Project description:To gain genome wide information on the association of EZH2 with promoter regions in HeLa cells, DamID experiments and subsequent analysis by promoter arrays (Affymetrix GeneChip Human Promoter 1.0R ) were performed. The DamID method uses fusions of the bacterial Dam DNA methylase and the protein of interest, to direct the enzymatic activity to the proteinM-bM-^@M-^Ys genomic binding sites, where the DNA is methylated. Methylated DNA is then extracted, enriched and further analysed by microarray. EZH2 is the enzymatic subunit of the Polycomb Repressive Complex 2, which deposits the H3K27me3 mark on chromatin. This mark is associated with low gene expression, either in polycomb repressed regions or, in combination with methylation of H3K4, at poised promoters. An EZH2-T416A mutant (EZH2-mTP5) fails to bind to NIPP1, a factor implied in the regulation of PRC2 binding to a subset of target regions. To obtain a genome wide picture of differential binding of EZH2-WT and the EZH2-mTP5 mutant to promoter regions, the mutant was subjected to DamID/microanalysis as well. DamID of EZH2-WT (2 replicates) and EZH2-mTP5(T416A)(2 replicates) vs. control (Dam without fused protein)(4 samples)
Project description:To gain genome wide information on the association of EZH2 with promoter regions in HeLa cells, DamID experiments and subsequent analysis by promoter arrays (Affymetrix GeneChip Human Promoter 1.0R ) were performed. The DamID method uses fusions of the bacterial Dam DNA methylase and the protein of interest, to direct the enzymatic activity to the protein’s genomic binding sites, where the DNA is methylated. Methylated DNA is then extracted, enriched and further analysed by microarray. EZH2 is the enzymatic subunit of the Polycomb Repressive Complex 2, which deposits the H3K27me3 mark on chromatin. This mark is associated with low gene expression, either in polycomb repressed regions or, in combination with methylation of H3K4, at poised promoters. An EZH2-T416A mutant (EZH2-mTP5) fails to bind to NIPP1, a factor implied in the regulation of PRC2 binding to a subset of target regions. To obtain a genome wide picture of differential binding of EZH2-WT and the EZH2-mTP5 mutant to promoter regions, the mutant was subjected to DamID/microanalysis as well.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.