Journal: PLoS Genetics

Article Title: TDP-43 mutations link Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated DNA damage

doi: 10.1371/journal.pgen.1009260

Figure Lengend Snippet: A) Screnshoots of genome windows showing co-occurrence between TDP43 (red), DRIPc-seq (green), untreated (dark blue) and RNH-treated (light blue) DRIP-seq and RNA-seq (yellow) data. Genome location is indicated in the top left corner and scale in the top right. Coverage scale indicated in the top left of each track. Biological replicates are also indicated (R1, R2). B) Venn diagram showing correlation between genes bind by TDP-43 (TDP43; red circle), genes forming R loops (DRIPc-seq; green) and expressed genes (RNA-seq; yellow). Numbers refer to genes co-occurring between conditions. C) TDP-43 ChIP-seq average coverage (log10) across silent (-) and expressed (+) genes. ****, P < 0,0001 (Mann-Whitney U test, two-tailed). D) Metagene analysis showing TDP-43 ChIP-seq coverage (red line) along gene body (+/- 2kb) of silent (-) and expressed (+) genes. Mean coverage is plotted in the upper panel and heatmap intensities for the entire gene population is shown below. Scale is also indicated.

Article Snippet: SH-SY5Y cells were stably transfected using Fugene HD Transfection Reagent (Promega) with C-terminally GFP-tagged TDP-43 WT vector (PS100010 pCMV6-AC-GFP), C-terminally GFP-tagged TDP-43 A382T mutation vector (CW303334 mutated ORF of RC210639 at nt position 1144, changed from G to A, to obtain A382T, inserted in PS100001) and a C-terminally GFP-tagged TDP-43 G294V mutation vector (CW303287 mutated ORF of RG210639 at nt position 881, changed from G to T, to obtain G294V, inserted in PS100010), purchased from OriGene.

Techniques: RNA Sequencing Assay, ChIP-sequencing, MANN-WHITNEY, Two Tailed Test

Journal: PLoS Genetics

Article Title: TDP-43 mutations link Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated DNA damage

doi: 10.1371/journal.pgen.1009260

Figure Lengend Snippet: A) IF of LCL-CTL, LCL-TDP382, LCL-SALS using an anti-TDP-43 antibody and an anti-S9.6 antibody after methanol fixation. The scatter plots show the increase of S9.6 signal intensity and the decrease in TDP-43 nuclear content in SH-TDP382. Median values are indicated. Scale bar: 25μm. *, P< 0,05 (Mann-Whitney U test, two-tailed). When no asterisk is shown indicates that is not significant. B) Flow cytometry plot reports the amount of R-loops in LCLs (blue) in comparison to LCL-TDP382 (orange) and LCL-SALS (blue). The RNaseH action on LCL-TDP382 decreases the presence of R-loops signal (orange peak vs green peak). Histogram shows S9.6 mean fluorescence of LCL-CTL, LCL-TDP382, LCL-SALS in presence (+) and in absence (-) of RNaseH. The value represented is the mean ± SEM of three biological experiments. ANOVA, Newman-Keuls Multiple Comparison Test, *P <0,05. When no asterisk is shown indicates that is not significant. C ) Model showing the link between TDP-43 and R loop metabolism. Functional TDP43 activity in the nucleus protects genome integrity by preventing R-loop accumulation. However, TDP-43 nuclear dysfunctions results in R loop-dependent DNA damage that eventually might lead to genome instability that could aggravate ALS phenotype.

Article Snippet: SH-SY5Y cells were stably transfected using Fugene HD Transfection Reagent (Promega) with C-terminally GFP-tagged TDP-43 WT vector (PS100010 pCMV6-AC-GFP), C-terminally GFP-tagged TDP-43 A382T mutation vector (CW303334 mutated ORF of RC210639 at nt position 1144, changed from G to A, to obtain A382T, inserted in PS100001) and a C-terminally GFP-tagged TDP-43 G294V mutation vector (CW303287 mutated ORF of RG210639 at nt position 881, changed from G to T, to obtain G294V, inserted in PS100010), purchased from OriGene.

Techniques: MANN-WHITNEY, Two Tailed Test, Flow Cytometry, Fluorescence, Functional Assay, Activity Assay