Journal: Nature Communications

Article Title: Pre-established ATF4 occupancy and chromatin organization instruct selective transcription activation during integrated stress response

doi: 10.1038/s41467-025-64577-7

Figure Lengend Snippet: a Western blot analysis of ATF4 in C2C12 cells treated with 100 nM Thap for 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h, respectively. β-actin was used as the loading control. Experiments were repeated independently at least three times with similar results. b Immunofluorescence images showing ATF4 signals (red) in C2C12 cells treated with 100 nM Thap for 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h, respectively. DAPI staining highlights nuclei (blue). Scale bar = 25μm. Experiments were repeated independently at least three times with similar results. c PCA plot of RNA-seq data sets of C2C12 cells treated with DMSO and Thap for different durations. d – f Volcano plots depicting differentially expressed genes (|log2FC | > 1 and p adj <0.05) between the C2C12 treated with DMSO and Thap for 2 h ( d ), 6 h ( e ), and 12 h ( f ). The p adj are calculated using the Wald test and adjusted for multiple testing by performing Benjamini-Hochberg correction. g – i Bar graphs indicate the top 10 most enriched Gene Ontology (GO) biological process (BP) terms for upregulated genes of C2C12 treated with Thap versus DMSO for 2 h ( g ), 6 h ( h ), and 12 h ( i ). Enrichment analyses were performed using the ClusterProfiler R package with a one-tailed hypergeometric test followed by a Benjamini-Hochberg correction. j Heatmap of mRNA expression levels of 83 stress-related genes that were differentially expressed in C2C12 cells treated with DMSO versus Thap at at least one time point (2, 6, or 12 h). k Boxplot comparing the expression changes of the stress-related genes shown in ( j ). Boxes represent the interquartile range (IQR), with bounds corresponding to the first and third quartiles; the center line indicates the median. Whiskers extend to the most extreme values within 1.5× IQR. P values were calculated using a two-tailed t-test. Each group includes 83 genes.

Article Snippet: The nuclear extracts were incubated overnight at 4 °C with rabbit anti-ATF4 antibody (Cell Signaling Technology, #11815, 1:50) or rabbit IgG as a negative control, followed by incubation with protein A agarose beads for 1 h at 4 °C.

Techniques: Western Blot, Control, Immunofluorescence, Staining, RNA Sequencing, One-tailed Test, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Pre-established ATF4 occupancy and chromatin organization instruct selective transcription activation during integrated stress response

doi: 10.1038/s41467-025-64577-7

Figure Lengend Snippet: a Venn diagram showing the overlap of ATF4 peaks identified in C2C12 cells treated with either DMSO or Thap for 6 h. b Averaged line graph and heatmaps displaying ATF4 occupancy (RPKM) around the center (±5 kb) of pre-occupied and gained ATF4 peaks in C2C12 cells treated with DMSO or Thap for 6 h. c CUT&Tag tracks illustrating ATF4 occupancy (RPKM) in C2C12 cells treated with DMSO or Thap for a representative region. Two replicates for each condition are shown. Representative examples of pre-occupied ATF4 enhancers, pre-occupied ATF4 promoters, gained ATF4 enhancers, and gained ATF4 promoters are shown at the bottom. d Gene Ontology (GO) enrichment analysis for genes associated with pre-occupied ATF4 promoters, pre-occupied ATF4 enhancers, gained ATF4 promoters, and gained ATF4 enhancers. GO terms related to protein homeostasis are highlighted with red triangles. GO terms related to stress response are highlighted with asterisks. Enrichment analyses were performed using the ClusterProfiler R package with a one-tailed hypergeometric test followed by a Benjamini-Hochberg correction. e Boxplots comparing the extent of expression changes for genes associated with different classes of ATF4 peaks. In the boxplots, the center line indicates the median, box limits represent the upper and lower quartiles, and whiskers extend to 1.5× the interquartile range. P values were calculated using a two-tailed t-test. The number of genes and the median expression changes for each group are shown at the bottom. Genes associated with pre-occupied ATF4 promoters exhibit the most robust transcription activation.

Article Snippet: The nuclear extracts were incubated overnight at 4 °C with rabbit anti-ATF4 antibody (Cell Signaling Technology, #11815, 1:50) or rabbit IgG as a negative control, followed by incubation with protein A agarose beads for 1 h at 4 °C.

Techniques: One-tailed Test, Expressing, Two Tailed Test, Activation Assay

Journal: Nature Communications

Article Title: Pre-established ATF4 occupancy and chromatin organization instruct selective transcription activation during integrated stress response

doi: 10.1038/s41467-025-64577-7

Figure Lengend Snippet: a Tracks showing ATF4 CUT&Tag (orange), H3K27Ac CUT&Tag (green), and ATAC-seq (blue) signals in a representative region in C2C12 cells treated with DMSO or Thap for 6 h. b , c Volcano plots showing the differential changes in H3K27Ac ( b ) and ATAC-seq ( c ) peaks between C2C12 cells treated with Thap and DMSO for 6 h. Differential peaks (FDR < 0.05 and |log2FC | > 0.5) are highlighted in green. d Overlaps between pre-occupied ATF4 promoter peaks and H3K27Ac peaks in C2C12 treated with DMSO for 6 h. e Pie charts depicting the proportions of up, down, and unchanged H3K27Ac peaks at the pre-occupied ATF4 promoter, the gained ATF4 promoter, and the control promoter. f Boxplots comparing the log2 fold changes of genes associated with pre-occupied ATF4 promoters, gained ATF4 promoters, and control promoters, categorized by changes in H3K27Ac signals. In the boxplots, the center line indicates the median, box limits represent the upper and lower quartiles, and whiskers extend to 1.5× the interquartile range. The values beyond the whiskers are not shown in the boxplots. P values were calculated using a two-tailed t test. The number of genes and the median expression changes for each group are shown at the bottom. g – i Analysis of ATAC-seq data, conducted similarly to the H3K27Ac analyses in d – f .

Article Snippet: The nuclear extracts were incubated overnight at 4 °C with rabbit anti-ATF4 antibody (Cell Signaling Technology, #11815, 1:50) or rabbit IgG as a negative control, followed by incubation with protein A agarose beads for 1 h at 4 °C.

Techniques: Control, Two Tailed Test, Expressing

Journal: Nature Communications

Article Title: Pre-established ATF4 occupancy and chromatin organization instruct selective transcription activation during integrated stress response

doi: 10.1038/s41467-025-64577-7

Figure Lengend Snippet: a Bar graph showing the fractions of 10 kb genomic bins that remain in A or B compartments (AA and BB), switch from A to B compartment (AB), or switch from B to A compartment (BA) during ISR. b, c Venn diagrams depicting the overlap between TAD boundaries ( b ) and chromatin loops ( c ) detected in C2C12 cells treated with DMSO or Thapsigargin (Thap) for 6 h. d , e Bar plots illustrating the proportions of non-ATF4 promoters, pre-occupied ATF4 promoters, and gained ATF4 promoters at TAD boundaries ( d ) or loop anchors ( e ) that are stable or altered upon ISR induction. P -values were calculated using the pairwise chi-square test. f Hi-C contact heatmaps at 10 kb resolution and corresponding tracks for ATF4 CUT&Tag (orange), H3K27Ac CUT&Tag (green), ATAC-seq (blue), RNA-seq (purple) at Cebpb, Plk3, and Xbp1 gene loci in C2C12 treated with DMSO and Thap for 6 h. Circles in the heatmaps highlight the loops connecting promoters and enhancers. Gene promoter regions are highlighted in gray.

Article Snippet: The nuclear extracts were incubated overnight at 4 °C with rabbit anti-ATF4 antibody (Cell Signaling Technology, #11815, 1:50) or rabbit IgG as a negative control, followed by incubation with protein A agarose beads for 1 h at 4 °C.

Techniques: Hi-C, RNA Sequencing

Journal: Nature Communications

Article Title: Pre-established ATF4 occupancy and chromatin organization instruct selective transcription activation during integrated stress response

doi: 10.1038/s41467-025-64577-7

Figure Lengend Snippet: a CUT&Tag tracks showing ATF4 (orange), CHOP (green), CEBPβ (blue), and CEBPγ (pink) binding in C2C12 cells treated with DMSO or Thap. b Averaged line plots and heatmaps depicting the log2 ratio of ATF4 or CEBPγ occupancy between Thap and DMSO treatments, centered around pre-occupied ATF4 promoters (±5 kb). Pre-occupied ATF4 peaks are classified into two groups based on the CEBPγ log2 ratio by performing k-means clustering: class 1 sites with increased CEBPγ occupancy and class 2 sites where CEBPγ occupancy remains unchanged or decreases. c Average intensity profiles and heatmaps showing normalized ATF4 and CEBPγ signals in DMSO- or Thap-treated C2C12 cells at class 1 and class 2 pre-occupied ATF4 promoter sites identified in ( b ). d , e Changes in ATF4 or CEBPγ occupancy at the gained ATF4 promoter peaks, analyzed similarly to ( b , c ). f , g Boxplots (top) and line plots (bottom) illustrating log2 fold changes (log2FC) in gene expression associated with class 1 and class 2 sites for pre-occupied ATF4 promoters ( f ) and newly gained ATF4 promoters ( g ). Genes linked to Class 1 sites exhibit stronger transcriptional activation. In the boxplots, the center line indicates the median, box limits represent the upper and lower quartiles, and whiskers extend to 1.5× the interquartile range. P values were calculated using a two-tailed t test. For pre-occupied ATF4 promoters ( f ): Class 1, n = 149; Class 2, n = 184. For gained ATF4 promoters ( g ): Class 1, n = 856; Class 2, n = 2460. h , i Scatter plots with linear regression (red line) depicting the correlation between the log2 ratio of CEBPγ occupancy and the log2FC of gene expression for class 1 and class 2 sites at pre-occupied ATF4 promoters ( h ) and newly gained ATF4 promoters ( i ). Statistical analysis was performed using two-sided Spearman correlations. Spearman correlation coefficients ( r ) and associated P values are displayed. j PCA plot of RNA-seq data sets of CEBPγ knockdown C2C12 cells treated with DMSO and Thap for different durations. k Boxplots showing the fold changes of transcript levels for stress genes and random genes in control cells and CEBPγ knockdown C2C12 cells. In the boxplots, the center line indicates the median, box limits represent the upper and lower quartiles, and whiskers extend to 1.5× the interquartile range. P -values were calculated using a two-tailed t-test. For both stress genes and random genes, n = 83. l Boxplots showing the fold changes of transcript levels for genes associated with pre-occupied ATF4 promoter class 1 sites, pre-occupied ATF4 promoter class 2 sites, gained ATF4 promoter class 1 sites, and gained ATF4 promoter class 2 sites in control cells and CEBPγ knockdown C2C12 cells. In the boxplots, the center line indicates the median, box limits represent the upper and lower quartiles, and whiskers extend to 1.5× the interquartile range. P values were calculated using a two-tailed t test. Pre-occupied ATF4 promoter class 1, n = 151; pre-occupied ATF4 promoter class 2, n = 185; gained ATF4 promoter class 1, n = 877; gained ATF4 promoter class 2, n = 2479.

Article Snippet: The nuclear extracts were incubated overnight at 4 °C with rabbit anti-ATF4 antibody (Cell Signaling Technology, #11815, 1:50) or rabbit IgG as a negative control, followed by incubation with protein A agarose beads for 1 h at 4 °C.

Techniques: Binding Assay, Gene Expression, Activation Assay, Two Tailed Test, RNA Sequencing, Knockdown, Control

Journal: Nature Communications

Article Title: Pre-established ATF4 occupancy and chromatin organization instruct selective transcription activation during integrated stress response

doi: 10.1038/s41467-025-64577-7

Figure Lengend Snippet: a – c Boxplots showing the intensity of ATF4 motifs ( a ), CEBPγ motifs ( b ), and ATAC-Seq signals ( c ) at class 1 and class 2 sites within pre-occupied ATF4 promoters, newly gained ATF4 promoters, and control promoters. In the boxplots, the center line indicates the median, box limits represent the upper and lower quartiles, and whiskers extend to 1.5× the interquartile range. Values beyond the whiskers are not displayed. P values were calculated using a two-tailed t test. The number of motifs or peaks and the corresponding median scores/signals for each group are indicated below. d Local pileups of Hi-C interaction heatmaps around the class 1 and class 2 sites within pre-occupied ATF4 promoters, gained ATF4 promoters, and control promoters in C2C12 treated with DMSO and Thap. e A schematic model depicting how pre-established ATF4 occupancy and chromatin organization modulate the binding of ATF4/CEBPγ heterodimers, thereby influencing transcriptional activation during ISR.

Article Snippet: The nuclear extracts were incubated overnight at 4 °C with rabbit anti-ATF4 antibody (Cell Signaling Technology, #11815, 1:50) or rabbit IgG as a negative control, followed by incubation with protein A agarose beads for 1 h at 4 °C.

Techniques: Control, Two Tailed Test, Hi-C, Binding Assay, Activation Assay