cells.
A. Diagram of FXN gene. Black boxes are exons, white boxes are 5′ and 3′UTRs, lines are introns, red triangle is (GAA)n expansion. TSS2 is the major transcriptional start site in lymphoblastoid cells. qPCR amplicons are shown below the diagram. Numbers indicate the distances to TSS2 in kilobases. B. Cell lines used in the study. The repeat sizes are indicated. C. RT-qPCR analysis of γ-actin, β-actin, GAPDH and FXN mRNAs in control (GM15851) and FRDA (GM15850) cells. Values are normalised to 5S rRNA and relative to control cells. D. RNA Pol II ChIP in control (GM15851) and FRDA (GM15850) cells. E. RT-qPCR analysis of FXN nascent RNA in control (GM15851) and FRDA (GM15850) cells, normalised to 5S rRNA and relative to ex1 RNA in control cells. F. DIP on endogenous FXN gene in control (GM15851) and FRDA (GM15851) cells. γ-actin is positive control. G. R-loops are sensitive to RNase H digestion. DIP samples were treated with 25 U of recombinant E.coli RNase H (NEB, M0297S) for 6 hours at 37°C. γ-actin is positive control. Bars in C–G are average values +/− SEM (n>3).