Journal: International Journal of Molecular Sciences
Article Title: Pdx1 Is Transcriptionally Regulated by EGR-1 during Nitric Oxide-Induced Endoderm Differentiation of Mouse Embryonic Stem Cells
doi: 10.3390/ijms23073920
Figure Lengend Snippet: EGR-1 binding to Pdx1 promoter is dependent on DNA methylation and histoneP300 acetyltransferase activity. ( A , B ) Analysis of Pdx1 promoter methylation status. a) Schematic depiction of the Pdx1 promoter. Short vertical lines represent the 11 CpG dinucleotides studied. Results are shown of bisulfite PCR sequencing of 10 individual clones in control and DETA-NO conditions. Presence of a methylated (black square) or unmethylated (white square) cytosine is indicated. ( B ) Specific CpG site methylation analysis of EGR-1bs (CpG referred to as 7) and neighboring CpG sites on Pdx1 promoter, analyzed by pyrosequencing. ( C ) EGR-1 and ( D ) Pdx1 expression by real-time PCR in control cells and cells treated with DETA-NO, p300i, and valproic acid (VPA), alone or in combinations. Graph shows the average ± SD of five independent experiments. ( E ) EGR-1 binding analysis of Pdx1 promoter by chromatin immunoprecipitation (ChIP) assay. Graph shows the average ± SD of three independent experiments. Y-axis corresponds to the percentage input relative to IgG. ( F ) Stacked bars for bivalent mark dynamics of H3K27me3 and H3K4me3 analyzed by ChIP assays. They represent an average of two independent experiments. Data with * p < 0.05 or ** p < 0.01 were considered statistically significant.
Article Snippet: When appropriate, cells were exposed after three days of culture over 20 h to 50 μM C646 (P300i) (Millipore, Darmstadt, Germany) and 10 μM VPA (Sigma-Aldrich).
Techniques: Binding Assay, DNA Methylation Assay, Activity Assay, Methylation, Sequencing, Clone Assay, Control, Expressing, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation
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