R-loops are formed over (CGG)_n expanded repeats of <i>FMR1</i> gene.

<p>A. Diagram of <i>FMR1</i> 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 <i>FMR1</i> mRNA in control and FXS cells, treated with 1 µM 5-azadC for 7 days, normalized to GAPDH. C. DIP analysis on endogenous <i>FMR1</i> 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. <i>FMR1</i> 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 <i>FMR1</i> 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 <i>FMR1</i> 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 <i>FMR1</i> mRNA in control cells is taken as 1. Bars in B–D are average values +/− SEM (n>3).