Unknown,Transcriptomics,Genomics,Proteomics

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Expresion profile of TGR-1 (Myc+/+) and HO15.19 (Myc-/-) infected with a retrovirus expressing Hhex or GFP (controls)

ABSTRACT: The aim of this experiment is to determine Hhex targets in the presence and absence of Myc. TGR-1 or HO15.19 cell lines were infected with a retrovirus expressing IRES-GFP (control) or Hhex-IRES-GFP. After infection, cells were GFP-sorted and total RNA extracted by a combination of Trizol and Rneasy columns.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Roque Bort

PROVIDER: E-GEOD-51467 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Cardiac muscle differentiation in vivo is guided by sequential growth factor signals, including endoderm-derived diffusible factors, impinging on cardiogenic genes in the developing mesoderm. Previously, by RNA interference in AB2.2 mouse embryonic stem cells (mESCs), we identified the endodermal transcription factor Sox17 as essential for Mesp1 induction in primitive mesoderm and subsequent cardiac muscle differentiation. However, downstream effectors of Sox17 remained to be proven functionally. In this study, we used genome-wide profiling of Sox17-dependent genes in AB2.2 cells, RNA interference, chromatin immunoprecipitation, and luciferase reporter genes to dissect this pathway. Sox17 was required not only for Hhex (a second endodermal transcription factor) but also for Cer1, a growth factor inhibitor from endoderm that, like Hhex, controls mesoderm patterning in Xenopus toward a cardiac fate. Suppressing Hhex or Cer1 blocked cardiac myogenesis, although at a later stage than induction of Mesp1/2. Hhex was required but not sufficient for Cer1 expression. Over-expression of Sox17 induced endogenous Cer1 and sequence-specific transcription of a Cer1 reporter gene. Forced expression of Cer1 was sufficient to rescue cardiac differentiation in Hhex-deficient cells. Thus, Hhex and Cer1 are indispensable components of the Sox17 pathway for cardiopoiesis in mESCs, acting at a stage downstream from Mesp1/2. Keywords: Cardiac development, Embryonic stem cells, Endoderm, Myogenesis, RNA interference Genome-wide expression profiling of Sox17-dependent genes. Mouse embryonic stem cells expressing Sox17 or luciferase shRNA were differentiated for up to 10 days by the embryoid body method [PMID:8155574], then were analysed using Affymetrix microarrays. ESCs were transduced with lentiviral vectors coexpressing enhanced green fluorescent protein (eGFP) with shRNA against Sox17, or against firefly luciferase. Transduced cells were flow-sorted based on GFP fluorescence, grown as embryoid bodies, and transferred to tissue culture plates after 4.5 days [PMID:17360443]. Cells were harvested at days 0, 2, 4, 5, 6, 8 and 10 in two biological replicates, except where noted.

Dataset Information

Expresion profile of TGR-1 (Myc+/+) and HO15.19 (Myc-/-) infected with a retrovirus expressing Hhex or GFP (controls)

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