Investigation Title Genome-Wide Promoter Analysis of Epigenetic Regulation by Cocaine (MM8 data) Comment[Submitted Name] Genome-Wide Promoter Analysis of Epigenetic Regulation by Cocaine (MM8 data) Experimental Design ChIP-chip by tiling array Experimental Design Term Source REF EFO Comment[SecondaryAccession] GSE16184 Comment[ArrayExpressReleaseDate] 2010-05-18 Comment[AEMIAMESCORE] 3 Comment[Publication DOI] 10.1016/j.neuron.2009.03.026 Comment[ArrayExpressAccession] E-GEOD-16184 Comment[MAGETAB TimeStamp_Version] 2010-08-02 16:59:17 Last Changed Rev: 13058 Experimental Factor Name ANTIBODY TREATMENT Experimental Factor Type antibody treatment Experimental Factor Term Source REF Person Last Name xiao Arvind William Person First Name guanghua Kumar Renthal Person Mid Initials Person Email guanghua.xiao@utsouthwestern.edu Person Phone (214)6484553 Person Fax Person Address Psychiatry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX, USA Person Affiliation University of Texas Southwestern Medical Center Person Roles submitter Person Roles Term Source REF The MGED Ontology Quality Control Type Quality Control Term Source REF Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2010-05-18 PubMed ID 19447090 Publication DOI 19447090 Publication Author List Renthal W, Kumar A, Xiao G, Wilkinson M, Covington HE 3rd, Maze I, Sikder D, Robison AJ, LaPlant Q, Dietz DM, Russo SJ, Vialou V, Chakravarty S, Kodadek TJ, Stack A, Kabbaj M, Nestler EJ Publication Title Genome-wide analysis of chromatin regulation by cocaine reveals a role for sirtuins. Publication Status journal_article Publication Status Term Source REF The MGED Ontology Experiment Description Changes in gene expression contribute to the long-lasting regulation of the brain's reward circuitry seen in drug addiction, however, the specific genes regulated and the transcriptional mechanisms underlying such regulation remain poorly understood. Here, we used chromatin immunoprecipitation coupled with promoter microarray analysis to characterize genome-wide epigenetic changes in the mouse nucleus accumbens, a crucial brain reward region, after repeated cocaine administration. Our findings reveal several interesting principles of gene regulation by cocaine and of the role of ΔFosB and CREB, two prominent cocaine-induced transcription factors, in this brain region. Mice were treated with cocaine or saline. Chromatin immunoprecipitation was performed for methylated histone H3 lysine 9 and 27, ∆FosB, and phospho-CREB as described previously (Kumar et al., 2005) with minor modifications. Immunoprecipitated DNA was amplified via ligation-mediated PCR and hybridized to Nimblgen mouse MM8 promoter arrays. 2-3 biological replicates per condition. Protocol Name P-GSE16184-2 P-GSE16184-3 P-GSE16184-4 P-GSE16184-5 P-GSE16184-6 P-GSE16184-7 P-GSE16184-1 Protocol Type specified_biomaterial_action nucleic_acid_extraction labeling hybridization image_aquisition feature_extraction bioassay_data_transformation Protocol Description 7 days of saline or cocaine (20mg/kg). Fresh nucleus accumbens (NAc) punches were cross-linked and lysed, and the chromatin was fragmented by sonication to an average length of 700 bp. For each sample, bilateral NAc punches were pooled from ~8 mice. Aliquots of sonicated chromatin were incubated with 4 µg of the following polyclonal antibodies for 8 hr at 4°C: anti-dimethyl K9/K27 H3 (cat. #ab7312, Abcam, Cambridge, UK); anti-FosB(C-terminus) antibody generated at UT Southwestern; anti-pan FosB/deltaFosB (cat. #SC-048, Santa Cruz Biotechnology, Santa Cruz, CA); and anti-phospho-CREB (cat. #06-519, Upstate). Note that for deltaFosB ChIP, chromatin was first immunoprecipitated with anti-FosB (C-terminus) to precipitate any full-length FosB-bound chromatin, followed by anti-pan FosB/deltaFosB which then precipitates deltaFosB-bound chromatin. These conditions have been shown to selectively precipitate deltaFosB-bound chromatin (Kumar et al., Neuron 2005). Immunprecipitates were reverse cross-linked, and both the immunoprecipitated (enriched) and total (input or non-enriched) DNA from each treatment group was amplified and labeled with a fluorescent dye (Cy5 and Cy3) with the use of ligation-mediated-polymerase chain reaction (Sikder et al., JBC 2006). NimbleGen standard protocol. NimbleGen standard protocol. Samples of Cy5-labeled immunoprecipitated DNA and Cy3-labeled input DNA were mixed and hybridized to NimbleGen (Madison, WI) MM8 mouse promoter microarrays. NimbleGen standard protocol. The log2 ratios of signal intensities between the immunoprecipitated samples and control genomic DNA (input).These ratios were scaled by Tukey's Bi-weight method. ID_REF =
VALUE = log2 ratio (immunoprecipitated sample/control genomic DNA) of signal intensities Protocol Parameters Protocol Hardware Protocol Software Protocol Contact Protocol Term Source REF The MGED Ontology The MGED Ontology SDRF File E-GEOD-16184.sdrf.txt Term Source Name NCBI Taxonomy ArrayExpress EFO The MGED Ontology Term Source File http://www.ncbi.nlm.nih.gov/Taxonomy/ http://www.ebi.ac.uk/arrayexpress http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php Term Source Version