Over-expressed RNase H1 resolves R-loops formed on <i>FXN</i> expanded repeats in HEK293 cells.

<p>A. Diagram of the FXN-Luc gene, containing 6 (<i>FXN-Luc</i>) or 310 GAA repeats (<i>FXN-GAA-Luc</i>), integrated on the chromosome 1 of HEK293 cells. Frataxin gene was fused to the luciferase at the beginning of the <i>FXN</i> exon 5. Black boxes are exons, white boxes are 5′ and 3′UTRs, lines are introns, red triangle is (GAA)_n expansion. TSS is the transcriptional start site. qPCR amplicons are shown below the diagram. Numbers indicate the distances to TSS in kilobases. B. Size of GAA expansion determined by PCR analysis on genomic DNA from <i>FXN-Luc</i> and <i>FXN-GAA-Luc</i> cell lines, using GAA104F and GAA629R primers. PCR products were run on 1% agarose gel. M denotes the marker lane. <i>FXN-Luc</i> and <i>FXN-GAA-Luc</i> cells contain endogenous wild type <i>FXN</i> gene <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004318#pgen.1004318-Lufino1" target="_blank">[23]</a>, giving rise to the PCR product of 0.5 kb. C. <i>FXN</i> and γ-actin nascent RNA levels in <i>FXN-Luc</i> (white bars) and <i>FXN-GAA-Luc</i> (black bars) HEK293 cells, determined by RT-qPCR and normalised to 5S rRNA. The level of <i>FXN</i> and γ-actin nascent RNA in <i>FXN-Luc</i> cells was taken as 1. LucR primer was used for the reverse transcription reaction. qPCR was carried using in4F and ex5R primers, shown in A. D. DIP analysis on <i>FXN-Luc</i> gene in <i>FXN-Luc</i> (white bars) and <i>FXN-GAA-Luc</i> (black bars) HEK293 cells using RNA/DNA hybrid-specific S9.6 antibody. E. RT-qPCR analysis of RNase H1 mRNA from <i>FXN-Luc</i> and <i>FXN-GAA-Luc</i> cells, treated with control and RNase H1 siRNAs. Values are normalised to GAPDH mRNA and are relative to <i>FXN-Luc</i> cells, treated with control siRNA. F. DIP analysis on <i>FXN-Luc</i> gene in <i>FXN-Luc</i> and <i>FXN-GAA-Luc</i> HEK293 cells, treated with control and RNase H1 siRNAs. G. Western blot analysis of 50 µg of protein extracts obtained from <i>FXN-Luc</i> and <i>FXN-GAA-Luc</i> cells transfected with Flag and RNase H1-Flag expression plasmids. Western blot was probed with anti-RNase H1 antibody. * denotes endogenous RNase H1 protein. H. DIP analysis on <i>FXN-Luc</i> gene in <i>FXN-Luc</i> and <i>FXN-GAA-Luc</i> HEK293 cells transfected with Flag or RNase H1-Flag expression plasmids. Bars in C–F and H represent the average values from at least three independent experiments +/− SEM.</p

Model for the role of R-loops in mediating <i>FXN</i> and <i>FMR1</i> gene silencing.

<p>Background R-loop level on wild type allele allows efficient transcriptional elongation and gene expression. Transcribed (GAA)_n and (CGG)_n expanded repeats form R-loops resulting in decreased initiation and elongation of RNA Pol II. This leads to downregulation of <i>FXN</i> and <i>FMR1</i> expression, associated with formation of repressive DNA and chromatin marks.</p

R-loops are stable and impede Pol II transcription on <i>FXN</i> gene.

<p>A. RT-qPCR analysis of nascent γ-actin and <i>FXN</i> RNA from control and FRDA cells treated with 5 µg/ml of actinomycin D for 21 hours. Values are relative to untreated control cells. B. DIP on <i>FXN</i> gene in control and FRDA cells treated with 5 µg/ml of actinomycin D for 21 hours. γ-actin is positive control. C. H3K9me2 ChIP in control and FRDA cells. H3K9me2 levels were normalized to the total H3 levels. γ-actin is used as background control. D. Diagram depicting the Br-UTP nuclear run-on (NRO) method. E. Br-UTP nuclear run-on in two control (GM15851, GM14926) and two FRDA (GM15850 and GM16243) cells, normalised to the region B in control cells. Bars in A–C and E are average values +/− SEM (n>3).</p

R-loops are not affected by changes in H3K9 dimethylation.

<p>A. H3K9me2 ChIP in control and FRDA cells, treated with 4 µM BIX-01294 for 72 h. H3K9me2 levels were normalized to the total H3 levels and relative to amplicon <i>FXN</i> A, not affected by the treatment. B. DIP analysis in control and FRDA cells, treated with 4 µM BIX-01294 for 72 h. C. RT-qPCR analysis of <i>FXN</i> nascent RNA in control and FRDA cells, treated with 4 µM BIX-01294 for 72 h. Values are relative to untreated control cells and normalized to γ-actin nascent RNA. Bars in A–C are average values +/− SEM (n>3).</p

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).</p

R-loops are formed over expanded repeats of <i>FXN</i> gene in FRDA cells.

<p>A. Diagram of <i>FXN</i> 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 <i>FXN</i> 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 <i>FXN</i> 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 <i>FXN</i> 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 <i>E.coli</i> 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).</p

R-loops trigger transcriptional repression of <i>FXN</i> gene.

<p>A. DIP analysis on <i>FXN</i> gene in control and FRDA cells, treated with 10 µM camptothecin (CPT) for 6 hours. B. H3K9me2 ChIP on <i>FXN</i> 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 <i>FXN</i> 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 <i>FXN</i> 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 <i>FXN-Luc</i> and <i>FXN-GAA-Luc</i> cells, treated with control and Top1 siRNAs. Western blot was probed with anti-Top1 and anti-actin antibody. F. DIP analysis on <i>FXN</i> gene in <i>FXN-Luc</i> and <i>FXN-GAA-Luc</i> HEK293 cells, treated with control and Top1 siRNAs. Bars in A–D and F are average values +/− SEM (n>3).</p

Functional characterisation of the maturation of human midbrain neuronal cultures.

<p>(A) Representative current-clamp recordings (400 ms current pulses and inj 40–50 pA) from hiPSC-derived neurons at specified time points (from left panel: 6, 7, 8, 9, and 10 weeks in culture). Neurons show changes in resting membrane potentials reflecting maturation (6 weeks: ∼ −35–40 mV, 10 week: ∼ −65–70 mV). These were recorded in neurons that were not showing pace-making activity. (B) Representative pace-making firing traces in whole-cell patch current clamp configuration and (C) representative traces of the spontaneous EPSCs recorded from 10 week old neuronal cultures (n >6 neurons showed spontaneous pace-making activity). Inset shows immunostaining on fixed cells of co-expression of TH and synaptic vesicle protein, SV2 in differentiated neurons. Similar recordings were obtained from NHDF-1 and -2 lines.</p

Physiological Characterisation of Human iPS-Derived Dopaminergic Neurons

<div><p>Human induced pluripotent stem cells (hiPSCs) offer the potential to study otherwise inaccessible cell types. Critical to this is the directed differentiation of hiPSCs into functional cell lineages. This is of particular relevance to research into neurological disease, such as Parkinson’s disease (PD), in which midbrain dopaminergic neurons degenerate during disease progression but are unobtainable until post-mortem. Here we report a detailed study into the physiological maturation over time of human dopaminergic neurons <i>in vitro.</i> We first generated and differentiated hiPSC lines into midbrain dopaminergic neurons and performed a comprehensive characterisation to confirm dopaminergic functionality by demonstrating dopamine synthesis, release, and re-uptake. The neuronal cultures include cells positive for both tyrosine hydroxylase (TH) and G protein-activated inward rectifier potassium channel 2 (Kir3.2, henceforth referred to as GIRK2), representative of the A9 population of substantia nigra pars compacta (SNc) neurons vulnerable in PD. We observed for the first time the maturation of the slow autonomous pace-making (<10 Hz) and spontaneous synaptic activity typical of mature SNc dopaminergic neurons using a combination of calcium imaging and electrophysiology. hiPSC-derived neurons exhibited inositol tri-phosphate (IP3) receptor-dependent release of intracellular calcium from the endoplasmic reticulum in neuronal processes as calcium waves propagating from apical and distal dendrites, and in the soma. Finally, neurons were susceptible to the dopamine neuron-specific toxin 1-methyl-4-phenylpyridinium (MPP+) which reduced mitochondrial membrane potential and altered mitochondrial morphology. Mature hiPSC-derived dopaminergic neurons provide a neurophysiologically-defined model of previously inaccessible vulnerable SNc dopaminergic neurons to bridge the gap between clinical PD and animal models.</p></div

Functional dopamine synthesis and homeostasis in differentiated neuronal cultures.

<p>A) Western blot analysis of proteins involved in dopamine synthesis and homeostasis in cells at different stages of maturation (EB: day 0, Rosette: day 16, Neuron: day 35). hiPSCs do not express TH, DAT or AADC but these were expressed as the cells differentiated in culture. DAT is expressed only in differentiated neurons. B) Immunostaining for dopamine in differentiated neurons. This labelling co-localised with TH expression. Scale bar: 70 µm. C) Dopamine content of differentiated neurons was measured by HPLC. Differentiated neurons (35 days total in culture) produced dopamine (48.98 pmol/mg ±0.389) and were responsive to L-DOPA (933.14 pmol/mg ±220.71), indicating high AADC activity. Data shown are from 4–6 wells each of 2 independent experiments and is expressed as mean ± SEM. D) Functional dopamine transporter activity was assessed by ³HDA uptake in differentiated neurons. DAT-specific uptake was demonstrated by inhibition by mazindol. Data are expressed as mean ± SEM.</p