Comment[ArrayExpressAccession]	E-GEOD-48344
MAGE-TAB Version	1.1						
Public Release Date	2013-06-27
Investigation Title	Pruning of the adipocyte cistrome by hematopoietic master regulator PU.1 (expression)						
Comment[Submitted Name]	Pruning of the adipocyte cistrome by hematopoietic master regulator PU.1 (expression)
Experiment Description	Master transcription factors are the gatekeepers of lineage identity. As such, they have been a major focus of efforts to manipulate cell fate for therapeutic purposes. The ETS transcription factor PU.1 has a potent ability to confer macrophage phenotypes on cells already committed to a different lineage, but how it overcomes the presence of other master regulators is not known. The nuclear receptor PPARγ is the master regulator of the adipose lineage, and its genomic binding pattern is well characterized in adipocytes. Here, we show that when expressed at macrophage levels in mature adipocytes, PU.1 bound a large fraction of its macrophage sites, where it induced chromatin opening and the expression of macrophage target genes. Strikingly, PU.1 markedly reduced the genomic binding of PPARγ without changing its abundance. PU.1 expression repressed genes with nearby adipocyte-specific PPARγ binding sites, while a common macrophage-adipocyte gene expression program was retained. Together, these data reveal unexpected lability within the adipocyte PPARγ cistrome and show that even in terminally differentiated cells, PU.1 can remodel the cistrome of another master regulator. Microarray expression profiling was performed on 3T3-L1 adipocytes from two treatment groups: (1) adipocytes transduced with a control adenovirus expressing beta-galactosidase (LACZ-Ads) and (2) adipocytes transduced with an adenovirus expressing full-length murine PU.1 cDNA (PU.1-Ads). Each sample group consists of four biological replicates which are here defined as separate differentiations of mature 3T3-L1 adipocytes and adenoviral infections. Each replicate was hybridized to an individual array for a total of eight arrays.						
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	DiSpirito	DiSpirito	Fang	Wang	Lazar		
Person First Name	Joanna	J	B	F	M		
Person Mid Initials	R	R			A		
Person Email	rdo2@mail.med.upenn.edu						
Person Affiliation	University of Pennsylvania						
Person Address	IDOM, University of Pennsylvania, Smilow Center for Translational Research, 3400 Civic Center Boulevard, Philadelphia, PA, USA						
Person Roles	submitter						
Protocol Name	P-GSE48344-1	P-GSE48344-5	P-GSE48344-6	P-GSE48344-2	P-GSE48344-3	P-GSE48344-4	P-GSE48344-7
Protocol Description	Data was quantile normalized in R with the limma package's function normalizeBetweenArray. FDRs were calculated using the SAMR package. ID_REF =  VALUE = Quantile-normalized signal intensity, log2	One-color RNA spike-in was diluted at 1:7500 in Dilution Buffer (Agilent, Cat # 5188-5282). T7 promoter primer from Low Input Quick Amp Labeling Kit one-color (Agilent 5190-2305) was diluted at 0.8 ul of T7 primer plus 1 ul of H2O. The reaction mixture was made by combining 2 ul of diluted spike-in, 1.5 ul of RNA (100 ng/ul) and 1.8 ul of diluted T7 primer. The rest of labeling and purification were done according to the manufacturer's protocol for Cy3 one-color labeling.	Cy3-labeled cRNA was fragmented at 60 oC for 30 minutes with the following combination 720 ng of Cy3 cRNA, 6 ul of 10X Blocking Agent, 1.2 ul of 25X fragmentation buffer and H2O to 30 ul. The hybridization cocktail was made by mixing 25 ul of fragmented Cy3 cRNA with 25 ul of 2x GEx hybridization buffer HI-RPM. Following the manufacturer's protocol, 45 ul of the cocktail was loaded onto each array of the 8X array/slide. The assembled array was incubated at 65 oC on a rotator at 10 rpm for 17 hours. Array slides were disassembled in GE Wash Buffer 1 at room temperature (RT). They were washed in GE Wash Buffer 1 at RT for 1 minute. They were washed again in GE Wash Buffer 2 at 37 oC for 1 minute. Slides were put in slide holder for scanning.	For adipocyte infections, purified adenoviruses were incubated in low-serum media (maintenance media with 0.5% FBS) containing 0.5μg/mL poly-L-lysine for two hours at 25C. Adipocytes were washed once with PBS then incubated with adenovirus (5mL of diluted virus per 10cm dish). After four hours, additional low-serum media was added (5mL per 10cm dish). After 16 hours, low serum media was replaced with growth media and cells were incubated under standard conditions until harvest (24-72 hours post-infection).	3T3-L1 pre-adipocytes were maintained in DMEM (Hi glucose) + 10% FBS + 1% PennStrep. For differentiation, cells were grown to confluence and media was changed (day -2). Two days later (day 0), a DMI cocktail was added consisting of 1uM dexamethasone, 0.5mM 3-isobutyl-1-methylxanthine and 10ug/mL insulin. On day 2, media was changed to maintenance media + 10ug/mL insulin. On day 4, media was changed to maintenance media. By day 7, differentiation to mature adipocytes was considered complete and cells were infected with adenovirus.	Total RNA was isolated from adipocytes using TRIzol (Invitrogen) followed by purification with the RNeasy mini kit (Qiagen).	Slides were scanned using Agilent SureScan at 3 um following manufacturer's instruction. Raw data were extracted using Agilent Feature Extraction software.
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	TREATMENT						
Experimental Factor Type	treatment						
Comment[SecondaryAccession]	GSE48344						
Comment[GEOReleaseDate]	2013-06-27						
Comment[ArrayExpressSubmissionDate]	2013-06-26						
Comment[GEOLastUpdateDate]	2013-06-28						
Comment[AEExperimentType]	transcription profiling by array						
SDRF File	E-GEOD-48344.sdrf.txt