Unknown,Transcriptomics,Genomics,Proteomics

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FunGenES: INS-3-1: Expression profiling of CGR8 cells after downregulation of Klf4 or Klf5.

ABSTRACT: This experiment is part 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.

ORGANISM(S): Mus musculus

SUBMITTER: Oliver Hummel

PROVIDER: E-MTAB-1838 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:DNA methylation changes dynamically during development and is essential for embryogenesis in mammals. However, how DNA methylation affects developmental gene expression and cell differentiation remains elusive. During embryogenesis, many key transcription factors are used repeatedly, triggering different outcomes depending on the cell type and developmental stage. Here, we report that DNA methylation modulates transcription-factor output in the context of cell differentiation. Using a drug-inducible Gata4 system and a mouse embryonic stem (ES) cell model of mesoderm differentiation, we examined the cellular response to Gata4 in ES and mesoderm cells. The activation of Gata4 in ES cells is known to drive their differentiation to endoderm. We show that the differentiation of wild-type ES cells into mesoderm blocks their Gata4-induced endoderm differentiation, while mesoderm cells derived from ES cells that are deficient in the DNA methyltransferases Dnmt3a and Dnmt3b can retain their response to Gata4, allowing lineage conversion from mesoderm cells to endoderm in part. Transcriptome analysis of the cells' response to Gata4 over time revealed groups of endoderm and mesoderm developmental genes whose expression was induced by Gata4 only when DNA methylation was lost, suggesting that DNA methylation restricts the ability of these genes to respond to Gata4, rather than controlling their transcription per se. Our data indicate that epigenetic regulation by DNA methylation functions as a heritable safeguard to prevent transcription factors from activating inappropriate downstream genes, thereby contributing to the restriction of the differentiation potential of somatic cells. To understand how mouse cells differentially respond to Gata4 in cell type- and DNA methylation-dependent manners and to examine what genes are differentially regulated, we analyzed temporal change of gene expression profile in response to Gata4 in ES or Flk1(+) mesoderm cells in WT and Dnmt3a-/-Dnmt3b-/- (DKO) background, in total 108 samples.

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FunGenES: INS-3-1: Expression profiling of CGR8 cells after downregulation of Klf4 or Klf5.

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