Comment[ArrayExpressAccession]	E-GEOD-42360
MAGE-TAB Version	1.1					
Public Release Date	2013-04-24
Investigation Title	Genome-wide control of RNA polymerase II activity by cohesin (tiling)					
Comment[Submitted Name]	Genome-wide control of RNA polymerase II activity by cohesin (tiling)
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 cohesinM-bM-^@M-^Ys 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. We performed ChIP-chip of Rpb3 (representing total Pol II), Ser2P-Pol II (representing elongating Pol II), and Cdk12 and CycT Pol II kinase components in Mock RNAi-treated and cohesin subunit Rad21 RNAi-treated ML-DmBG3-c2 cells, which revealed that cohesin depletion has a variety of effects on Pol II occupancy and modification, as well as on occupancy of Pol II kinases.					
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	Dorsett			
Person First Name	Cheri	Cheri	Dale			
Person Mid Initials	A	A				
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-GSE42360-4	P-GSE42360-5	P-GSE42360-1	P-GSE42360-2	P-GSE42360-3	P-GSE42360-6
Protocol Description	Immunoprecipitated DNA was amplified using the GenomePlex Complete Whole Genome Amplification Kit (Sigma) and fragmented using controlled DNAse I reactions. Fragmented DNA was labeled with bio-11-ddATP (Perkin Elmer) using a Terminal Transferase labeling kit (Roche).	Hybridization cocktails were generated using the labeled DNA fragments (2 M-NM-<g labeled DNA, 1x MES, 3M TMAC, 40 pM Affymetrix control oligo B2, 100 M-NM-<g/mL salmon sperm DNA, and 0.02% Triton X-100). Each chip was pre-hybridized with 200 M-NM-<l of 1X MES-triton for 1 hour in the Affymetrix Hybridization 645 oven at 45 rpm and 45oC. Samples in hybridization cocktail were heated for 10 minutes at 99oC then incubated to a 45oC for 10 minutes. Samples are spun in a microcentrifuge at maximum speed for 3 min. 200 M-NM-<l of a sample was injected in to a chip. Hybridization was carry out in the hybridization oven at 45 rpm and 45oC for 18 hours. The arrays were washed and stained on the GeneChip Fluidics Station 450 series.	For RNAi, media was replaced with 1 ml of Express Five SFM (Invitrogen) with 1% FCS, (and 10 M-NM-<g per ml insulin for BG3 cells), for 2 hours. 10 M-NM-<g of dsRNA was added per well of a 6-well plate. Media was adjusted to 3 ml and 10% FCS withSchneider'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 M-bM-^IM-%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.	BG3 cells were cultured in Schneider's media with 10% FCS and 10 M-NM-<g per ml insulin.	BG3 cells were grown to a concentration of 5x106/mL, then crosslinked by addition of 36% formaldehyde directly to the cell culture media to a final concentration of 1%, and incubation for 10 minutes at room temperature on a rocking platform. The reaction was stopped by addition of glycine pH 7.0 to a final concentration of 0.125 M. Cells were washed once with 1x PBS and once each with Wash Buffers A (10 mM HEPES pH 7.6; 10 mM EDTA pH 8.0; 0.5 mM EGTA pH 8.0; 0.25% Triton X100) and B (10 mM HEPES pH 7.6; 200 mM NaCl; 1 mM EDTA pH 8.0; 0.5 mM EGTA pH 8.0; 0.01% Triton X-100) for 10 minutes each. Cells were resuspended in sonication buffer to a concentration of 1x109/3 mL, and sonicated using a BioRuptor Sonicator for 14-20 cycles of 30 s on, 30 s off. Na-lauroylsarcosine was added to 0.5% and incubated for 10 minutes, rotating. Chromatin was centrifuged at 12,000g for 5 minutes at 4M-0C to pellet insoluble material, and 200 uL chromatin aliquots, each representing ~50x106 cells, were snap frozen on dry ice. For immunoprecipitation, 200 M-NM-<L aliquots of chromatin (50 x 106 BG3 cells) were adjusted to RIPA buffer in a final volume of 500 M-NM-<L. Adjusted chromatin was pre-cleared for 2 hours with Protein A agarose beads, then incubated with primary antibody overnight at 4M-0C. Antibody-chromatin complexes were pulled down by incubation with Protein A/G agarose beads for 4 hours at 4M-0C, and beads were washed consecutively with RIPA, LiCl buffer, and TE. Samples were treated with RNAse A for 30 minutes at 37M-0C, then DNA crosslinks were reversed by overnight treatment at 65M-0C with Proteinase K, and DNA was recovered using Qiagen MinElute columns.	Arrays were scanned on an Affymetrix GeneChipM-. Scanner 3000 7G.
Protocol Type	labelling protocol	hybridization protocol	sample treatment protocol	growth protocol	nucleic acid extraction protocol	array scanning protocol
Experimental Factor Name	TREATMENT	ANTIBODY				
Experimental Factor Type	treatment	antibody				
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]	GSE42360					
Comment[GEOReleaseDate]	2013-04-24					
Comment[ArrayExpressSubmissionDate]	2012-11-16					
Comment[GEOLastUpdateDate]	2013-04-24					
Comment[AEExperimentType]	ChIP-chip by tiling array					
Comment[AdditionalFile:Data1]	GSE42360_Input_Repl1.CEL					
Comment[AdditionalFile:Data2]	GSE42360_Input_Repl2.CEL					
Comment[AdditionalFile:Data3]	GSE42360_Input_Repl3.CEL					
SDRF File	E-GEOD-42360.sdrf.txt