Journal: Nature

Article Title: Replication-stress-induced chromatin loops protect fork stability

doi: 10.1038/s41586-026-10695-1

Figure Lengend Snippet: a , DNA FISH validation of a HU-specific loop in Hap1 cells. Left, representative images of left (green) and right (red) anchor probes (DAPI, blue). Right, the interprobe distance distribution. n = 108 (UT) and n = 119 (HU) S phase nuclei; n = 102 (UT), n = 81 (HU) non-S-phase nuclei. Statistical analysis was performed using two-sided Mann–Whitney U -tests; P = 0.0001 (S phase) and P = 0.4295 (non-S phase). Data were pooled from two independent experiments and normalized to their respective UT conditions (set to 1) to enable direct comparison across experiments. Scale bars, 5 μm. b , Representative HU-specific, G9a-dependent chromatin loops on chromosomes 9 (top) and 2 (bottom). The bottom tracks show H3K9me3 Rep-ChIC peaks, IZs and annotated genes. c , The fractions of IZs and TZs located within HU-unique loop bodies, anchors or neither. d , The aggregate average CTCF Rep-ChIC signal within HU-unique loops and ±10 kb around their anchors. Statistical analysis was performed using the two-sided Mann–Whitney U -test; P = 1.96 × 10 −16 . The results shown are from one biological replicate. e , The distribution of IZs and CTCF motifs within HU-specific loops and flanking regions. Consensus CTCF motifs at loop anchors enriched in the CTCF Rep-ChIC signal and their orientations (convergent versus tandem) are shown below. Heat maps of the CTCF Rep-ChIC signal around loop anchors (±1 kb) in UT and HU-treated MRC5 cells are also shown. f , g , APA of HU-specific loops in HCT116 CTCF-mAID2-mClover cells ( f ) and MRC5 cells ( g ). The enrichment relative to the background is indicated in the top-right corners. Results shown are from one biological replicate. h , Schematic of a loop with CTCF sites (top). Bottom, heat maps of IZs, H3K9me3, FANCD2 Rep-ChIC and the strand-specific fork pausing signal (TrAEL-seq) within HU-unique loops and flanking regions (±3.5 kb). C, Crick strand; W, Watson strand. i , Fork pausing signal within and flanking HU-unique loops with (+CTCF) or without (−CTCF) CBSs at the loop anchor, and UT-HU common loops. Results shown are a representative dataset out of two independent biological replicate. j , The average TrAEL-seq Watson (blue) and Crick (orange) strand profiles across HU-unique loops ±5 kb. All experiments used MRC5 cells unless otherwise stated.

Article Snippet: In experiments using HCT116 cells expressing CTCF-mAID2-mClover, the culture medium was replaced with medium containing 1 μM 5-Ph-IAA (HY-134653, MedChemExpress) for at least 2 h before the experiment to induce transient depletion of CTCF.

Techniques: Biomarker Discovery, MANN-WHITNEY, Comparison

Journal: Nature

Article Title: Replication-stress-induced chromatin loops protect fork stability

doi: 10.1038/s41586-026-10695-1

Figure Lengend Snippet: a , Distribution of TADs by replication timing (early, late, TTR: Timing Transition Regions) ± HU for two replicates (MRC5 cells). b, c , Insulation score ( b ) and CTCF Rep-ChIC signal ( c ) ± 10 kb of HU-unique loops overlapping (blue/top) or not overlapping (green/bottom) TAD borders. Aggregate average (top) and tornado plots (bottom) are shown. A1 and A2 indicate the loop anchors. Data shown are from one biological replicate. d , Top: CTCF-EdU PLA signal (red) in nuclei (blue). Bottom: Distribution of total PLA spot intensity per nucleus. S-phase nuclei analysed n = 986, 1416, 1556, 1524, 1204 and 846 (left to right) from one representative experiment which has been performed 2 times with similar results. ****P ≤ 0.0001, ns=non-significant (Kruskal–Wallis, Dunn’s test, P values from left to right: <0.0001, <0.0001, <0.0001, 0.3113). Experiments in HCT116-CTCF-mAID2-mClover cells. e , Western blot of CTCF depletion via 5-Ph-IAA, in HCT116-CTCF-mAID2-mClover cells. For gel source data, see Supplementary Fig. [n = 3] independent replicates. f , Heatmaps of average CTCF/FANCD2 Rep-ChIC, Fork-Deg-seq (WT vs. shBRCA2, in RPE1-shBRCA2 cells), γH2AX , and tumour SNVs across HU-unique loops with (+CTCF) or without (-CTCF) CTCF Binding Sites (CBSs) at the anchors for 3 biological replicates. g, h , Heatmaps of IZs (TrAEL-seq MRC5 IZ (g) or HCT116 IZ (h)), H3K9me3, FANCD2 Rep-ChIC, and strand-specific fork pausing signal (W for Watson strand and C for Crick strand, MRC5 cells) within HU-unique loops ± 3.5 kb in MRC5 ( g ) and HCT116-CTCF-mAID2-mClover ( h ) cells. i , Average TrAEL-seq Watson (blue) and Crick (orange) profiles ± 5 kb of HU-unique loops in HCT116-CTCF-mAID2-mClover cells (see Fig. ). All experiments in MRC5 cells unless otherwise stated.

Article Snippet: In experiments using HCT116 cells expressing CTCF-mAID2-mClover, the culture medium was replaced with medium containing 1 μM 5-Ph-IAA (HY-134653, MedChemExpress) for at least 2 h before the experiment to induce transient depletion of CTCF.

Techniques: Insulation, Western Blot, Binding Assay

Journal: Nature

Article Title: Replication-stress-induced chromatin loops protect fork stability

doi: 10.1038/s41586-026-10695-1

Figure Lengend Snippet: a , Rolling mean of z-scored DAPI intensity vs. S-phase progression from scEdU-seq tracks. Ribbon indicates s.d. b , Number of forks per cell vs. S-phase progression. Line is median; ribbon is 95% CI. c , Heatmap of maximum normalized scEdU-seq log counts binned per 40 kb along a 60 Mb region of chromosome 2, ordered by S-phase progression. Colour scale: Normalized read coverage across the region. d, e , DNA replication speeds at indicated loop anchors and background regions. Number of regions analysed: d, [n = 345 and 192 for UT and 0.5mMHU respectively] and e, n = 302, 283, 302, 214, 203 and 215 (left to right). Results shown are from one representative experiment which has been performed 2 times with similar results. f, g , Schematic of DNA fibre degradation assay in HCT116-CTCF-mAID2-mClover (top). IdU/EdU track length ratio distributions. Means ± s.d. shown (bottom). Number of tracks analysed: Panel f: n = 1015, 1058, 1057, 1057, 1116, 1080, 1117, 1079, 1101, 1086, 1069 and 1061 (left to right), pooled from 3 independent biological replicates and overlaid in three different colours in the plot. Panel g: n = 681, 726, 708, 724, 641, 669, 643, 676, 682, 684, 683, 666, 703, 676, 676, 689, 673, 686, 713 and 665 (left to right) pooled from 2 independent biological replicates and overlaid in two different colours. ****P ≤ 0.0001, **P ≤ 0.01, ns=non-significant (Kruskal–Wallis, Dunn’s test, panel f: All P values < 0.0001, panel g: P values: (left to right) >0.9999, <0.0001, <0.0001, <0.0001, <0.0001, 0.0095, 0.0061, <0.0001, >0.9999, <0.0001, 0.4485, >0.9999, 0.0019, >0.9999, >0.9999, >0.9999). h , Schematic of Fork-Deg-seq approach to map nucleolytic degradation at newly replicated regions. The diagram was created using BioRender; Taneja, N. https://BioRender.com/d7kf3t0 (2026). i , Fork-Deg-seq signal in WT and shBRCA2-induced RPE1 cells after 8 h 4 mM HU, alongside BrdU IP signal and replication timing for the indicated region on chromosome 3. Dotted square highlights a loop-poor region with enhanced Fork-Deg-seq enrichment. Loops of bidirectional replicon (fountain)- scale are shown in dark red; smaller loops are shown in light red. j , Aggregate heatmap of CTCF (CTCF-Rep-ChIC, in MRC5 cells), ForkDeg-seq signal (in RPE1-shBRCA2 cells), γH2AX , and Single Nucleotide Variant (SNV ) distribution within and in a +/− 1 kb region flanking the HU-specific loops identified in MRC5 cells. Schematics: loop body and flanking regions. Orange triangles mark loop anchors position based on CTCF-binding sites. Colour scale: Normalized read coverage across the region. All experiments in MRC5 cells unless otherwise stated.

Article Snippet: In experiments using HCT116 cells expressing CTCF-mAID2-mClover, the culture medium was replaced with medium containing 1 μM 5-Ph-IAA (HY-134653, MedChemExpress) for at least 2 h before the experiment to induce transient depletion of CTCF.

Techniques: Degradation Assay, Variant Assay, Binding Assay

Journal: Nature

Article Title: Replication-stress-induced chromatin loops protect fork stability

doi: 10.1038/s41586-026-10695-1

Figure Lengend Snippet: a , Schematic of the replication fork degradation DNA fibre assay in HCT116 CTCF-mAID2-mClover cells, involving CTCF depletion (dep; 5-Ph-IAA) and G9a inhibition (UNC0642) (top). Middle, representative fibres. Bottom, the IdU/EdU track length ratio. Data are mean ± s.d. From left to right, numbers of forks analysed per condition: n = 1,014, 1,015, 1,034, 1,039, 1,032, 1,053, 1,070 and 1,005, pooled from three independent replicates and overlaid in three different colours in the plot. Statistical analysis was performed using Kruskal–Wallis tests followed by Dunn’s test; from left to right, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.0001, P > 0.9999, P > 0.9999, P > 0.9999. Scale bar, 5 μm. b , Representative locus (chromosome 16: 81.3–82.75 Mb). Top, Hi-C heat map (the red squares highlight the positions of the loop anchors). The Fork-deg-seq signal in HCT116-CTCF-mAID2-mClover cells UT or treated with G9ai (4 h), 5-Ph-IAA (4 h, CTCF-depleted) or both after 4 mM HU (5 h or 8 h). MRC5 CTCF and H3K9me3 Rep-ChIC signals are shown below, alongside IZs and fragile sites. The black arrowheads indicate high Fork-deg-seq signal. The shaded area shows a loop-dense region with reduced degradation; unshaded areas show enhanced Fork-deg-seq enrichment. c , BrdU-enriched 5-kb bins classified by HU-unique loop coverage: loop-poor (0–1 loop, left, n = 4,398 bins) and loop-dense (≥2 loops, right, n = 6,844 bins). The fold change in Fork-deg-seq signal relative to the WT is shown. Data are mean ± s.d. Statistical analysis was performed using two-sided Mann–Whitney U -tests; loop-free region, from top to bottom: P = 4.4 × 10 −33 , P = 2.4 × 10 −132 , P = 2.4 × 10 −132 , P = 4.1 × 10 −33 , P = 2.4 × 10 −132 , P = 2.7 × 10 −34 ; loop-dense region, from left to right: P = 1.4 × 10 −130 , P = 2.0 × 10 −130 , P = 1.8 × 10 −130 , P = 3.2 × 10 −1 , P = 7.8 × 10 −2 , P = 8.4 × 10 −1 . Results shown are from one biological replicate. d , Aggregate analysis of the mean ± s.d. Fork-deg-seq signal within HU-unique loops and 5 kb flanking regions after 3 h of treatment with 4 mM HU alone (top row, left four plots) or with mirin and DNA2i followed by 4 mM HU (bottom row). Ionizing radiation (10 Gy) was included as a control without further treatment (top right plot). The results shown are from one biological replicate. All of the experiments described in this figure were performed in HCT116 CTCF-mAID2-mClover cells, unless otherwise stated.

Article Snippet: In experiments using HCT116 cells expressing CTCF-mAID2-mClover, the culture medium was replaced with medium containing 1 μM 5-Ph-IAA (HY-134653, MedChemExpress) for at least 2 h before the experiment to induce transient depletion of CTCF.

Techniques: Inhibition, Hi-C, MANN-WHITNEY, Control

Journal: Nature

Article Title: Replication-stress-induced chromatin loops protect fork stability

doi: 10.1038/s41586-026-10695-1

Figure Lengend Snippet: a , Representative locus (chr8: 122.8-133.9 Mb). Top: Hi-C heatmap (red squares highlight positions of loop anchors). Below: Fork-Deg-seq signal (HCT116-CTCF-mAID2-mClover cells) untreated or treated with G9ai (4 h), 5-Ph-IAA (4 h, CTCF-dep) or both upon 4 mM HU (5 h or 8 h), alongside MRC5 CTCF and H3K9me3 Rep-ChIC signals, IZs and fragile sites. Black arrowheads indicate high Fork-Deg-seq signal. Shaded area: loop-dense region with reduced degradation; unshaded areas show increased degradation. b , Aggregate mean Fork-Deg-seq signal ± s.d. after 5 h 4 mM HU within HU-unique loops ± 5 kb in HCT116-CTCF-mAID2-mClover cells. Results shown are from one representative experiment which has been performed 2 times with similar results. c-e , Distribution of IZs at early [n = 2119] and late [n = 2193] replicating fragile sites. ( c ) HU-unique loops overlapping IZs (MRC5), ****P ≤ 0.0001, (Two-sided Mann–Whitney U test, p-value: 7.858e-38). ( d ) Fork-Deg-seq signal in HCT116 IZs, ****P ≤ 0.0001, (Two-sided Mann–Whitney U test, p-values: 1.222e-07, 6.247e-06, 1.161e-05, 6.247e-06 left to right). ( e ) Fork-Deg-seq signal in RPE1 IZs (RPE1-shBRCA2 cells), ****P ≤ 0.0001, (Two-sided Mann–Whitney U test, p-values: 3.119e-37 WT + HU and 2.080e-55 shBRCA2+HU). f , Representative electron micrographs showing a reversed fork with ssDNA gaps on both daughter strands and intact reversed arms (HCT116-CTCF-mAID2-mClover). P, parental strand; D, daughter strand; R, reversed arms. Scale bars: 250 nm or 1183 bp (main), 50 nm or 473 bp (insets). g , Ranked gap length distribution per condition. h , qPCR analysis of HU-unique loop formation in WT and TKO (SMARCAL1, ZRANB3, HLTF knockout) U2OS cells ± 4 mM HU. Means ± s.e.m. [n = 4 independent biological replicates]. ****P < 0.0001, ***P < 0.001, **P < 0.01, ns=non-significant (Ordinary one-way ANOVA, Tukey’s test, P value from top to bottom: a: <0.0001, 0.0026, 0.4735; b: 0.0003, 0.0095, 0.5794; c: 0.004, 0.1237, 0.3688; d: <0.0001, 0.0471, 0.1211; e: <0.0001, 0.0003, 0.0752; f: 0.0004, 0.0003, 0.5844; g: <0.0001, 0.0042, 0.4301; h: 0.0003, 0.0004, 0.935; i: <0.0001, 0.0019, 0.4734,; j: 0.0006, 0.0001, 0.6204; k: 0.0005, 0.0206, 0.4417; l: <0.0001, 0.0032, 0.3017; m: <0.0001, 0.0173, 0.269; n: 0.0013, 0.0074, 0.8887; o: 0.0013, 0.0074, 0.7701; p: 0.0003, 0.095, 0.1693). i , Top: Schematic showing that only H3K9me3 signal intensity overlapping with EdU was measured. Bottom: Distribution of H3K9me3 intensity in the region of interest and for the indicated conditions. Number of replication sites analysed per condition across two independent experiments: n = 148, 98, 111, 107, 103 and 84 from left to right. (*: p < 0.05, ****: p < 0.0001, ns: non-significant. Kruskal–Wallis test followed by Dunn’s test, P values: 0.0160, <0.0001, <0.0001, >0.9999, 0.7905, >0.9999, 0.0946, >0.9999 and 0.0156 (top to bottom)). j , Top: Schematic showing that only the H3K9me3 signal intensity just outside of the EdU track was measure (H3K9me3 intensity over a region covering 20% of the total length of the EdU track was measured on both side of the EdU track). Bottom: Distribution of H3K9me3 intensity in the region of interest and for the indicated conditions. Number of replication sites analysed per condition across two independent experiments: n = 98, 119, 107 and 103 from left to right. (***: p < 0.001, ****: p < 0.0001, ns: non-significant. Kruskal–Wallis test followed by Dunn’s test, P values: <0.0001, 0.7726, 0.4606, 0.0003, <0.0001 and <0.0001 (top to bottom). k , Total intensity distribution of CTCF-EdU PLA spots. S-phase nuclei analysed: n = 642 for all conditions imaged from one representative experiment, which has been performed 2 times with similar results. Red line marks mean. ****P ≤ 0.0001, **P ≤ 0.01, ns=non-significant (Kruskal–Wallis, Dunn’s test, P values from left to right: <0.0001, <0.0001, 0.0011, <0.0001, <0.0001, 0.1690).

Article Snippet: In experiments using HCT116 cells expressing CTCF-mAID2-mClover, the culture medium was replaced with medium containing 1 μM 5-Ph-IAA (HY-134653, MedChemExpress) for at least 2 h before the experiment to induce transient depletion of CTCF.

Techniques: Hi-C, MANN-WHITNEY, Knock-Out

Journal: Bioactive Materials

Article Title: Gynostemma pentaphyllum -derived extracellular vesicles alleviate skin aging by destabilizing STING

doi: 10.1016/j.bioactmat.2026.03.010

Figure Lengend Snippet: The extraction process and proposed mechanisms for GPEVs in treating UVB-induced Skin aging. Extracellular vesicles derived from Gynostemma pentaphyllum (GPEVs), isolated and purified from fresh whole herb via differential ultracentrifugation, exhibit anti-photoaging properties. UVB radiation triggers the up-regulation of STING, activating the TBK1-CTCF pathway and causing photoaging. GPEVs effectively promote STING degradation, reducing CTCF-mediated aging gene transcription, protecting against UVB-induced skin aging.

Article Snippet: The primary antibodies used included γ-H2AX and CTCF (1:1,000, Cell Signaling), p21 and STING (diluted 1:1,000, Cell Signaling).

Techniques: Extraction, Derivative Assay, Isolation, Purification

Journal: bioRxiv

Article Title: A Promoter Competition Hub Orchestrates Runx1 Alternative Promoter Usage during Skeletal Muscle Stem Cell Activation

doi: 10.64898/2026.07.13.738242

Figure Lengend Snippet: A dynamic promoter competition hub comprising PP, SP, and a set of KEs orchestrates Runx1 AP usage within a TAD maintained by CTCF and cohesin during MuSC activation. During the QSC-to-FISC transition, the PP is selectively activated to initiate MuSC activation. A subset of KEs, assisted by TFs, interacts with the PP within a multi-connected interaction hub, while the SP remains in a closed, transcriptionally silent state. During this phase, USF1 acts as a pioneer factor by binding to the SP to prime it for subsequent activation. During the FISC-to-ASC transition, USF1 facilitates the opening of the SP, which, together with other TFs, drives SP-Runx1 expression to promote MuSC proliferation. Due to the competitive relationship between the two promoters, KEs that previously interacted with the PP switch their associations to the SP. Concurrently, additional KEs initially located outside the hub are recruited to interact with the SP to boost its rapid expression, while a small subset of these KEs retains interaction with the PP to maintain a basal level of PP-Runx1 expression.

Article Snippet: Following antibodies were used: H3K4me3(), CTCF (ABclonal, A1133), USF1(Santa Cruz Biotechnology, sc-390027x), H3K27ac (Abcam, ab4729), PolII (Santa Cruz Biotechnology, sc-899).

Techniques: Activation Assay, Binding Assay, Expressing

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Pik3cd E1020K reshapes the epigenetic landscape of CD4 + T cells. (A and B) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice were polarized under Th2 conditions and evaluated by ATACseq ( n = 3). A total of 71,040 peaks were detected. (A) Venn diagram of WT-specific, Pik3cd E1020K/+ -specific, and common peaks. (B) WT and Pik3cd E1020K/+ -specific peaks examined by motif enrichment analysis. Enrichment P values were plotted for both groups. Red: motifs specifically enriched in WT peaks; blue: motifs specifically enriched in Pik3cd E1020K/+ peaks; orange: motifs enriched in both groups. (C) Peak heatmap of CTCF CUT&Tag peaks from WT and Pik3cd E1020K/+ naïve, Th1, and Th2 cells organized into six clusters (1–6) specific to each indicated population, as described. (D) CTCF motif enrichment P value (top) and fold enrichment (bottom) in clusters 1–6. (E) DEGs (WT vs Pik3cd E1020K/+ ) and non-DEGs from bulk RNAseq data were compared in the indicated populations for percentages of genes showing differential CTCF peaks (WT versus Pik3cd E1020K/+ ). (F) Frequencies of CTCF peaks repressed, induced, or both induced and repressed in Pik3cd E1020K/+ Th2 cells (versus WT Th2) near DEGs (WT Th2 versus Pik3cd E1020K/+ Th2). (G) Th2 DEGs (WT Th2 versus Pik3cd E1020K/+ Th2) were organized into two categories: DEGs showing no change in CTCF (WT versus Pik3cd E1020K/+ ) and DEGs showing repressed CTCF peaks in Pik3cd E1020K/+ relative to WT. Pathway enrichment analysis (Enrichr; ) of TFTs (ChEA; ) was performed using these two categories of DEGs. Adjusted P values (−log 10 ) of the top 25 enriched TF signatures in each category plotted against each other. (H) Western blot evaluating CTCF and Zap70 in lysates from WT and Pik3cd E1020K/+ Th2-polarized cells, cultured in the presence or absence of Cal101 (10 nM) or rapamycin (200 nM). Data are representative of three independent experiments ( n = 3), quantified in . (I) Western blot evaluating CTCF and Zap70 in lysates from Th2-polarized NC and Foxo1 gRNA-Cas9–nucleofected WT CD4 T cells, compared with Pik3cd E1020K/+ cells. Data are representative of three independent experiments ( n = 3), . NC, negative control. Source data are available for this figure: .

Article Snippet: CUT&Tag utilized primary antibodies against CTCF (D31H2; Cell Signaling) and rabbit mAB IgG XP isotype control (DA1E; Cell Signaling).

Techniques: RNA sequencing, Western Blot, Cell Culture, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: A PI3Kδ-Foxo1-FasL signaling amplification loop rewires CD4 + T cell signaling and differentiation

doi: 10.1084/jem.20252154

Figure Lengend Snippet: Altered chromatin accessibility and CTCF activity in Pik3cd E1020K/+ Th2 cells. Supporting data for . (A and B) Naïve CD4 T cells from WT and Pik3cd E1020K/+ mice underwent Th2 polarization and were examined by ATACseq. n = 3. Volcano plot showing WT-specific peaks in red and Pik3cd E1020K/+ -specific peaks in blue (fold change >1.5, P < 0.05) (B) CTCF CUT&Tag tracks of Id3 , Bcl2 , Tbx21 , and Eomes loci. (C) Quantification of CTCF protein expressed as a ratio of CTCF/Zap70. Supporting data for . n = 3 for each group, from three independent experiments. (D) Quantification of CTCF protein expressed as a ratio of CTCF/Zap70. Supporting data for . n = 3 for each group, from three independent experiments. Statistical comparisons were made using ratio paired t tests. *P < 0.05.

Article Snippet: CUT&Tag utilized primary antibodies against CTCF (D31H2; Cell Signaling) and rabbit mAB IgG XP isotype control (DA1E; Cell Signaling).

Techniques: Activity Assay