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题目:: siRNA profiling of human embryonic fibroblasts transfected with mock, EZH2, EED, SUZ12, and BMI-1 siRNA reveals genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions
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状态:: 发布时间June 14, 2008 , 更新时间 March 27, 2012 , 提交时间 Oct. 11, 2006,
物种:: Homo sapiens
摘要:: The Polycomb group (PcG) proteins form chromatin-modifying complexes that are essential for embryonic development and stem cell renewal and are commonly deregulated in cancer. Here, we identify their target genes using genome-wide location analysis in human embryonic fibroblasts. We find that Polycomb-Repressive Complex 1 (PRC1), PRC2, and tri-methylated histone H3K27 co-occupy >1000 silenced genes with a strong functional bias for embryonic development and cell fate decisions. We functionally identify 40 genes derepressed in human embryonic fibroblasts depleted of the PRC2 components (EZH2, EED, SUZ12) and the PRC1 component, BMI-1. Interestingly, several markers of osteogenesis, adipogenesis, and chrondrogenesis are among these genes, consistent with the mesenchymal origin of fibroblasts. Using a neuronal model of differentiation, we delineate two different mechanisms for regulating PcG target genes. For genes activated during differentiation, PcGs are displaced. However, for genes repressed during differentiation, we paradoxically find that they are already bound by the PcGs in nondifferentiated cells despite being actively transcribed. Our results are consistent with the hypothesis that PcGs are part of a preprogrammed memory system established during embryogenesis marking certain key genes for repressive signals during subsequent developmental and differentiation processes. Experiment Overall Design: Total RNA was extracted from mock, EZH2, EED, SUZ12, and BMI-1 siRNA-transfected cells. For each treatment, RNA was prepared from six independent experiments and was pooled into one sample to reduce the experimental variation. Targets for microarray hybridization were synthesized accordingly to the supplier’s instructions (Affymetrix). The human GeneChip array U133A (Affymetrix), which interrogates 33,000 transcripts, was used for gene expression profiling. Fifteen GeneChip arrays U133A were used for this analysis; for each different condition, three separate arrays were hybridized with pooled cRNA. Hybridization, washing, staining, scanning, and data analysis were performed at The Affymetrix Microarray Unit at the IFOM-IEO campus, Milan, Italy, according to the manufacturer’s instructions. Expression levels were analyzed using Microarray Analysis Suite (MAS) 5.0 statistical algorithm software (Affymetrix), using the default parameters and scaling (TGT Value) signal intensities for all the GeneChip arrrays to a value of 500. The mock siRNA treatment was used as a baseline condition for comparison with the polycomb siRNA-treated samples.
实验种类:: RNAi profiling by array
样本量:: 15
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: 无设计数据
数据号:: E-GEOD-6015, GSE6015
数据状态:: 无法自动分析，您可以尝试手动分析数据。

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