Fig 1: MRE11-dependent recruitment of CTCF at sites of DNA damage. (A) MRE11-depleted (shMRE11) or control (shCTL) U2OS cells were presensitized with BrdU and subjected to laser micro-irradiation. Cells were fixed and stained with the indicated antibodies; scale bar: 10 μm. (B) Immunofluorescence was performed 4 h after induction of double-strand breaks by ER-mCherry-lacR-FokI-DD in the FokI-U2OS reporter cells transfected with shRNA targeting MRE11 (shMRE11) or control shRNA (shCTL); scale bar: 10 μm. The plot represents the percentage of cells positive for CTCF co-localized at mCherry-FokI foci. Data are the means ± SD of three independent experiments. More than 100 cells were counted in each experiment; *P≤ 0.05. (C) ChIP-qPCR was performed with an antibody to γ-H2AX or CTCF in the FokI-U2OS DSB reporter cells transfected with the indicated shRNA (shCTL as a control or shMRE11 targeting MRE11), with (+) or without (−) induction of DSBs by mCherry-LacI-FokI. The values of recruitment to DSBs were relative to those of cells without the induction of DSBs. All qPCR reactions were performed in triplicate, with the SEM values calculated from at least three independent experiments; *P≤ 0.05; ***P≤ 0.001. (D) AsiSI-ER-U2OS cells were transfected with the indicated shRNA, with (+) or without (−) induction of DSBs by AsiSI. CTCF and chromatin were immunoprecipitated with anti-CTCF antibody. The fold enrichment values were relative to those of cells without induction of DSBs. Primers on chromosome 22 (no DSB) were used as negative controls. Data are the means ± SD of at least three independent experiments, and all qPCR reactions were performed in triplicate. (E) Immunofluorescence was performed 4 h after induction of double-strand breaks (DSBs) by mCherry-LacI-FokI in the CTCF-depleted (shCTCF) or control (shCTL) FokI-U2OS cells; scale bar: 10 μm. Bar graph represents the percentage of cells positive for MRE11 co-localized at mCherry-LacI-FokI foci. Data are the means ± SD of three independent experiments. More than 100 cells were counted in each experiment. ns, not significant. (F) Recruitment of MRE11 (green) to DSBs induced by laser micro-irradiation in the CTCF-depleted (shCTCF) and control (shCTL) U2OS cells; scale bar: 10 μm. (G) ChIP-qPCR was performed with an antibody to γ-H2AX or MRE11 in FokI-U2OS cells (left and center), and AsiSI-ER-U2OS cells (right) transfected with the control (shCTL) or CTCF shRNA (shCTCF), with (+) or without (−) induction of DSBs by FokI (FokI-U2OS) or AsiSI (AsiSI-ER-U2OS). The fold enrichment values were relative to those of cells without induction of DSBs. Data are presented as means ± SD of three independent experiments, and all qPCR reactions were performed in triplicate; **P≤ 0.01; ***P≤ 0.001.
Fig 2: CTCF interacts with MRE11 in response to DNA damage. (A) Selected protein list obtained from LC-MS/MS analysis after interactome tandem affinity purification of FLAG-SFB-tagged CTCF with or without γ-irradiation. The previously known and novel hits from MS results are shown. (B and C) Forward (B) and reciprocal (C) co-immunoprecipitation (co-IP) between endogenous CTCF and MRE11 in 293T cells, after the addition of DNase Benzonase, was performed with anti-CTCF (B) or anti-MRE11 (C) antibody, without (left, DMSO) and with (right, Etoposide) etoposide treatment. Immunoblot (IB) analysis was performed with the indicated antibodies. IgG immunoprecipitation (IP) was used as a negative control. See also Supplementary Figure S1. (D and E) Schematic representations of CTCF (D) and MRE11 (E) constructs used in this study (Top). The 293T cells were transfected with the indicated HA-tagged CTCF truncation constructs (D) or GFP-tagged MRE11 truncation constructs (E). Cell lysates were immunoprecipitated with anti-MRE11 (D) or anti-GFP (E) antibody, and immunoblot (IB) analyses were performed with the indicated antibodies. (D) Positions of molecular weight makers are denoted on the left of the INPUT SDS-PAGE gel. The interaction strengths between MRE11-HA and fusion CTCF proteins were normalized to the expression levels of HA fusion CTCF proteins, i.e. input. The interaction strength between MRE11-HA full-length CTCF was set as one, and the interaction strength of each HA fusion CTCF protein was calculated (bottom right panel); PAR, poly ADP-ribosylation; NLS, nuclear localization signal; N+GAR, N-terminus and glycine arginine rich motif.
Fig 3: Signature of chromatin accessibility changes upon acute CTCF loss. A Volcano plots of motif enrichment analysis of ATAC-seq comparing control nucleosome-free regions (NFRs) versus decreased differential accessibility regions (DARs). Fisher exact tests comparing motif frequency generated the p values and odds ratios. Each dot represents a motif in the database. Dots in the top left corner indicate motifs enriched for decreased DARs. B Volcano plots of motif enrichment analysis of ATAC-seq for increased DARs versus control NFRs. Fisher exact tests comparing motif frequency generated the p values and odds ratios. Each dot represents a motif in the database. Dots in the top right corner indicate motifs enriched for increased DARs. C ATAC-seq footprint profiles of the top motifs enriched for decreased DARs. Ratios between the nearest summit and the center indicate the probability of motifs protected from Tn5 insertion. Stronger dips in the center indicate higher confidence in binding. The height of the nearest summit to the center indicates chromatin accessibility. The number of matched motifs we used for each footprint profiling was attached at the end of each TF motif. D ATAC-seq footprint profiles of the top motifs enriched for increased DARs. E Log2 fold change of normalized contact numbers from Hi-C (+IAA versus -IAA) at loops grouped by whether the loop anchors overlapped the DARs or control NFRs. ***p value < 0.001; ****p value < 0.0001, Student’s t test. F Density plot measures the distance from DARs to the closest TAD boundaries
Fig 4: Sub-TAD looping mediated enhancer-promoter interaction of scar-6/prozb locus.(A) Hi-C heatmap representation of Human SCAR-6 locus in HepG2 at 10 kb resolution from ENCODE database (ENCODE Project Consortium et al, 2020; Wang et al, 2018) and zebrafish scar-6 locus in brain tissue at 5 kb resolution from (Yang et al, 2020). (B) UCSC genome browser snapshot of data for CTCF binding peaks at 24 hpf of zebrafish for at scar-6 and prozb locus (Pérez-Rico et al, 2020). (C) Bar plot representing ChIP-qPCR quantifying fold enrichment using CTCF antibody for scar-6 locus in wild type and scar-6gib007Δ12/Δ12 mutant zebrafish. Data from 3 independent biological replicates plotted as mean fold enrichment ± standard deviation; *P < 0.05, **P < 0.01 (two-tailed unpaired t-test). (D) Schematic and western blot of DNA-pulldown assay was performed using streptavidin tagged scar-6 gene DNA in zebrafish, and immuno blotting was done using prdm14 antibody. N = 3.
Fig 5: Epigenetic regulation of prozb through scar-6 locus is important for vascular function and hemostatic process.Hypothetical schematic of scar-6 locus in wild-type zebrafish, where scar-6 elncRNA and prdm14-PRC2 complex hypermethylated the proximal CpG island and inhibits CTCF binding. In scar-6gib007Δ12/Δ12 mutant zebrafish. The 12 bp deletion affects the binding of the scar-6 elncRNA and prdm14-PRC2 complex leading to a partial change in methylation. This change allows the CTCF occupancy at the locus further mediating sub-TAD looping of the enhancer-promoter of prozb. This causes upregulation of prozb leading to endothelial cell activation via the PAR2-NF-κB pathway and causing vascular dysfunction leading to hemorrhage.
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