Comment[ArrayExpressAccession]	E-GEOD-8472
Public Release Date	2007-08-23
Investigation Title	Chip-chip from pro-B cells with H3K4me3, H3K4me2 and H3K9ac													
Comment[Submitted Name]	Chip-chip from pro-B cells with H3K4me3, H3K4me2 and H3K9ac
Experiment Description	The transcription factor Pax5 represses B-lineage-inappropriate genes and activates B-cell-specific genes in B-lymphocytes. Here we have identified 170 novel Pax5-activated genes. Conditional mutagenesis demonstrated that the Pax5-regulated genes require continuous Pax5 activity for normal expression in pro-B and mature B cells. Expression of half of the Pax5-activated genes is either absent or significantly reduced upon Pax5 loss in plasma cells. Direct Pax5 target genes were identified based on their protein synthesis-independent activation by a Pax5-estrogen receptor fusion protein. Chromatin immunoprecipitation (ChIP) of Pax5 together with chromatin profiling by ChIP-on-chip analysis demonstrated that Pax5 directly activates the chromatin at promoters or putative enhancers of Pax5 target genes. The novel Pax5-activated genes code for key regulatory and structural proteins involved in B cell signaling, adhesion, migration, antigen presentation and germinal center B cell formation, thus revealing a complex regulatory network, which is activated by Pax5 to control B cell development and function. Keywords: Chip-chip, cell type comparison comparison of Pax5-/-Rag2-/- vs Rag2-/- pro-B cells													
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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	Busslinger	McManus	Busslinger											
Person First Name	Meinrad	Shane	Meinrad											
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Person Email	busslinger@imp.ac.at													
Person Affiliation	Research Institute of Molecular Pathology													
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Person Address	Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, Vienna, Austria													
Person Roles	submitter													
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Protocol Name	P-GSE8472-1	P-GSE8472-10	P-GSE8472-7	P-GSE8472-14	P-GSE8472-3	P-GSE8472-12	P-GSE8472-11	P-GSE8472-6	P-GSE8472-2	P-GSE8472-13	P-GSE8472-4	P-GSE8472-8	P-GSE8472-5	P-GSE8472-9
Protocol Description	ID_REF = <br>VALUE = log2(Signal Cy5/Signal Cy3) - tukey.biweight(Signal Cy5/Signal Cy3)<br>Cy5 = Cy5 signal, 635nm<br>Cy3 = Cy5 signal, 532nm	The labeled ChIP DNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, and hybridized in 45 ul of buffer containing 20% formamide, 1.2 M betaine, 0.1 ug/ul herring sperm DNA and 10 ug of human COT1 DNA (Invitrogen). Arrays were hybridized in Maui hybridization stations for 16-18 h at 42C, and then washed in 42C 0.2% SDS/0.2x SSC, room temperature 0.2x SSC, and 0.05x SSC. Hybridization buffers and washes were completed using manufacturer's protocols (http://www.nimblegen.com/products/lit/lit.html)	see chanel 1	Pax5-/- Rag2-/- pro-B cells were cultured on gamma-irradiated ST2 cells in IL-7 containing IMDM medium.	Rag2-/- pro-B cells were cultured for 5 days in the presence of IL-7 on OP9 feeder cells.	log2(Signal Cy5/Signal Cy3) - tukey.biweight(Signal Cy5/Signal Cy3)	Arrays were scanned on an Axon 4000B scanner per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).	see chanel 1	Rag2-/- pro-B cells were MACs sorted from the bone marrow of 3-5 week old mice.	Pax5-/- Rag2-/- pro-B cells are an established cell line.	5x10E6 cells were fixed with 1% formaldehyde in culture medium for 10 min at room temperature followed by quenching with 0.125 M glycine for 5 min. The cells were washed twice with ice-cold PBS and lysed in 1 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH8.0) for at least 30 min on ice. The crosslinked chromatin was sheared to an average size of 500 bp by ten 30-second pulses using a BiorupterTM sonicator (Diagenode). The lysate was then centrifuged to remove cell debris, and the chromatin was quantified in a UV spectrophotometer. The chromatin (400 µg) was diluted 10-fold in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA 16.7 mM Tris pH8.10, 167 mM NaCl) and pre-cleared with 25 µl protein A sepharose beads (pre-absorbed with salmon sperm DNA and BSA) for 2 h at 4C. The chromatin supernatant was then incubated overnight with 5 µg of purified antibody at 4C. After incubation with protein A sepharose beads (50 µl) for 2h at 4C, the immune complexes were collected by centrifugation and washed with the following buffers each for 10 min at 4C; RIPA buffer (150 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS), high-salt buffer (500 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS, 1% NP40) LiCl buffer (250 mM LiCl, 50 mM Tris pH8.0, 0.5% Na deoxycholate, 1% NP40) and 2x TE (20 mM Tris pH8.0, 2 mM EDTA). The protein-DNA complexes were eluted from the beads in 450 µl elution buffer (1% SDS, 100 mM NaHCO3) at 55C for 2 h followed by the addition of proteinaseK to 500 µg/ml and overnight incubaton at 65C. Genomic DNA was isolated from the precipitated material as well as from the sheared chromatin input (1/100 of the material used for ChIP) by phenol extraction and ethanol precipitation.	see chanel 1	1 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy5 nonamers (ChIP DNA isolated from normal rabbit IgG-immunoprecipitated cells) per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).	1 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers (ChIP DNA isolated from normal rabbit IgG-immunoprecipitated cells) per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
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Protocol Type	bioassay_data_transformation	hybridization	grow	grow	grow	feature_extraction	image_aquisition	specified_biomaterial_action	specified_biomaterial_action	specified_biomaterial_action	nucleic_acid_extraction	nucleic_acid_extraction	labeling	labeling
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Publication Title	Transcription factor Pax5 activates the chromatin of key genes involved in B cell signaling, adhesion, migration, and immune function.													
Publication Author List	Schebesta A, McManus S, Salvagiotto G, Delogu A, Busslinger GA, Busslinger M													
PubMed ID	17658281													
Publication DOI	10.1016/j.immuni.2007.05.019													
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Comment[SecondaryAccession]	GSE8472													
Comment[GEOLastUpdateDate]	2007-08-22
Comment[AEExperimentType]	ChIP-chip by tiling array													
Comment[GEOReleaseDate]	2007-08-22
Comment[ArrayExpressSubmissionDate]	2007-07-12
SDRF File	E-GEOD-8472.sdrf.txt