Unknown,Transcriptomics,Genomics,Proteomics

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Comparison of Hob6-RFP positive and negative Flk-1+ cells (RFP reporter)

ABSTRACT: ES derived Flk-1+ cells were separated by sorting into Hoxb6 positive and negative populations by RFP Hoxb6 reporter. Gene expression was compared between these two groups. Duplicate analysis of Hoxb6-RFP positive and negative Flk-1+ cells.

ORGANISM(S): Mus musculus

SUBMITTER: Hiroshi Kataoka

PROVIDER: E-GEOD-45192 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Although differentiation of mice embryonic stem cells into vascular endothelial cells (ECs) gives a model for investigating molecular mechanisms of vascular development in vivo, temporal dynamics of gene expressions and chromatin modifications have not been studied until now. Here, we interrogated transcriptome and two histone modifications, H3K4me3 and H3K27me3, with a genome-wide scale during ECs differentiation and elucidated epigenetic switch peculiar to ECs. We find Gata2, Fli1, Sox7, and Sox18 are master regulators from genetic and epigenetic data, these genes were induced after Etv2 activation. These genes have specific histone modification pattern which is repressed by H3K27me3 modification at Flk-sorted mesoderm and changed to the bivalent (H3K4me3 and H3K27me3 both positive) state rapidly after vascular endothelial cells growth factor (VEGF) stimuli. Using a previously reported ECs differentiation model, we demonstrate that four transcription factors are critical for ECs specific gene expressions and efficient differentiation. Moreover, from knockdown experiments using si-RNA, we discovered these factors inhibited not only TGFÎ² signaling pathway, that is endothelial mesenchymal transition pathway, but also other near lineage commitment, including blood cells, skeletal muscle cells, vascular smooth muscle cells, and cardiomyocytes. We further identify each factor specific target genes during ECs differentiation by microarray, including both activating and repressing genes. Together, our findings from a detailed epigenetic approach provide a basic understanding temporal regulated chromatin signatures and resulting gene expression profile during ECs commitment, which is applicable to other models of differentiation and production of mature and long lasting ECs for regenerative medicine. Total 17 samples were derived from [1] ES cells, Flk-sorted mesoderm cells, and in the absense or presence of VEGF (6, 12, 24, and 48h) to determine VEGF activated genes during endothelial cells differentiation, [2] control si-RNA, si-Gata2, si-Fli1, si-Sox7, or si-Sox18 transfected cells under VEGF stimuli, [3] control si-RNA or si-Mix (si-Gata2, si-Fli1, si-Sox7, and si-Sox18) transfected cells under VEGF stimuli for the identification of each transcription factor dependent genes during endothelial cells differentiation.

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Comparison of Hob6-RFP positive and negative Flk-1+ cells (RFP reporter)

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