Comment[ArrayExpressAccession] E-GEOD-39912 MAGE-TAB Version 1.1 Public Release Date 2013-10-18 Investigation Title Genome Wide Profiling of p53 Response to Differentiation or DNA Damage of Human Embryonic Stem Cells Comment[Submitted Name] Genome Wide Profiling of p53 Response to Differentiation or DNA Damage of Human Embryonic Stem Cells Experiment Description Tumor suppressor p53 promotes differentiation of human embryonic stem cells (hESCs), but an in-depth understanding of mechanism is lacking. Here, we define p53 functions in hESCs by genome wide profiling of p53 chromatin interactions and intersection with gene expression during early differentiation and in response to DNA damage. During differentiation, p53 targets and regulates a unique collection of genes, many of which encode transcription factors and developmental regulators with chromatin structure poised by OCT4 and NANOG and marked by repressive H3K27me3 in pluripotent hESCs. In contrast, genes associated with cell migration and motility are bound by p53 specifically after DNA damage. Surveillance functions of p53 in regulation of cell death and cell cycle genes are conserved during both DNA damage and differentiation. Our findings expand the registry of p53 -regulated genes in hESCs and define specific functions of p53 in opposing pluripotency, which are highly distinct from stress-induced p53 response in stem cells. Identification of p53 binding sites in hESC under three conditions : Pluripotent, DNA damaged, Differentiating 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 Akdemir Akdemir Person First Name Kadir Kadir Person Mid Initials Caner C Person Email kcakedemir@mdanderson.org Person Affiliation MD Anderson Cancer Center Person Address MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX, USA Person Roles submitter Protocol Name P-GSE39912-4 P-GSE39912-1 P-GSE39912-3 P-GSE39912-2 Protocol Description Sequence reads were obtained and mapped to the February 2006 human reference sequence (hg18) using the Illumina Genome Analyzer Pipeline using Illumina’s ELAND software. Enriched regions for each condition were normalized to input DNA and detected with MACS (Zhang et al., 2008) with a cut-off P < 10-8 for Damage and Differentiation P< 10-10 for Untreated. Genome_build: hg18 Supplementary_files_format_and_content: peak and aligned bed files For differentiating hESCs cultured on mTeSR1, 1 μM Retinoic Acid (RA) was added to homemade MEF-CM (Conditioned Media, without additional FGF). CM media was prepared in our facility by culturing γ-irradiated MEFs in complete hESC culture media for 24 hours, and collected every day, filtered and freezed at -20 degrees. FGF was added to CM media before use to a final concentration of 10 ng/ml to culture the cells grown on Matrigel under pluripotent conditions. Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols. Human Embryonic Stem (WA09) Cells were obtained from National Stem Cell Bank (Madison, WI) and cultured according to the protocol from WiCell Research Insitute. Briefly, WA09 cells were maintained in hESC culture medium on γ-irradiated Mouse Embryonic Fibroblasts (MEFs) prepared using WiCell instructions. hESCs ranging from passage number 32-38 were used for all of our experiments. hESC complete culture medium is composed of DMEM/F12 supplemented with 20% knockout serum replacement, 1 mM L-glutamine, 1% nonessential amino acids, 4 ng/ml human FGF2 (all from Invitrogen), and 0.1 mM 2-mercaptoethanol (Sigma). The medium was changed daily, and cells were passaged every 4–6 days with 1 mg/ml Collagen IV (Invitrogen). For differentiation studies hESCs were cultured in differentiation media (hESC media without FGF) containing 1 μM Retinoic Acid (RA) with fresh medium change daily. hESCs were also maintained as feeder-free cultures on hESC qualified Matrigel (BD Biosciences) in mTeSR1 media (Stem Cell Technologies) and CM (MEF conditioned media). Passage 32 hESCs were grown on mTeSR1 media for 5 passages. hESCs were cultured on Matrigel (BD Biosciences) following manufacturer’s instructions, received fresh mTeSR1 media everyday and cells were passaged every 4-6 days with 1 mg/ml Dispase (Stem Cell Technologies). Protocol Type normalization data transformation protocol sample treatment protocol nucleic acid library construction protocol growth protocol Experimental Factor Name ANTIBODY VENDOR CHIP ANTIBODY CELL TYPE Experimental Factor Type antibody vendor chip antibody cell type Publication Title Genome-wide profiling reveals stimulus-specific functions of p53 during differentiation and DNA damage of human embryonic stem cells. Publication Author List Akdemir KC, Jain AK, Allton K, Aronow B, Xu X, Cooney AJ, Li W, Barton MC PubMed ID 24078252 Publication DOI 10.1093/nar/gkt866 Comment[SecondaryAccession] GSE39912 Comment[GEOReleaseDate] 2013-10-18 Comment[ArrayExpressSubmissionDate] 2012-08-06 Comment[GEOLastUpdateDate] 2013-11-11 Comment[AEExperimentType] ChIP-seq Comment[SecondaryAccession] SRP015876 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR575903-SRR575945 SDRF File E-GEOD-39912.sdrf.txt