Description

\ \pard\tx566\tx1133\tx1700\tx2267\tx2834\tx3401\tx3968\tx4535\tx5102\tx5669\tx6236\tx6803\pardeftab720\pardirnatural\partightenfactor0 \cf2 \kerning1\expnd0\expndtw0 The carboxy-terminal domain (CTD) of the RNA polymerase II (RNAPII) subunit POLR2A is a platform for modifications specifying the recruitment of factors that regulate transcription, mRNA processing, and chromatin remodelling. \cf0 We previously found that symmetrical demethylation (me2s) of a CTD Arginine residue (R1810 in human) causes recruitment of the Tudor domain of SMN, which interacts with Senataxin. SMN is mutated in spinal muscular atrophy (SMA), and Senataxin is sometimes mutated in Amyotrophic Lateral Sclerosis (ALS). R1810me2s and SMN, like Senataxin, are important for resolving R-loops (DNA:RNA hybrids) at transcription terminators. FUS and TDP-43 (TARDBP) are DNA/RNA binding proteins that are sometimes mutated in ALS and FTD (Frontotemporal dementia). Here we show that TDP-43 and, to some extent, FUS are recruited by the R1810me2s-SMN pathway. Defects in FUS and TDP-43 recruitment influence RNAPII termination and R-loop accumulation, leading to elevated DNA damage at terminators that may contribute to neurodegenerative disorders like ALS and FTD.\expnd0\expndtw0\kerning0 \ \pard\pardeftab720\partightenfactor0 \cf0 \

Methods

\ \pard\tx566\tx1133\tx1700\tx2267\tx2834\tx3401\tx3968\tx4535\tx5102\tx5669\tx6236\tx6803\pardeftab720\pardirnatural\partightenfactor0 \cf0 \kerning1\expnd0\expndtw0 ChIP-Seq of RNA polymerase II in control, FUS and TDP-43 knockout HEK293 cells\expnd0\expndtw0\kerning0 \ \pard\pardeftab720\partightenfactor0 \cf0 Reads were extracted and demultiplexed from BCL files with blc2fastq version 1.8.3. Reads were aligned to the human genome, build hg19, using Bowtie2 with settings -5 1 -3 2 \'96local. Duplicate reads were removed to generate BAM files.\kerning1\expnd0\expndtw0 To generate 'meta-gene' alignment plots, reads were extended to 300 bp. Pileups \'96 the number of fragments overlapping each genomic bp \'96 were calculated, and were normalized by million mappable reads in the ChIP-seq library. Normalized pileups from different replicates (done with different antibodies) were then averaged to create FPM (fragments per million reads). Figures showing the average (across represented genes) density of aligned ChIP fragments per million in the library, for each aligned base. Plots were generated directly from BAM files.\ \expnd0\expndtw0\kerning0 \

Credits

\ \itap1\trowd \taflags1 \trgaph108\trleft-108 \trbrdrt\brdrnil \trbrdrl\brdrnil \trbrdrr\brdrnil \clvertalc \clshdrawnil \clbrdrt\brdrs\brdrw20\brdrcf3 \clbrdrl\brdrs\brdrw20\brdrcf3 \clbrdrb\brdrs\brdrw20\brdrcf3 \clbrdrr\brdrs\brdrw20\brdrcf3 \clpadl100 \clpadr100 \gaph\cellx8640 \pard\intbl\itap1\pardeftab720\partightenfactor0 \cf0 \kerning1\expnd0\expndtw0 Dorothy,Y,Zhao\cell \row \itap1\trowd \taflags1 \trgaph108\trleft-108 \trbrdrl\brdrnil \trbrdrr\brdrnil \clvertalc \clshdrawnil \clbrdrt\brdrs\brdrw20\brdrcf3 \clbrdrl\brdrs\brdrw20\brdrcf3 \clbrdrb\brdrs\brdrw20\brdrcf3 \clbrdrr\brdrs\brdrw20\brdrcf3 \clpadl100 \clpadr100 \gaph\cellx8640 \pard\intbl\itap1\pardeftab720\partightenfactor0 \cf0 Zuyao,Ni\cell \row \itap1\trowd \taflags1 \trgaph108\trleft-108 \trbrdrl\brdrnil \trbrdrr\brdrnil \clvertalc \clshdrawnil \clbrdrt\brdrs\brdrw20\brdrcf3 \clbrdrl\brdrs\brdrw20\brdrcf3 \clbrdrb\brdrs\brdrw20\brdrcf3 \clbrdrr\brdrs\brdrw20\brdrcf3 \clpadl100 \clpadr100 \gaph\cellx8640 \pard\intbl\itap1\pardeftab720\partightenfactor0 \cf0 Shuye,Pu\cell \row \itap1\trowd \taflags1 \trgaph108\trleft-108 \trbrdrl\brdrnil \trbrdrr\brdrnil \clvertalc \clshdrawnil \clbrdrt\brdrs\brdrw20\brdrcf3 \clbrdrl\brdrs\brdrw20\brdrcf3 \clbrdrb\brdrs\brdrw20\brdrcf3 \clbrdrr\brdrs\brdrw20\brdrcf3 \clpadl100 \clpadr100 \gaph\cellx8640 \pard\intbl\itap1\pardeftab720\partightenfactor0 \cf0 Guoqing,Zhong\cell \row \itap1\trowd \taflags1 \trgaph108\trleft-108 \trbrdrl\brdrnil \trbrdrr\brdrnil \clvertalc \clshdrawnil \clbrdrt\brdrs\brdrw20\brdrcf3 \clbrdrl\brdrs\brdrw20\brdrcf3 \clbrdrb\brdrs\brdrw20\brdrcf3 \clbrdrr\brdrs\brdrw20\brdrcf3 \clpadl100 \clpadr100 \gaph\cellx8640 \pard\intbl\itap1\pardeftab720\partightenfactor0 \cf0 Frank,Schmitges\cell \row \itap1\trowd \taflags1 \trgaph108\trleft-108 \trbrdrl\brdrnil \trbrdrr\brdrnil \clvertalc \clshdrawnil \clbrdrt\brdrs\brdrw20\brdrcf3 \clbrdrl\brdrs\brdrw20\brdrcf3 \clbrdrb\brdrs\brdrw20\brdrcf3 \clbrdrr\brdrs\brdrw20\brdrcf3 \clpadl100 \clpadr100 \gaph\cellx8640 \pard\intbl\itap1\pardeftab720\partightenfactor0 \cf0 Ulrich,Braunschweig\cell \row \itap1\trowd \taflags1 \trgaph108\trleft-108 \trbrdrl\brdrnil \trbrdrr\brdrnil \clvertalc \clshdrawnil \clbrdrt\brdrs\brdrw20\brdrcf3 \clbrdrl\brdrs\brdrw20\brdrcf3 \clbrdrb\brdrs\brdrw20\brdrcf3 \clbrdrr\brdrs\brdrw20\brdrcf3 \clpadl100 \clpadr100 \gaph\cellx8640 \pard\intbl\itap1\pardeftab720\partightenfactor0 \cf0 Benjamin,J,Blencowe\cell \row \itap1\trowd \taflags1 \trgaph108\trleft-108 \trbrdrl\brdrnil \trbrdrt\brdrnil \trbrdrr\brdrnil \clvertalc \clshdrawnil \clbrdrt\brdrs\brdrw20\brdrcf3 \clbrdrl\brdrs\brdrw20\brdrcf3 \clbrdrb\brdrs\brdrw20\brdrcf3 \clbrdrr\brdrs\brdrw20\brdrcf3 \clpadl100 \clpadr100 \gaph\cellx8640 \pard\intbl\itap1\pardeftab720\partightenfactor0 \cf0 Jack,F,Greenblatt\cell \lastrow\row \pard\pardeftab720\partightenfactor0 \cf0 \expnd0\expndtw0\kerning0 \ \ track name="Control-RNAPII-ChIPseq.bam" type=bam bigDataUrl=http://vulture.ccbr.utoronto.ca/greenblatt/T4-Scr-RNAPII-2.bam\ track name="FUS-KO-RNAPII-ChIPseq.bam" type=bam bigDataUrl=http://vulture.ccbr.utoronto.ca/greenblatt/T5-Fus-KO-RNAPII-2.bam\ track name="TDP43-KO-RNAPII-ChIPseq.bam" type=bam bigDataUrl=http://vulture.ccbr.utoronto.ca/greenblatt/T6-Tdp-KO-RNAPII-2.bam\ track name="Control-input.bam" type=bam bigDataUrl=http://vulture.ccbr.utoronto.ca/greenblatt/T7-Scr-RNAPII-input.bam\ track name="FUS-KO-input.bam" type=bam bigDataUrl=http://vulture.ccbr.utoronto.ca/greenblatt/T8-Fus-KO-RNAPII-input.bam\ track name="TDP43-KO-input.bam" type=bam bigDataUrl=http://vulture.ccbr.utoronto.ca/greenblatt/T9-Tdp-KO-RNAPII-input.bam}