Journal: bioRxiv

Article Title: CTCF-RNA interactions orchestrate cell-specific chromatin loop organization

doi: 10.1101/2025.03.19.643339

Figure Lengend Snippet: (A) Schematic showing CTCF-AID2 degron and dox-inducible rescue system generated in ESCs. (B) Western blot of CTCF-AID2 and rescue lines after 24 hours with no treatment, or treatment with 5Ph-IAA, or 5Ph-IAA and dox. (C) CTCF-AID2 and dox-inducible rescue lines were differentiated into NPCs for 2 days. Cells were immunostained with anti-Sox1 antibody and percentages of Sox1+ cells were quantified by flow cytometry analysis. Neither 5-Ph-IAA nor dox was added. Data are represented as mean ± SEM, N=2. (D) Diagram of experimental approach. ESCs or Day 2 NPCs were treated with 5-Ph-IAA and dox, then harvested for downstream analysis after 24 hours. Image generated using Biorender ( biorender.com ).

Article Snippet: E14Tga2 (ATCC, CRL-1821) mouse ESCs (E14) were cultured in N2B27 media [Advanced Dulbecco’s modified eagle medium (DMEM)/F12:Neurobasal-A (1:1) medium (ThermoFisher Scientific 12634028 and 10888022, respectively), with 3% ESC-grade fetal bovine serum (v/v, Gemini 900-108), 1x N2 (ThermoFisher Scientific 17502-048), 1x B27 (Thermo Fisher 17504-044), 2 mM L-glutamine (Sigma G7513), 0.1 mg/mL Penicillin-Streptomycin (Sigma P0781), 0.1 mM β-mercaptoethanol (ThermoFisher Scientific 21985023)] supplemented with leukemia inhibitory factor (homemade), 3 μM CHIR99021 (Tocris 4423) and 1 μM PD0325901 (Sigma PZ0162).

Techniques: Generated, Western Blot, Flow Cytometry

Journal: bioRxiv

Article Title: CTCF-RNA interactions orchestrate cell-specific chromatin loop organization

doi: 10.1101/2025.03.19.643339

Figure Lengend Snippet: (A, B) Aggregate peak analysis (APA) plots (top) of all WT CTCF loop anchors in (A) ESCs and (B) NPCs, comparing WT and ΔZF1. The numbers indicate the APA score. Lower-left is the difference in APA enrichment between ΔZF1 and WT. Lower-right are box plots quantifying the pixel enrichment of each loop, comparing WT and ΔZF1. p-values were determined using Wilcoxon signed-rank test. **** p-value < 1×10 - . (C) Bar plots showing the number of WT CTCF loop anchors and the proportions that are lost or retained in the ΔZF1 mutant. Loops were classified as ΔZF1-lost if they were found to be significant only in WT but not in ΔZF1 (q-value cutoff of 0.01), and if contact counts had log 2 fold-change ≤-1 (see Methods). (D) Bar plot showing the proportions of cell-type-specific and common loops among ΔZF1-lost or ΔZF1-retained loops. The numbers indicate the absolute numbers of loops. p-values were determined using Fisher’s Exact test. (E, F) Micro-C contact heatmaps (top) at the (E) Podxl and (F) Grb10 loci. Arrows point to NPC-specific CTCF-bound loops. Below the heatmaps is the view of one of the loop boundaries (indicated by the yellow bar), which is located in the gene body of (E) Podxl and (F) Grb10 . Below the gene annotation are CTCF ChIP-seq tracks, and the annotation of conserved CTCF motifs with their orientation. The distance of the CTCF loop boundary sites to the gene TSS is indicated.

Article Snippet: E14Tga2 (ATCC, CRL-1821) mouse ESCs (E14) were cultured in N2B27 media [Advanced Dulbecco’s modified eagle medium (DMEM)/F12:Neurobasal-A (1:1) medium (ThermoFisher Scientific 12634028 and 10888022, respectively), with 3% ESC-grade fetal bovine serum (v/v, Gemini 900-108), 1x N2 (ThermoFisher Scientific 17502-048), 1x B27 (Thermo Fisher 17504-044), 2 mM L-glutamine (Sigma G7513), 0.1 mg/mL Penicillin-Streptomycin (Sigma P0781), 0.1 mM β-mercaptoethanol (ThermoFisher Scientific 21985023)] supplemented with leukemia inhibitory factor (homemade), 3 μM CHIR99021 (Tocris 4423) and 1 μM PD0325901 (Sigma PZ0162).

Techniques: Mutagenesis, ChIP-sequencing

Journal: bioRxiv

Article Title: CTCF-RNA interactions orchestrate cell-specific chromatin loop organization

doi: 10.1101/2025.03.19.643339

Figure Lengend Snippet: (A, B) DiffBind MA plot of differentially called peaks comparing ΔZF1 vs WT in (A) ESCs and (B) NPCs. Adjusted p-value cutoff: ≤ 0.05, log 2 fold-change cutoff: ≥ 1, ≤-1, N=4. (C) Bar plot showing the proportion of loops that exhibit decreased or no change in CTCF chromatin binding at the anchors. Numbers indicate the absolute numbers of loops. p-values were determined using Fisher’s Exact test (n.s. = not significant). (D, E) (Left) The APA plots in (D) ESCs and (E) NPCs show the comparison between WT and ΔZF1 for the lost and retained loop subsets. Numbers indicate APA scores. (Right) CTCF ChIP-seq heatmaps comparing CTCF chromatin binding in (D) ESC-WT and ESC-ΔZF1 and (E) NPC-WT and NPC-ΔZF1. Each row is a loop anchor coordinate, and the heatmap is clustered based on whether the anchor is ΔZF1-lost or ΔZF1-retained. (F) Micro-C contact heatmap (above) and ChIP-seq tracks for CTCF and cohesin subunits, Rad21 and Smc3 (below) at a representative Podxl locus. Loop boundaries are highlighted in yellow. CTCF motifs with their orientation are annotated.

Article Snippet: E14Tga2 (ATCC, CRL-1821) mouse ESCs (E14) were cultured in N2B27 media [Advanced Dulbecco’s modified eagle medium (DMEM)/F12:Neurobasal-A (1:1) medium (ThermoFisher Scientific 12634028 and 10888022, respectively), with 3% ESC-grade fetal bovine serum (v/v, Gemini 900-108), 1x N2 (ThermoFisher Scientific 17502-048), 1x B27 (Thermo Fisher 17504-044), 2 mM L-glutamine (Sigma G7513), 0.1 mg/mL Penicillin-Streptomycin (Sigma P0781), 0.1 mM β-mercaptoethanol (ThermoFisher Scientific 21985023)] supplemented with leukemia inhibitory factor (homemade), 3 μM CHIR99021 (Tocris 4423) and 1 μM PD0325901 (Sigma PZ0162).

Techniques: Binding Assay, Comparison, ChIP-sequencing

Journal: bioRxiv

Article Title: CTCF-RNA interactions orchestrate cell-specific chromatin loop organization

doi: 10.1101/2025.03.19.643339

Figure Lengend Snippet: (A, B) ESCs (A) and NPCs (B) were UV-crosslinked and Flag-halo tagged WT or ΔZF1 rescue CTCF was immunoprecipitated using anti-Flag beads. RNA was purified and cDNA library was sequenced. Reproducible CLIP peaks were called (see Methods) in WT and ΔZF1 mutant. Bar graphs of the number of reproducible peaks are shown (N=3-4). (C, D) Heatmaps of ESC-CTCF CLIP peak enrichment in the non-crosslinking negative control, and crosslinked WT and ΔZF1. CLIP peaks are shown for the (C) plus (+) and (D) minus (-) strands. (E, F) Heatmaps of NPC-CTCF CLIP peak enrichment in the non-crosslinking negative control, and crosslinked WT and ΔZF1. CLIP peaks are shown for the (E) plus (+) and (F) minus (-) strands.

Article Snippet: E14Tga2 (ATCC, CRL-1821) mouse ESCs (E14) were cultured in N2B27 media [Advanced Dulbecco’s modified eagle medium (DMEM)/F12:Neurobasal-A (1:1) medium (ThermoFisher Scientific 12634028 and 10888022, respectively), with 3% ESC-grade fetal bovine serum (v/v, Gemini 900-108), 1x N2 (ThermoFisher Scientific 17502-048), 1x B27 (Thermo Fisher 17504-044), 2 mM L-glutamine (Sigma G7513), 0.1 mg/mL Penicillin-Streptomycin (Sigma P0781), 0.1 mM β-mercaptoethanol (ThermoFisher Scientific 21985023)] supplemented with leukemia inhibitory factor (homemade), 3 μM CHIR99021 (Tocris 4423) and 1 μM PD0325901 (Sigma PZ0162).

Techniques: Immunoprecipitation, Purification, cDNA Library Assay, Mutagenesis, Negative Control

Journal: bioRxiv

Article Title: CTCF-RNA interactions orchestrate cell-specific chromatin loop organization

doi: 10.1101/2025.03.19.643339

Figure Lengend Snippet: (A) Deseq2 volcano plot showing differential CTCF-RNA interactions comparing NPCs vs ESCs (see all in Table S7 ). Each dot is a gene. Total CLIP-seq read counts from the TSS to TES of genes were compared. Adjusted p-value cutoff: ≤ 0.05, log 2 fold-change cutoff: ≥ 1, ≤-1, N=3-4. (B) RNA-seq heatmaps of NPC-specific, CLIP-seq annotated genes, comparing ESC-WT, ESC-ΔZF1, NPC-WT, and NPC-ΔZF1. Each row is a gene and is annotated based on whether or not gene expression in NPC-ΔZF1 is significantly downregulated compared to WT. (C) Venn diagram of i) Genes upregulated in NPCs relative to ESCs, ii) genes colocalized at NPC-specific loop anchors, and iii) genes annotated to be NPC-specific, CTCF-interactors by CLIP-seq. Genes at the intersection of the three gene lists were considered for functional validation with Podxl and Grb10 being selected. (D, E) RNA-seq tracks (above) and CLIP-seq tracks (below) at genes of interest: (D) Podxl and (E) Grb10 . CLIP-seq tracks are zoomed in for better visualization of nucleotide-level crosslinks.

Article Snippet: E14Tga2 (ATCC, CRL-1821) mouse ESCs (E14) were cultured in N2B27 media [Advanced Dulbecco’s modified eagle medium (DMEM)/F12:Neurobasal-A (1:1) medium (ThermoFisher Scientific 12634028 and 10888022, respectively), with 3% ESC-grade fetal bovine serum (v/v, Gemini 900-108), 1x N2 (ThermoFisher Scientific 17502-048), 1x B27 (Thermo Fisher 17504-044), 2 mM L-glutamine (Sigma G7513), 0.1 mg/mL Penicillin-Streptomycin (Sigma P0781), 0.1 mM β-mercaptoethanol (ThermoFisher Scientific 21985023)] supplemented with leukemia inhibitory factor (homemade), 3 μM CHIR99021 (Tocris 4423) and 1 μM PD0325901 (Sigma PZ0162).

Techniques: RNA Sequencing, Gene Expression, Functional Assay, Biomarker Discovery