Comment[ArrayExpressAccession] E-GEOD-42397 MAGE-TAB Version 1.1 Public Release Date 2013-04-24 Investigation Title Genome-wide control of RNA polymerase II activity by cohesin (sequencing) Comment[Submitted Name] Genome-wide control of RNA polymerase II activity by cohesin (sequencing) Experiment Description Cohesin is a well-known mediator of sister chromatid cohesion, but it also influences gene expression and development. These non-canonical roles of cohesin are not well understood, but are vital: gene expression and development are altered by modest changes in cohesin function that do not disrupt chromatid cohesion. To clarify cohesin’s roles in transcription, we measured how cohesin controls RNA polymerase II (Pol II) activity by genome-wide chromatin immunoprecipitation and precision global run-on sequencing. On average, cohesin-binding genes have more transcriptionally active Pol II and promoter-proximal Pol II pausing than non-binding genes, and are more efficient, producing higher steady state levels of mRNA per transcribing Pol II complex. Cohesin depletion frequently increases pausing at cohesin-binding genes, indicating that cohesin often facilitates transition of paused Pol II to elongation. In many cases this likely reflects a role for cohesin in transcriptional enhancer function. Strikingly, more than 95% of predicted extragenic enhancers bind cohesin, and cohesin depletion can reduce their association with Pol II, indicating that cohesin facilitates enhancer-promoter contact. Cohesin directly promotes transcription of the myc gene, and cohesin depletion reduces Pol II activity at most Myc target genes. The multiple transcriptional roles of cohesin revealed by these studies likely underlie the growth and developmental deficits caused by minor changes in cohesin activity. The PRO-seq method was used to measure transcriptionally engaged Pol II genome-wide in two replicates each of mock RNAi-treated, Nipped-B RNAi-treated, and Rad21 RNAi-treated ML-DmBG3-c2 cells. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Schaaf Schaaf Kwak Misulovin Koenig Lis Dorsett Person First Name Cheri Cheri Hojoong Ziva Amanda John Dale Person Mid Initials A A T Person Email cheri.schaaf@gmail.com Person Affiliation Saint Louis University School of Medicine Person Phone 314-977-9224 Person Address Biochemistry & Molecular Biology, Saint Louis University School of Medicine, 1100 South Grand Ave, St Louis, MO, USA Person Roles submitter Protocol Name P-GSE42397-4 P-GSE42397-1 P-GSE42397-3 P-GSE42397-2 Protocol Description Raw sequenced reads were trimmed for adaptor sequence using fastx_clipper of the FASTX-Toolkit. The first 26 bases of each read were trimmed using fastx_trimmer of the FASTX-Toolkit, and sequence reads shorter than 16 bp were removed. Reverse complementation of the sequence reads, which were the sense sequences of nascent RNA, were generated using fastx_reverse_complement of the FASTX-Toolkit. Sequence reads were aligned to dm5.22 whole genome using bowtie allowing 2 mismatches and excluding any non-uniquely aligned reads. Abundance measurements were generated in bedgraph format. Genome_build: dm5.22 Supplementary_files_format_and_content: bedgraph files provide abundance measurements. For RNAi, media was replaced with 1 ml of Express Five SFM (Invitrogen) with 1% FCS, (and 10 μg per ml insulin for BG3 cells), for 2 hours. 10 μg of dsRNA was added per well of a 6-well plate. Media was adjusted to 3 ml and 10% FCS with Schneider's media after 2 hrs of RNAi treatment. Cells were replated as needed. Templates for dsRNA synthesis were made by PCR from cDNA or genomic DNA templates using primers with T7 promoters. In most experiments, equal amounts of two dsRNAs against each target were used. Both individual dsRNAs knocked down the targets, but knockdown was generally more efficient with a mixture. Templates were designed to avoid off-target matches of ≥19 nucleotides using Drosophila RNAi Screening Center (http://www.flyrnai.org/) online tools. Cohesin RNAi-treated cells were screened for sister chromatid cohesion defects, and none were found, though a mild cell division delay occurred immediately following RNAi treatment. Nuclei Isolation: All steps were conducted at 4o unless indicated otherwise. 2.5 to 7.5 x 108 mock RNAi-treated control or RNAi-treated BG3 cells were collected by centrifugation (1000g for 5 min), suspended in 5 to 10 mL Phosphate Buffered Saline (PBS) pH 7.0, collected by centrifugation, suspended in 5 mL Buffer W [10 mM Tris-HCl pH 7.5,10 mM KCl, 150 mM sucrose 5 mM MgCl2, 0.5 mM CaCl2, 0.5 mM dithiothreitol (DTT)], and collected by centrifugation. The cells were suspended in 5 mL Buffer P (10mM Tris-HCl, pH 7.5, 10 mM KCl, 250 mM sucrose, 5 mM MgCl2, 1 mM EGTA, 0.05% Tween-20, 0.5 mM DTT), the suspension was adjusted to 0.14% NP-40, and then incubated on ice for 3 min. The nuclei were washed twice in 5 mL Buffer W, suspended in 1 mL Buffer W, and transferred to a siliconized 1.5 mL microcentrifuge tube. The nuclei were collected by centrifugation at 1000g for 5 min, suspended in 0.5 mL Buffer F (50 mM Tris-HCL pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT), and counted using a hemacytometer. The nuclei were then suspended in Buffer F to concentration of 40 to 50 x 105 per microliter, distributed into 100 microliter aliquots in siliconized 1.5 mL tubes, snap frozen in liquid nitrogen, and stored at -80o. Precision Run-On sequencing (PRO-seq): biotin-NTP nuclear run-on; RNA extraction; streptavidin affinity purification; base-hydrolysis; 3' RNA adaptor ligation; 5' end repair; 5' RNA adaptor ligation; reverse transcription; PCR amplification. BG3 cells were cultured in Schneider's media with 10% FCS and 10 μg per ml insulin. Protocol Type normalization data transformation protocol sample treatment protocol nucleic acid library construction protocol growth protocol Experimental Factor Name TREATMENT Experimental Factor Type treatment Publication Title Genome-Wide Control of RNA Polymerase II Activity by Cohesin. Publication Author List Schaaf CA, Kwak H, Koenig A, Misulovin Z, Gohara DW, Watson A, Zhou Y, Lis JT, Dorsett D PubMed ID 23555293 Publication DOI 10.1371/journal.pgen.1003382 Comment[SecondaryAccession] GSE42397 Comment[GEOReleaseDate] 2013-04-24 Comment[ArrayExpressSubmissionDate] 2012-11-19 Comment[GEOLastUpdateDate] 2013-04-24 Comment[AEExperimentType] RNA-seq of coding RNA Comment[SecondaryAccession] SRP017251 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR618498-SRR618503 SDRF File E-GEOD-42397.sdrf.txt