Comment[ArrayExpressAccession]	E-MTAB-1839
MAGE-TAB Version	1.1
Investigation Title	FunGenES: INS-4-1: Expression profiling of CGR8 cells after upregulation of Klf4 or Klf5 expression.
Comment[Submitted Name]	FunGenES: INS-4-1: Expression profiling of CGR8 cells after upregulation of Klf4 or Klf5 expression.
Experiment Description	This experiment is a follow-up experiment of the FunGenES project (FunGenES - Functional Genomics in Embryonic Stem Cells partially funded by the 6th Framework Programme of the European Union, http://www.fungenes.org). The experiment was conducted at Inserm U846, Bron, France. Aim: Kru_ppel-like factors (Klf) 4 and 5 are two closely related members of the Klf family, known to play key roles in somatic cell reprogramming and in self-renewal of pluripotent stem cells. In this study, we focused on the functional divergence between Klf4 and Klf5. We showed that Klf4 and Klf5 regulate the expression of distinct subsets of genes. Klf4 negatively regulates the expression of endodermal markers, some of which encode transcription factors involved in the commitment of pluripotent system cells to endoderm differentiation. In contrast, Klf5 negatively regulates the expression of mesodermal markers, some of which controls commitment to the mesoderm lineage. Functional studies with reporter cell lines indicate that knockdown of Klf4 enhances differentiation toward visceral endoderm, mesendoderm, and definitive endoderm, whereas knockdown of Klf5 specifically enhances differentiation toward mesoderm. Thus, additive functions of Klf4 and Klf5 secure pluripotent stem cell propagation by inhibiting endoderm and mesoderm differentiation.
Experimental Design	strain_or_line_design, in_vitro_design, co-expression_design
Comment[AEExperimentType]	transcription profiling by array
Comment[AEExperimentDisplayName]	Transcription profiling by array of mouse CGR8 ES cells with overexpression of Klf4 or Klf5 against GFP-overexpressing controls and wild-type controls to study the functional divergence of Klf4 and Klf5
Experimental Factor Name	genotype
Experimental Factor Type	genotype
Person Last Name	Hummel
Person First Name	Oliver
Person Email	o.hummel@mdc-berlin.de
Person Affiliation	The Max Delbrück Center for Molecular Medicine, Berlin
Person Roles	submitter
Quality Control Type	biological replicate	technical replicate
Quality Control Term Source REF	EFO	EFO
Public Release Date	2014-04-28
Comment[ArrayExpressSubmissionDate]	2013-08-10
Publication Author List	Irene Aksoy, Pierre-Yves Bourillot, Pierre Savatier et al.
Publication Title	Klf4 and Klf5 differentially inhibit mesoderm and endoderm differentiation in embryonic stem cells
Protocol Name	P-MTAB-34708	P-MTAB-34709	P-MTAB-34710	P-MTAB-34711	P-MTAB-34712	P-MTAB-34713	P-MTAB-34714
Protocol Type	growth protocol	treatment protocol	treatment protocol	treatment protocol	nucleic acid extraction protocol	normalization data transformation protocol	growth protocol
Protocol Description	CGR8 mouse embryonic stem (ES) cell line, used here as unmodified control.	CGR8-GFP mouse embryonic stem (ES) cell line on the basis of CGR8 cell line, which has been lentivirally transfected with a construct facilitating GFP expression (as control).	CGR8-KLF4_OE mouse embryonic stem (ES) cell line on the basis of CGR8 cell line, which has been lentivirally transfected with a construct facilitating KLF4 overexpression.	CGR8-KLF5_OE mouse embryonic stem (ES) cell line on the basis of CGR8 cell line, which has been lentivirally transfected with a construct facilitating KLF5 overexpression.	Total RNA was extracted from cells using Trizol reagent (Invitrogen) and purified using RNeasy Mini kit (Qiagen) in accordance with the manufacturer's protocol with on-column DNase treatment (RNase-free DNase Set; Qiagen).	RMA (background adjustment, quantile normalization, median polish as summarization method)	All ES cell lines were routinely cultured in Glasgow's Modified Eagle's Medium (GMEM) supplemented with 10% fetal calf serum (PerbioScience CRC0406) and 1000 U/ml of LIF. To induce differentiation, cells were allowed to form aggregates in hanging drops in ES cell medium without LIF (100 cells/drop). After 2 days, embryoid bodies were collected and further grown in suspension for 1 to 10 days in non-adherent Petri dishes. Differentiation into visceral endoderm was induced by culturing the cells on gelatin-coated dishes in SF03 serum- free culture medium at high cell density (10e4 cells/cm2). Differentiation into mesendoderm/definitive endoderm was induced by culturing cells on collagen IV-coated dishes in SF03 supplemented with 10 ng/ml human activin A at low cell density of 10e3 cells/cm2. Differentiation was performed in alpha-Modified Eagle's Medium to enhance mesoderm differentiation.
Protocol Term Source REF	EFO	EFO	EFO	EFO	EFO	EFO	EFO
Term Source Name	EFO	ArrayExpress
Term Source File	http://www.ebi.ac.uk/efo	http://www.ebi.ac.uk/arrayexpress
SDRF File	E-MTAB-1839.sdrf.txt