Comment[ArrayExpressAccession]	E-GEOD-29364							
MAGE-TAB Version	1.1							
Public Release Date	2013-03-26							
Investigation Title	Identification of WUSCHEL direct targets.							
Comment[Submitted Name]	Identification of WUSCHEL direct targets.							
Comment[AEExperimentDisplayName]	Transcription profiling of Arabidopsis WUSCHEL targets							
Experiment Description	Cell-cell communication is critical for stem cell maintenance. Shoot apical meristem (SAM) located at the shoot tip harbors stem cells within the central zone (CZ). Their progeny differentiate in the adjacent peripheral zone (PZ). WUSCHEL (WUS) is a homeodomain transcription factor produced in a few cells of the organizing center (OC), located beneath the CZ. It has been shown to specify stem cell fate and also activate CLAVATA3 (CLV3) expression in cells of the CZ. CLV3 is a secreted peptide that activates a membrane bound receptor kinase-CLAVATA1 to restrict WUS transcription to the OC. It has been hypothesized that WUS activates CLV3 expression and stem cell fate in adjacent cells of the CZ by activating a non-cell autonomous signal. Contrary to this hypothesis, here we show that the WUS protein after being synthesized in cells of the OC, migrates into the superficial cell layers of the CZ where it activates CLV3 transcription by binding to its promoter elements. WUS also migrates laterally into the PZ to repress the expression of differentiation promoting transcription factors by binding to their regulatory regions. Migration of a stem cell inducing transcription factor into adjacent cells to activate a negative regulator, whereby restricting its own accumulation is unique to plant stem cell niches. While stem cell promoting transcription factor directly repressing differentiation promoting transcription factors to prevent premature differentiation of stem cell progenitors is conserved among diverse stem cell niches. A dexamethasone inducible version of WUSCHEL was used to identify its direct tragets. To analyze WUS-regulated gene expression programs at a higher spatial resolution, we introduced a dexamethasone (Dex)-inducible form of WUS consisting of the WUS protein-coding region fused to sequences coding for the ligand-binding domain of the rat glucocorticoid receptor (GR), expressed under a ubiquitous promoter (35S::WUS-GR) into apetala1-1;cauliflower1-1 (ap1-1;cal1-1) double mutant background. 35S::WUS-GR; ap1-1;cal1-1 SAMs were either treated with 10μM Dex or mock solution for four hours and RNA samples were hybridized to Arabidopsis ATH1 gene Chip (Affymmetrix). To identify putative genes that are directly regulated by WUS, in independent experiments, SAMs were simultaneously treated with 10μM Dex and 10μM Cycloheximide (Cyc), protein synthesis inhibitor and Cyc alone for four hours. A comparison of Dex-treated samples with mock identified 641 genes as differentially expressed [DEGs] (≥/≤2 fold; p < 0.01) and comparison of Dex+Cyc with Cyc identified 457 genes as DEGs (≥/≤2 fold; p < 0.01).							
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	Reddy	Yadav	Reddy					
Person First Name	Venu 	R	G					
Person Mid Initials		K	V					
Person Email	ryadav@iisermohali.ac.in							
Person Affiliation	University of California Riverside							
Person Phone	919463998780							
Person Fax	951-827-4437							
Person Address	Plant Biology, University of California Riverside, 2150 Batchelor Hall, Riverside, CA, USA							
Person Roles	submitter							
Protocol Name	P-GSE29364-1	P-GSE29364-6	P-GSE29364-3	P-GSE29364-8	P-GSE29364-7	P-GSE29364-2	P-GSE29364-4	P-GSE29364-5
Protocol Description	ID_REF =  VALUE = MAS 5 signal	Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on ATH1 chip. GeneChips were washed and stained in the Affymetrix Fluidics Station 450. Affymetrix Hybridization oven 640	Arabidopsis thaliana 35S::WUS-GR ap1-1;cal1-1 plants were grown in growth room for 28 days. The shoot apices from these plants were used for RNA isolation after respective treatments.	The probe set-to-locus mappings for the ATH1 chip were obtained from TAIR (2009). The normalization of the raw data Cel files was performed with the MAS5 algorithm using the default settings of the corresponding R function of the affy package (Gautier et al., 2004). The quality of the Affymetrix GeneChips was assessed with analysis routines provided by the affyPLM and arrayQualityMetrics packages (Bolstad et al., 2005; Kauffmann et al., 2009).	GeneChips were scanned using the Affymetrix Scanner 3000 with 7G upgrade	35S::WUS-GR; ap1-1;cal1-1 SAMs were either treated with 10μM Dex or mock solution for four hour. SAMs were simultaneously treated with 10μM Dex and 10μM Cycloheximide (Cyc), protein synthesis inhibitor and Cyc alone for four hours.	RNA was isolated using RNeasy kit (Qiagen).	Biotinylated cRNA were prepared according to the standard Affymetrix protocol.
Protocol Type	bioassay_data_transformation	hybridization	grow	feature_extraction	image_aquisition	specified_biomaterial_action	nucleic_acid_extraction	labeling
Experimental Factor Name	treatment							
Experimental Factor Type	treatment							
Comment[SecondaryAccession]	GSE29364							
Comment[GEOReleaseDate]	2013-03-26							
Comment[ArrayExpressSubmissionDate]	2011-05-17							
Comment[GEOLastUpdateDate]	2013-03-26							
Comment[AEExperimentType]	transcription profiling by array							
SDRF File	E-GEOD-29364.sdrf.txt							
Publication Title	Plant stem cell maintenance involves direct transcriptional repression of differentiation program.							
Publication Author List	Yadav RK, Perales M, Gruel J, Ohno C, Heisler M, Girke T, Jönsson H, Reddy GV.							
PubMed ID	23549482							
Publication DOI	10.1038/msb.2013.8