Journal: Molecular cell

Article Title: The Integrator complex attenuates promoter-proximal transcription at protein-coding genes

doi: 10.1016/j.molcel.2019.10.034

Figure Lengend Snippet: (A) IntS11 harbors RNA endonuclease activity (depicted as scissors). Cells were depleted of IntS11 and rescued using a stably integrated transgene expressing WT IntS11, or IntS11 with a mutation that disrupts endonuclease activity (E203Q). To specifically deplete endogenous IntS11 from the rescue cell lines, a dsRNA targeting the untranslated (UTR) regions of endogenous IntS11 (green) was used. Cells were treated for 60 h with control or IntS11 UTR RNAi (N=3, see STAR Methods), and RNA harvested for RNA-seq.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies IntS1 Ezzeddine et al., 2011 IntS9 Ezzeddine et al., 2011 IntS11 Ezzeddine et al., 2011 alpha-tubulin abcam ab15246 AB_301787 H3K4me3 EpiCypher 13-0028 Normal Rabbit serum Invitrogen 01-610-1 Chemicals, Peptides, and Recombinant Proteins AzNTPs TriLink Technologies Cat# K-1005 Biotin-11-NTPs Perkin Elmer Cat# NEL54(2/3/4/5)001 Critical Commercial Assays NEBNext Ultra II DNA library kit NEB Cat# E7103S Deposited Data Raw and analyzed data This paper GEO: {"type":"entrez-geo","attrs":{"text":"GSE114467","term_id":"114467"}} GSE114467 Start-seq from Triptolide-treated S2 cells Krebs et al., 2017 GEO: {"type":"entrez-geo","attrs":{"text":"GSE77369","term_id":"77369"}} GSE77369 H3K4me1, H3K4me3 and H3K36me3 ChIP-seq from S2 cells Henriques et al., 2018 GEO: {"type":"entrez-geo","attrs":{"text":"GSE85191","term_id":"85191"}} GSE85191 Start-seq from Control and Rrp40-depleted S2 cells Henriques et al., 2013 GEO: {"type":"entrez-geo","attrs":{"text":"GSE49078","term_id":"49078"}} GSE49078 Chromatin RNA-seq from IntS11-depleted HeLa cells Lai et al., 2015 GEO: {"type":"entrez-geo","attrs":{"text":"GSE68401","term_id":"68401"}} GSE68401 H3K4me1, H3K4me3 and H3K36me3 ChIP-seq from HeLa cells Gerstein et al., 2012 GEO: {"type":"entrez-geo","attrs":{"text":"GSE29611","term_id":"29611"}} GSE29611 PRO-seq from DMSO-treated HeLa cells Nilson et al. 2017 GEO: {"type":"entrez-geo","attrs":{"text":"GSE100742","term_id":"100742"}} GSE100742 Chromatin RNA-seq from Xrn2 or CPS73-depleted HeLa cells Nojima et al., 2015 GEO: {"type":"entrez-geo","attrs":{"text":"GSE60358","term_id":"60358"}} GSE60358 Experimental Models: Cell Lines DL1 cells Dr. Sara Cherry, UPenn S2-DGRC clone 6 DGRC Stock number 6 DL1 FLAG-eGFP This paper DL1 FLAG-IntS11WT This paper DL1 FLAG-IntS11E203Q This paper Oligonucleotides Table S3 This paper Recombinant DNA pUB-3xFLAG vector Chen et al., 2012 pMT-puro David Sabatini lab Addgene #17923 pMT-FLAG-dIntS11-WT-puro This paper pMT-FLAG-dIntS11-E203Q-puro This paper pMT-FLAG-eGFP-puro This paper Software and Algorithms bowtie 1.2.2 Langmead et al., 2009 R v3.3.1 www.r-project.org Rstudio v1.0.136 www.rstudio.com featureCounts Liao et al., 2014 DESeq2 Love et al. , 2014 MISO Katz et al., 2010 Prism v8.1.2 GraphPad Partek Genomics Suite v6.15.0127 www.partek.com Open in a separate window KEY RESOURCES TABLE Integrator inhibits transcription elongation at ~15% of mRNA genes and enhancers Integrator targets promoter-proximally paused Pol II for termination The RNA endonuclease of Integrator subunit 11 is critical for gene repression Integrator-depletion increases productive elongation and Histone H3 K4 methylation

Techniques: Activity Assay, Stable Transfection, Expressing, Mutagenesis, RNA Sequencing Assay

Journal: Molecular cell

Article Title: The Integrator complex attenuates promoter-proximal transcription at protein-coding genes

doi: 10.1016/j.molcel.2019.10.034

Figure Lengend Snippet:

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies IntS1 Ezzeddine et al., 2011 IntS9 Ezzeddine et al., 2011 IntS11 Ezzeddine et al., 2011 alpha-tubulin abcam ab15246 AB_301787 H3K4me3 EpiCypher 13-0028 Normal Rabbit serum Invitrogen 01-610-1 Chemicals, Peptides, and Recombinant Proteins AzNTPs TriLink Technologies Cat# K-1005 Biotin-11-NTPs Perkin Elmer Cat# NEL54(2/3/4/5)001 Critical Commercial Assays NEBNext Ultra II DNA library kit NEB Cat# E7103S Deposited Data Raw and analyzed data This paper GEO: {"type":"entrez-geo","attrs":{"text":"GSE114467","term_id":"114467"}} GSE114467 Start-seq from Triptolide-treated S2 cells Krebs et al., 2017 GEO: {"type":"entrez-geo","attrs":{"text":"GSE77369","term_id":"77369"}} GSE77369 H3K4me1, H3K4me3 and H3K36me3 ChIP-seq from S2 cells Henriques et al., 2018 GEO: {"type":"entrez-geo","attrs":{"text":"GSE85191","term_id":"85191"}} GSE85191 Start-seq from Control and Rrp40-depleted S2 cells Henriques et al., 2013 GEO: {"type":"entrez-geo","attrs":{"text":"GSE49078","term_id":"49078"}} GSE49078 Chromatin RNA-seq from IntS11-depleted HeLa cells Lai et al., 2015 GEO: {"type":"entrez-geo","attrs":{"text":"GSE68401","term_id":"68401"}} GSE68401 H3K4me1, H3K4me3 and H3K36me3 ChIP-seq from HeLa cells Gerstein et al., 2012 GEO: {"type":"entrez-geo","attrs":{"text":"GSE29611","term_id":"29611"}} GSE29611 PRO-seq from DMSO-treated HeLa cells Nilson et al. 2017 GEO: {"type":"entrez-geo","attrs":{"text":"GSE100742","term_id":"100742"}} GSE100742 Chromatin RNA-seq from Xrn2 or CPS73-depleted HeLa cells Nojima et al., 2015 GEO: {"type":"entrez-geo","attrs":{"text":"GSE60358","term_id":"60358"}} GSE60358 Experimental Models: Cell Lines DL1 cells Dr. Sara Cherry, UPenn S2-DGRC clone 6 DGRC Stock number 6 DL1 FLAG-eGFP This paper DL1 FLAG-IntS11WT This paper DL1 FLAG-IntS11E203Q This paper Oligonucleotides Table S3 This paper Recombinant DNA pUB-3xFLAG vector Chen et al., 2012 pMT-puro David Sabatini lab Addgene #17923 pMT-FLAG-dIntS11-WT-puro This paper pMT-FLAG-dIntS11-E203Q-puro This paper pMT-FLAG-eGFP-puro This paper Software and Algorithms bowtie 1.2.2 Langmead et al., 2009 R v3.3.1 www.r-project.org Rstudio v1.0.136 www.rstudio.com featureCounts Liao et al., 2014 DESeq2 Love et al. , 2014 MISO Katz et al., 2010 Prism v8.1.2 GraphPad Partek Genomics Suite v6.15.0127 www.partek.com Open in a separate window KEY RESOURCES TABLE Integrator inhibits transcription elongation at ~15% of mRNA genes and enhancers Integrator targets promoter-proximally paused Pol II for termination The RNA endonuclease of Integrator subunit 11 is critical for gene repression Integrator-depletion increases productive elongation and Histone H3 K4 methylation

Techniques:

Journal: Molecular cell

Article Title: The Integrator complex attenuates promoter-proximal transcription at protein-coding genes

doi: 10.1016/j.molcel.2019.10.034

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies IntS1 Ezzeddine et al., 2011 IntS9 Ezzeddine et al., 2011 IntS11 Ezzeddine et al., 2011 alpha-tubulin abcam ab15246 AB_301787 H3K4me3 EpiCypher 13-0028 Normal Rabbit serum Invitrogen 01-610-1 Chemicals, Peptides, and Recombinant Proteins AzNTPs TriLink Technologies Cat# K-1005 Biotin-11-NTPs Perkin Elmer Cat# NEL54(2/3/4/5)001 Critical Commercial Assays NEBNext Ultra II DNA library kit NEB Cat# E7103S Deposited Data Raw and analyzed data This paper GEO: {"type":"entrez-geo","attrs":{"text":"GSE114467","term_id":"114467"}} GSE114467 Start-seq from Triptolide-treated S2 cells Krebs et al., 2017 GEO: {"type":"entrez-geo","attrs":{"text":"GSE77369","term_id":"77369"}} GSE77369 H3K4me1, H3K4me3 and H3K36me3 ChIP-seq from S2 cells Henriques et al., 2018 GEO: {"type":"entrez-geo","attrs":{"text":"GSE85191","term_id":"85191"}} GSE85191 Start-seq from Control and Rrp40-depleted S2 cells Henriques et al., 2013 GEO: {"type":"entrez-geo","attrs":{"text":"GSE49078","term_id":"49078"}} GSE49078 Chromatin RNA-seq from IntS11-depleted HeLa cells Lai et al., 2015 GEO: {"type":"entrez-geo","attrs":{"text":"GSE68401","term_id":"68401"}} GSE68401 H3K4me1, H3K4me3 and H3K36me3 ChIP-seq from HeLa cells Gerstein et al., 2012 GEO: {"type":"entrez-geo","attrs":{"text":"GSE29611","term_id":"29611"}} GSE29611 PRO-seq from DMSO-treated HeLa cells Nilson et al. 2017 GEO: {"type":"entrez-geo","attrs":{"text":"GSE100742","term_id":"100742"}} GSE100742 Chromatin RNA-seq from Xrn2 or CPS73-depleted HeLa cells Nojima et al., 2015 GEO: {"type":"entrez-geo","attrs":{"text":"GSE60358","term_id":"60358"}} GSE60358 Experimental Models: Cell Lines DL1 cells Dr. Sara Cherry, UPenn S2-DGRC clone 6 DGRC Stock number 6 DL1 FLAG-eGFP This paper DL1 FLAG-IntS11WT This paper DL1 FLAG-IntS11E203Q This paper Oligonucleotides Table S3 This paper Recombinant DNA pUB-3xFLAG vector Chen et al., 2012 pMT-puro David Sabatini lab Addgene #17923 pMT-FLAG-dIntS11-WT-puro This paper pMT-FLAG-dIntS11-E203Q-puro This paper pMT-FLAG-eGFP-puro This paper Software and Algorithms bowtie 1.2.2 Langmead et al., 2009 R v3.3.1 www.r-project.org Rstudio v1.0.136 www.rstudio.com featureCounts Liao et al., 2014 DESeq2 Love et al. , 2014 MISO Katz et al., 2010 Prism v8.1.2 GraphPad Partek Genomics Suite v6.15.0127 www.partek.com Open in a separate window KEY RESOURCES TABLE Integrator inhibits transcription elongation at ~15% of mRNA genes and enhancers Integrator targets promoter-proximally paused Pol II for termination The RNA endonuclease of Integrator subunit 11 is critical for gene repression Integrator-depletion increases productive elongation and Histone H3 K4 methylation

Techniques: Recombinant, Plasmid Preparation, Software