Comment[ArrayExpressAccession]	E-GEOD-20213
Public Release Date	2010-05-14
Investigation Title	ChIP for H3K27me3 in Murine ES Cells: wild type and Ring1B-/- cells							
Comment[Submitted Name]	ChIP for H3K27me3 in Murine ES Cells: wild type and Ring1B-/- cells
Experiment Description	Native ChIP on chip for H3K27me3 in murine ES cells comparing WT and Ring1B-/- cells. Paper Abstract: How polycomb group proteins repress gene expression in vivo is not known. Whilst histone modifying activities of the polycomb repressive complexes have been studied extensively, in vitro data has suggested a direct activity of the PRC1 complex in compacting chromatin. Here, we investigate higher-order chromatin compaction of polycomb targets in vivo. We show that polycomb repressive complexes are required to maintain a compact chromatin state at Hox loci in embryonic stem (ES) cells. There is specific decompaction in the absence of PRC2 or PRC1. This is due to PRC1, since decompaction occurs in Ring1B null cells that still have PRC2-mediated H3K27 methylation. Moreover, we show that the ability of Ring1B to restore a compact chromatin state, and to repress Hox gene expression in ES cells, is not dependent on its histone ubiquitination activity. We suggest that Ring1B-mediated chromatin compaction acts to directly limit transcription in vivo. Biological replicates: 3 independently grown, harvested,preplated, micrococcal nuclease digested and ChIP for H3K27me3. 5 Technical replicates.							
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Term Source Name	EFO
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Term Source File	http://efo.svn.sourceforge.net/viewvc/efo/trunk/src/efoinowl/efo.owl							
Person Last Name	Grimes	Eskeland	Grimes	Bickmore				
Person First Name	Graeme	Ragnhild	Graeme	Wendy				
Person Mid Initials	Richard		R	A				
Person Email	Graeme.Grimes@hgu.mrc.ac.uk							
Person Affiliation	Medical Research Council							
Person Phone	+44 (0)131 332 2471							
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Person Address	Human Genetics Unit, Medical Research Council, Western General Hospital, Crewe Road, Edinburgh, UK							
Person Roles	submitter							
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Protocol Name	P-GSE20213-1	P-GSE20213-6	P-GSE20213-3	P-GSE20213-8	P-GSE20213-7	P-GSE20213-2	P-GSE20213-4	P-GSE20213-5
Protocol Description	ID_REF = <br>VALUE = Normalized log2 ratio (Cy5/Cy3 or Cy3/Cy5)) representing ChIP/Input	Using an in-house humidity chamber for 45 hours at 45 °C	Grown on mitomycin C-treated primary embryonic fibroblasts (PEFs) derived from E12.5 mouse embryos, in DMEM supplemented with 15% fetal calf serum (FCS), 1000 units/ml LIF, non-essential amino acids, sodium pyruvate, 2-mecaptoethanol, L-glutamine, and Penicillin/Streptomycin. For analysis of chromatin,ES cells were trypsinized and the PEFs allowed reattaching 2x 30 min. (Ref cells: J Cell Biol. 2007 Jul 16;178(2):219-29:PMID: 17620408 )	BlueFuse software package v3.3 (BlueGnome).Four replicate spots combined and no background subtractions.Normalised by Global Lowess (f = 1/4, iter = 3).	ProScan Array HT scanner (Perkin Elmer) using a laser power of 90% and 10 µm resolution.	Nuclei micrococcal nuclease digested and chromatin released. ChIP for H3K27me3 (ab6002).	RNase A and ProteinaseK digestion. Purified using Qiagen PCR purification kit (Qiagen).	100 ng input (micrococcal nuclease-digested chromatin) and 50-100 ng immuno-enriched material were labelled with Alexa3 and Alexa 5 (and dye swap) using Bioprime Kit according to manufacturers instructions (Invitrogen)
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Protocol Type	bioassay_data_transformation	hybridization	grow	feature_extraction	image_aquisition	specified_biomaterial_action	nucleic_acid_extraction	labeling
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Experimental Factor Name	GENOTYPE	ANTIBODY						
Experimental Factor Type	genotype	antibody						
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Publication Title	Ring1B compacts chromatin structure and represses gene expression independent of histone ubiquitination.							
Publication Author List	Eskeland R, Leeb M, Grimes GR, Kress C, Boyle S, Sproul D, Gilbert N, Fan Y, Skoultchi AI, Wutz A, Bickmore WA							
PubMed ID	20471950							
Publication DOI	10.1016/j.molcel.2010.02.032							
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Comment[SecondaryAccession]	GSE20213							
Comment[GEOLastUpdateDate]	2010-07-14
Comment[AEExperimentType]	ChIP-chip by tiling array							
Comment[GEOReleaseDate]	2010-05-13
Comment[ArrayExpressSubmissionDate]	2010-02-05
SDRF File	E-GEOD-20213.sdrf.txt