Journal: Cell reports

Article Title: A Role for FACT in RNA Polymerase II Promoter-Proximal Pausing.

doi: 10.1016/j.celrep.2019.05.099

Figure Lengend Snippet: Figure 4. Paused Pol II Half-Life (t1/2) Decreases in FACT-Depleted S2 Cells (A–D) Chromatin from cells that had been treated with control dsRNA targeting EGFP or dsRNA targeting SSRP1 were incubated with 500-mM triptolide for 5, 10, 15, or 30 min or with 2% DMSO as control and subjected to ChIP-nexus using anti-Rpb3 antibodies. For each promoter where a typical Pol II ChIP-nexus footprint was observed (distance between positive and negative strand peak < 50 bp, position of Pol II footprint < 150 bp downstream of the TSS), spike-in normalized Pol II signal in a 51-bp window centered on the midpoint between paused Pol II positive and negative summits was determined, and the half-life of paused Pol II was calculated based on an exponential decay model. (A) Pol II in the pausing window at 18w as a function of time after addition of triptolide, in cells treated with EGFP (red) or SSRP1 (blue) dsRNAs. (B) Boxplot showing decreased paused Pol II half-life following SSRP1 depletion. p value was calculated with a two-sample Wilcoxon test, n = 998. (C) Heatmaps of Pol II ChIP-nexus data at various times after triptolide addition in EGFP and SSRP1 dsRNA-treated cells. (D) Boxplot showing fold change in pause half-life across highly paused (n = 411), moderately paused (n = 358), and lowly paused (n = 229) genes. p values were calculated using Wilcoxon rank sum test of pairwise comparisons. (E) A model for FACT function in promoter-proximal pausing and transcription-coupled histone modifications. In control cells (top panel), the +1 nucleosome is stabilized by FACT (Ramachandran et al., 2017). The +1 nucleosome in turn helps to maintain Pol II in the vicinity of the promoter-proximal pause, and tran- scription-coupled histone modifications H3K4me3 and H3K36me3 are normally deposited. Upon depletion of FACT, the +1 nucleosome is destabilized, and Pol II spends less time at the promoter-proximal pause. Hence, methyltransferases associated with elongating Pol II are allowed less time to place marks, leading to a broadening of H3K4me3 and relative depletion of H3K36me3 from more 50 portions of genes.

Article Snippet: To control for the loss of Pol II ChIP signal after triptolide treatment, a spike-in control was prepared by incubating human chromatin extracts from GM12878 cells with a 1:1 mixture of Dynabeads Protein A and Dynabeads Protein G coupled to antibodies against human Pol II (N20, Santa Cruz) followed by washing with nexus washing buffers A to D. A fixed amount of Dynabeads and human Pol II ChIP mixture was then spiked into each S2 ChIP-nexus experiment.

Techniques: Control, Incubation

Journal: PLoS Genetics

Article Title: Drosophila TDP-43 RNA-Binding Protein Facilitates Association of Sister Chromatid Cohesion Proteins with Genes, Enhancers and Polycomb Response Elements

doi: 10.1371/journal.pgen.1006331

Figure Lengend Snippet: The boxplots show the average ChIP-seq enrichment of Ser5P Pol II, Nipped-B, TBPH and Lark at active promoters, extragenic enhancers, and PREs in control cells, and cells treated with 10 μM triptolide for 1, 2, and 4 hours. The genome browser tracks at the right show the log2 ChIP-seq enrichment for the same proteins at the string gene and enhancers over the same time course of triptolide treatment.

Article Snippet: The Ser5P Pol II rabbit monoclonal antibody was purchased from Cell Signaling Technology (#13523) and validated by western blots of cell extracts with and without triptolide treatments.

Techniques: ChIP-sequencing, Control

Journal: PLoS Genetics

Article Title: Drosophila TDP-43 RNA-Binding Protein Facilitates Association of Sister Chromatid Cohesion Proteins with Genes, Enhancers and Polycomb Response Elements

doi: 10.1371/journal.pgen.1006331

Figure Lengend Snippet: The log2 ChIP-seq enrichment for Ser5P Pol II, Nipped-B, TBPH and Lark at all individual active promoters, enhancers and PREs in control untreated (Mock) cells is plotted against the enrichment after treatment of cells with 10 μM triptolide for 1, 2 and 4 hours.

Article Snippet: The Ser5P Pol II rabbit monoclonal antibody was purchased from Cell Signaling Technology (#13523) and validated by western blots of cell extracts with and without triptolide treatments.

Techniques: ChIP-sequencing, Control

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Advances in nanomedicine and delivery systems for gastric cancer research

doi: 10.3389/fbioe.2025.1565999

Figure Lengend Snippet: Promising areas for nanomedicine development in gastric cancer.

Article Snippet: Biomimetic Nanoparticles , , PTX, Triptolide , NPs , RP (P/T).

Techniques:

Journal: Nucleic Acids Research

Article Title: ADAR3 modulates neuronal differentiation and regulates mRNA stability and translation

doi: 10.1093/nar/gkae753

Figure Lengend Snippet: ADAR3 influences mRNA stability. ( A ) Scatterplot comparing mRNA decay rates in N2A-C and N2A-A3 cells following treatment with triptolide for 8 h. For each transcript and each cell line, the ratio between the normalized abundances at 0 and at 8 h was calculated and expressed as a log2 fold change. The plot shows the average ratio of the log2 fold change ( n = 4) in each cell line. The x-axis shows the value for N2A-C and the y-axis shows the values for N2A-A3 cells. Only the top 10% of stabilized or destabilized transcripts were considered significant. Destabilized transcripts in N2A-A3 cells are shown in red and stabilized transcripts in blue. See Methods for details. ( B ) Gene ontology enrichment test (GO: Biological processes) of destabilized (right) and stabilized (left) transcripts. The x-axis indicates Fold Enrichment, color signifies –log 10 False Discovery Rate (FDR) and the size of the dot indicates the number of genes in the dataset that belong to the pathway. ( C ) Scatter plot comparing changes in mRNA stability (x-axis) with changes in mRNA abundance (y-axis). The plot includes all transcripts with available mRNA stability data in the triptolide experiment, except those showing increased abundance after triptolide treatment. Differentially expressed transcripts are shown in blue. Pearson's correlations are shown in the figure. n = 6304 transcripts. ( D ) Scatter plot as in C including only the mRNAs that showed extreme changes in mRNA stability in the triptolide experiment (stability residual > 0.5 and stability residual < –0.5). Differentially expressed transcripts (FDR < 0.05) are shown in blue. Pearson's correlation is shown in the figure.

Article Snippet: For triptolide treatment, cells were seeded at 300 000 cells per well in a 12-well plate and treated with 1 μM triptolide (Tocris) 24 h after seeding.

Techniques: