Journal: bioRxiv

Article Title: Cumulative TF binding and H3K27 Acetylation drive enhancer activation frequency

doi: 10.1101/2025.03.26.645413

Figure Lengend Snippet: (A) Targeted Locus Amplification (TLA) signal across the mouse genome. The chromosomes and the chromosomal position are shown. The landing pad is inserted on chr2:130,098,005-130,098,011. (B) Genome browser tracks displaying levels of H3K4Me3 and H3K27Ac and chromatin accessibility at the landing pad at the neutral ectopic locus (chr2: 130,075,508- 130,120,508 – 45kb window of observation). (C) Pearson correlation heatmap of the methylation rates of cytosines in different contexts (i.e., CpG and GpC) across biological replicates. Methylation rates were measured by SMF at the genomic regions inserted at the ectopic site in untreated (UT), steady state condition. The dendrogram (left) shows hierarchical clustering of methylation signal, using complete-linkage method. The sample order reflects their similarity in methylation signal, with branch lengths indicating the relative distance between clusters. The Pearson correlation coefficient R is annotated. (D) Pearson correlation heatmap of the methylation rates of cytosines in different contexts (i.e., CpG and GpC) across biological replicates in untreated (UT), DMSO or p300 inhibition (p300i) conditions. Methylation rates were measured by SMF at the 4 endogenous control loci in the DNA methyltransferase triple-knockout (DNMT TKO, reference mESCs) and in the RMCE TC-1 DNMT TKO cells. Same representation as (C). (E) Stacked bar plot showing the proportion of DNA fragments that are less or equally accessible at the ectopic locus compared to their endogenous counterpart. (F) Violin plots showing the frequency of chromatin accessibility at enhancers (left) and promoters (right) when measured at the endogenous loci (black) and at the ectopic locus (blue). (G) Violin plots showing the frequency of chromatin accessibility at CREs when measured at the endogenous loci (black) and at the ectopic locus (blue) as a function of the presence of specific TF motifs within the DNA fragments, as defined by ChIP-seq and motif annotation (see Methods: TFBS annotation). (H) H3K27Ac ChIP signal, normalized per ChIP input and spike-in ranked from high to low. The H3K27Ac signal is calculated in 2kb windows covering Transcription factor binding sites (TFBSs) in the genomic regions enriched during the SMF bait capture step (see Methods for TFBS annotation). Shown is the log10 of the H3K27Ac coverage. Classes were defined based on the enrichment pattern, as: low (<100), mid (between 100 and 500), and high (>500). (I) H3K4Me3 ChIP signal, normalized per ChIP input and spike-in ranked from high to low. The H3K4Me3 signal is calculated in 2kb windows covering Transcription factor binding sites (TFBSs) in the genomic regions enriched during the SMF bait capture step (see Methods for TFBS annotation). Shown is the log10 of the H3K4Me3 coverage. Classes were defined based on the enrichment pattern, as: low (<50), mid (between 50 and 300), and high (>300).

Article Snippet: For GpC methyltransferase treatment, freshly prepared GpC methyltransferase mix (1x M.GpC buffer, 300 mM sucrose, 64 µM SAM (NEB, #B9003S), M.CviPI (NEB,#M0227L)) was added and incubated at 37°C for 7.5 minutes.

Techniques: Amplification, Methylation, Inhibition, Control, Triple Knockout, ChIP-sequencing, Binding Assay

Journal: Frontiers in Chemistry

Article Title: Activation of GATA4 gene expression at the early stage of cardiac specification

doi: 10.3389/fchem.2014.00012

Figure Lengend Snippet: Valproic acid-enhanced cardiac differentiation. (A) P19 stem cells were treated with DMSO or increasing concentrations of valproic acid (VPA 0.5, 1, 2 mM) during EB formation, maintained in the tissue culture dishes for 3 additional days without treatments, and then stained for myosin heavy chain and cTnT. Quantification is presented as fractions of cells differentiated into cardiomyocytes relative to the total cell populations. Error bars represent the standard deviations of three independent experiments. (B) Shown are the representative microscopy images of the cells stained for cTnT (green). Hoechst was used to stain the DNA (blue) concomitantly (scale bars = 50 μm). (C) Western analysis of GATA4 protein expression and the levels of global H3 acetylation. The blots were then stripped and reprobed for β-tubulin as loading controls. Undifferentiated cells were included as a negative control. Shown are the cropped blot images representing indicated protein. (D) Occupancy of p300 at the GATA4 promoter (GATApro) and a control locus (GATActl) were examined by a real-time PCR based ChIP analysis. Quantification is presented as the fold variations of undifferentiated control.

Article Snippet: The P19 EBs were crosslinked with 1% formaldehyde and lysed using ChIP Lysis Buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1%SDS, 1X protease inhibitors, 1 mM DTT, 1 mM PMSF), and then sonicated with the Bioruptor system (Diagenode).

Techniques: Staining, Microscopy, Western Blot, Expressing, Negative Control, Control, Real-time Polymerase Chain Reaction

Journal: Frontiers in Chemistry

Article Title: Activation of GATA4 gene expression at the early stage of cardiac specification

doi: 10.3389/fchem.2014.00012

Figure Lengend Snippet: Occupancy of p300 at the GATA4 promoter at early stage of differentiation. (A) P19 cells were differentiated with DMSO and co-treatment of curcumin (10 μM) was during the first 2 days of EB formation. The cellular levels of H3 acetylation and p300 protein were analyzed by Western blotting on day 4. The blots were then stripped and reprobed for β-tubulin as loading controls. Undifferentiated cells were used as the negative control. Shown are the cropped blot images representing indicated protein. (B) Quantification of acetylated H3 blots is presented as fold variations of the undifferentiated control (mean ± SD , n = 3). (C) The levels of acetylated H3 at the GATA4 promoter were determined by the ChIP analysis. Quantification is presented as fold variations of the undifferentiated control. (D) Occupancy of p300 at the GATA4 promoter was examined in parallel. (E) Quantification of the p300 Western blots is presented as fold variations of the undifferentiated controls (mean ± SD , n = 3).

Article Snippet: The P19 EBs were crosslinked with 1% formaldehyde and lysed using ChIP Lysis Buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1%SDS, 1X protease inhibitors, 1 mM DTT, 1 mM PMSF), and then sonicated with the Bioruptor system (Diagenode).

Techniques: Western Blot, Negative Control, Control