Comment[ArrayExpressAccession]	E-GEOD-39762
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-GSE39762-1	P-GSE39762-5	P-GSE39762-6	P-GSE39762-2	P-GSE39762-3	P-GSE39762-4	P-GSE39762-7
Protocol Description	Robust multi-array average (RMA) method was used with default options (with background correction, quantile normalization, and log transformation) to normalize raw data from batches using R/Bioconductor‘s affy package (Irizarry et al., 2003). ID_REF =  VALUE = RMA normalized intensities	Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).	Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Drosophila Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.	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.	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).	RNA was isolated using TRIzol Reagent (Invitrogen) as per manufacturer’s specifications	GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.
Protocol Type	normalization data transformation protocol	labelling protocol	hybridization protocol	sample treatment protocol	growth protocol	nucleic acid extraction protocol	array scanning protocol
Experimental Factor Name	CELL TYPE						
Experimental Factor Type	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]	GSE39762						
Comment[GEOReleaseDate]	2013-10-18						
Comment[ArrayExpressSubmissionDate]	2012-07-31						
Comment[GEOLastUpdateDate]	2013-10-25						
Comment[AEExperimentType]	transcription profiling by array						
SDRF File	E-GEOD-39762.sdrf.txt