CTCF-RNA interactions orchestrate cell-specific chromatin loop organization

Abstract

CCCTC-binding factor (CTCF) is essential for chromatin organization. CTCF interacts with endogenous RNAs, and deletion of its ZF1 RNA-binding region (ΔZF1) disrupts chromatin loops in mouse embryonic stem cells (ESCs). However, the functional significance of CTCF-ZF1 RNA interactions during cell differentiation is unknown. Using an ESC-to-neural progenitor cell (NPC) differentiation model, we show that CTCF-ZF1 is crucial for maintaining cell-type-specific chromatin loops. Expression of CTCF-ΔZF1 leads to disrupted loops and dysregulation of genes within these loops, particularly those involved in neuronal development and function. We identified NPC-specific, CTCF-ZF1 interacting RNAs. Truncation of two such coding RNAs, Podxl and Grb10, disrupted chromatin loops in cis, similar to the disruption seen in CTCF-ΔZF1 expressing NPCs. These findings underscore the inherent importance of CTCF-ZF1 RNA interactions in preserving cell-specific genome structure and cellular identity.

Keywords: CTCF; RNA binding; chromatin loops; embryonic stem cells; gene expression; genome organization; neural progenitor cells.

Figure 1.. Acute CTCF depletion and rescue using the AID2 system.

(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).

Figure 2.. CTCF loop anchors induced after differentiation are disrupted in the ΔZF1 mutant.

(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 < 1×10⁻¹⁰⁰. (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 log₂ 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.

Figure 3.. ΔZF1 loop loss is not due to changes in CTCF chromatin binding.

(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, log₂ 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.

Figure 4.. Dysregulated genes in NPC-ΔZF1 mutant are enriched at disrupted loops.

(A) Deseq2 volcano plots showing gene expression changes in NPC-ΔZF1 compared to NPC-WT (see all in Table S4). Adjusted p-value cutoff: ≤ 0.05, log₂ fold-change cutoff: ≥ 0.5, ≤ −0.5, N=2. (B) Panther GO term analysis of NPC-ΔZF1 dysregulated genes (see all in Table S5). Shown are the top 10 terms related to neuronal function. (C) Bar plots showing the percentage of genes from each gene set (dysregulated, random genes 1, random genes 2) that overlap with CTCF ChIP-seq peaks in NPCs. p-values comparing each of the random gene sets to dysregulated genes were determined using Fisher’s Exact test. (D) CTCF ChIP-seq heatmaps at the TSS to TES of dysregulated genes and two sets of randomly generated genes in NPCs. (E) Quantification of CTCF ChIP-seq reads from TSS to TES of genes shown in (D). p-values were determined using t-test. (F) Bar plots showing the percentage of genes from each gene set (dysregulated, random genes 1, random genes 2) that are co-localized within ΔZF1-lost loops in NPCs. p-values comparing each of the random gene sets to dysregulated genes were determined using Fisher’s Exact test. (F) Bar plots showing the number of NPC-ΔZF1-lost anchors that overlapped with promoters and/or enhancers. (G) An example locus with decreased enhancer-promoter loop contact in ΔZF1 mutant. Micro-C contact heatmap (above) compares WT and ΔZF1 contact frequency at the loop anchors (highlighted in yellow). Dlx3 gene expression is shown in the RNA-seq tracks below. CTCF ChIP-seq tracks show CTCF-binding at the loop anchors, wherein the upstream anchor is at the Dlx3 promoter and the downstream anchor is at a putative enhancer marked by H3K27ac. H3K27ac ChIP-seq is reanalyzed data from Tiwari et al., 2018 (see Table S6). The presence of CTCF motifs and their orientation are annotated.

Figure 5.. Decreased CTCF-RNA crosslinking in the ΔZF1 mutant.

(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.

Figure 6.. Identification of NPC-specific CTCF-RNA interactions.

(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, log₂ 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.

Figure 7.. Truncation of NPC-specific RNAs interacting with CTCF leads to decreased chromatin loops in cis.

(A) Schematic of CRISPR-Cas9-mediated truncation of target RNAs, Podxl and Grb10. We generated a homozygous in-frame knock-in of T2A-eGFP-SV40 polyA (pA) downstream of the 1^st exon of the coding sequence (CDS) of each gene. (B, C) RNA-seq Deseq2 normalized read counts of (B) Podxl in WT and Podxl pA mutant and (C) Grb10 in WT and Grb10 pA mutant. Data are represented as mean ± SEM, N=3. (D) APA plots of NPC WT CTCF anchors, comparing WT, NPC Podxl pA, and NPC Grb10 pA. Numbers indicate APA score. (E, F) Micro-C contact heatmaps (above) and RNA-seq tracks and ChIP-seq tracks (CTCF and Smc3) (below) at (E) the Podxl locus or (F) the Grb10 locus. NPC WT is compared to either the Podxl pA mutant or the Grb10 pA mutant, respectively. Loop boundaries are highlighted in yellow. The presence of CTCF motifs and their orientation are annotated.