Journal: Stem Cells and Development
Article Title: Transcriptional Reprogramming and Chromatin Remodeling Accompanies Oct4 and Nanog Silencing in Mouse Trophoblast Lineage
Figure Lengend Snippet: Chromatin remodeling at the Oct-Sox binding motif in Oct4 and Nanog. (A) Schematic showing the location of the Oct-Sox binding motif and adjacent restriction enzyme cut site within the forward and reverse primers for Oct4 and Nanog . Shown are the effects of open and condensed chromatin on the accessibility of Dde I and Mse I at Oct4 and Nanog , respectively. (B) qPCR analysis of restriction enzyme accessibility at the Oct-Sox binding motif in Oct4 and Nanog at 0, 48, and 96 h after induction of Cdx2. As a negative control, the accessibility of Mse I at an intergenic region was assessed. Data are presented as the percentage of DNA cut by the respective restriction enzyme. An assay was performed on a total of three biological replicates. For each biological replicate, two PCR reactions were performed in duplicate. (C) Histone H3 ChIP analysis at the Oct-Sox binding motif in Oct4 and Nanog at 0, 48, and 96 h after induction of Cdx2. The enrichment of histone H3 on Gapdh was utilized as a control. As a negative control, a rabbit nonspecific IgG was used. A total of two ChIP replicates were performed for each biological replicate. * P
Article Snippet: Isolated nuclei were then treated with restriction endonucleases Dde I or Mse I (New England Biolabs) that were specific for single-cut sites located within the amplicons for Oct4 and Nanog , respectively.
Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Negative Control, Polymerase Chain Reaction, Chromatin Immunoprecipitation