DNA methylation insulates genic regions from CTCF loops near nuclear speckles
- Harvard University Department of Chemistry and Chemical Biology, United States
- Broad Institute of MIT and Harvard, United States
- Dana-Farber Cancer Institute, United States
- Harvard Medical School, United States
- University of California, Berkeley, United States
Figures
Figure 1 with 1 supplement
DNMT1 inhibition activates CTCF peaks and loops on gene bodies.
(A) Schematic demonstrating the experimental workflow. K562 and HCT116 cells were treated with DNMT1i for 3 days followed by CTCF ChIP-seq and CTCF HiChIP. (B) Replicate-averaged input-normalized reads within merged peaks for CTCF ChIP-seq in DNMT1i (y-axis) vs. DMSO (x-axis) for K562 cells (left) and HCT116 cells (right). DNMT1i-specific peaks are highlighted in red. (C) Example Integrated Genome Browser (IGV) CTCF ChIP-seq tracks ± DNMT1i at the MEG3 locus for K562 (top) and HCT116 (bottom). DNMT1i-specific peaks indicated with arrows. (D) Example IGV CTCF ChIP-seq and HiChIP tracks ± DNMT1i at the Protocadherin Alpha (PCDHA) locus (HCT116). DNMT1i-specific peaks are indicated below. Red indicates a looping DNMT1i-specific peak, whereas gray indicates a nonlooping DNMT1i-specific peak. (E) Boxplot showing the log2(fold-change) in loop strength DNMT1i vs. DMSO (y-axis) for loops with or without at least one anchor overlapping a DNMT1i-specific peak (x-axis) for K562 (left) and HCT116 (right). (F) Heatmaps depicting log2(mean observed/expected) Hi-C interaction frequency centered on DNMT1i-specific peaks for DMSO (left) and DNMT1i (right). K562 Hi-C data from Siegenfeld et al., 2022. (G) Proportion of looping and nonlooping peaks with each annotation (y-axis) for both DNMT1i-specific and all CTCF peaks in K562 in DNMT1i. In (E), the interquartile range (IQR) is depicted by the box with the median represented by the center line. Outliers are excluded. P-Values were calculated by a Mann-Whitney test and are annotated as follows: ns: not significant; *: 0.01<p≤0.05; **: 0.001<p≤0.01; ***: 0.0001<p≤0.001; ****: p≤0.0001.
Figure 1—figure supplement 1
DNMT1 inhibition activates CTCF peaks and loops on gene bodies.
(A) Dose-response curve in K562 (left) and HCT116 cells (right) of percent growth (y-axis) in varying doses of DNMT1i treatment (x-axis) relative to DMSO-treated control cells after 3 days of treatment. Data represent mean ± s.e.m. (B) Percent methylation in wild-type cells across CpG sites within 300 bp CTCF peak regions for DNMT1i-specific peaks and all other CTCF peaks. (C) Percent methylation status of CTCF motifs underlying DNMT1i-specific peaks clustered by K-means. Number of CTCF sites per cluster: K562 (Cluster 1: 139, Cluster 2: 351, Cluster 3: 221, Cluster 4: 738); HCT116 (Cluster 1: 405, Cluster 2: 1005, Cluster 3: 746, Cluster 4: 2249). Bisulfite data for HCT116 from GEO GSM3317488, K562 from ENCODE ENCFF459XNY. (D) Example Integrated Genome Browser (IGV) CpG methylation fraction±DNMT1i at the MEG3 locus for K562. K562 CpG methylation data extracted from LIMe-Hi-C methylation bigwigs published in Siegenfeld et al., 2022. (E) Boxplots showing normalized CpG methylation fraction (y-axis) averaged across all CpG sites within non-DNMT1i-specific and DNMT1i-specific peaks (x-axis) in the DMSO condition alone (right), the DNMT1i condition alone (middle), and the difference between DNMT1i and DMSO (left). K562 CpG methylation data extracted from LIMe-Hi-C methylation bigwigs published in Siegenfeld et al., 2022. (F) Proportion of looping and nonlooping peaks with each annotation (y-axis) for both DNMT1i-specific and all CTCF peaks in HCT116 in DNMT1i. (G) log2(fold-change) ratio of the fractions of each annotation within looping vs. nonlooping DNMT1i-specific peaks. Ratio of bars from F, Figure 1G, . (H) Number of different loops each CTCF peak makes (number of interaction partners) (y-axis) for DNMT1i-specific K562 (left) and DNMT1i-specific HCT116 (right) peaks in DNMT1i by peak annotation (x-axis). In (E), the interquartile range (IQR) is depicted by the violin with the median represented by the center dot. Outliers are excluded. For E and H, p-values were calculated by a Mann-Whitney test and are annotated as follows: ns: not significant; *: 0.01<p≤0.05; **: 0.001<p≤0.01; ***: 0.0001<p≤0.001; ****: p≤0.0001.
Figure 2 with 1 supplement
DNMT1i-specific CTCF peaks interact with highly looping partners near nuclear speckles.
(A) Number of different loops (loops to different partners) the maximally looping partner peak makes (y-axis) for all vs. DNMT1i-specific CTCF peaks (x-axis) in DNMT1i for K562 (left) and HCT116 (right). (B) Number of different loops (number of partner peaks) (y-axis) assigned to each peak by rank (x-axis) in DNMT1i for K562. Highly looping peaks reside above the red dashed line. (C) Integrated Genome Browser (IGV) representation of highly looping (yellow) and normal-looping CTCF peaks (gray) in K562 DNMT1i-treated cells. CTCF ChIP-seq and HiChIP are shown. (D) Aggregate profile (signal p-value, y-axis) of genomic features over highly looping peaks (blue) and all other CTCF peaks (gray). CTCF peaks were called in K562 DNMT1i treatment, and histone modifications/ATAC-seq data are from public datasets in K562 (see Methods). (E) Heatmap depicting log2(mean observed/expected) Hi-C interaction frequency centered on highly looping (left) and all other (right) peaks in the DMSO treatment condition in K562 cells. K562 Hi-C data from Siegenfeld et al., 2022. (F) Example IGV tracks depicting SON Cut&Tag signal (top), SON TSA-seq normalized counts (middle), CTCF ChIP-seq (bottom), and CTCF HiChIP for all CTCF peaks for a region on chromosome 11 for K562 DMSO. (G) Boxplot showing replicate-averaged DNMT1i SON Cut&Tag signal (RPKM, 20 kb bins, y-axis) in the respective cell type over DNMT1i-specific peaks vs. all other CTCF peaks called in DNMT1i for K562 and HCT116 cells broken down by whether the CTCF peak is in a loop anchor (x-axis). (H) Same as G, but for highly looping peaks vs. all other CTCF peaks. (I) Boxplot showing average log2(counts per million [CPM] loop strength) (y-axis) for CTCF HiChIP loops relative to SON Cut&Tag signal decile (x-axis). logCPM defined by Diffloop across all conditions (DMSO and DNMT1i). Loops are segregated into equally sized deciles by Cut&Tag signal in DMSO (RPKM, 20 kb bins). Pearson R=0.2. (J) Heatmap depicting log2(mean observed/expected) Hi-C interaction frequency centered on CTCF peaks at speckles (denoted by high SON signal, left) and not at speckles (right) in DMSO. K562 Hi-C data from Siegenfeld et al., 2022. In (A, G, H, and I), the interquartile range (IQR) is depicted by the box with the median represented by the center line. Outliers are excluded. P-Values were calculated by a Mann-Whitney test and are annotated as follows: ns: not significant; *: 0.01<p≤0.05; **: 0.001<p≤ 0.01; ***: 0.0001<p≤0.001; ****: p≤0.0001.
Figure 2—figure supplement 1
DNMT1i-specific CTCF peaks interact with highly looping partners near nuclear speckles.
(A) Number of different partner peaks the maximally looping partner interacts with (y-axis) for all K562 (left) and all HCT116 (right) peaks in DNMT1i by peak annotation (x-axis). (B) Number of different loops (partner peaks) (y-axis) assigned to each peak by rank (x-axis) in DNMT1i for HCT116. Highly looping peaks reside above the red dashed line. (C) Boxplot showing the log2(fold-change) in loop strength DNMT1i vs. DMSO (y-axis) for loops connecting DNMT1i-specific peaks to highly looping partners vs. all other loops (x-axis) for K562 and HCT116. (D) Histogram showing density (y-axis) of genomic distances (x-axis) of CTCF highly looping peaks (top) and all other CTCF peaks (bottom) in DNMT1i-treated K562 cells from stripe anchors identified from DNMT1i-treated K562 Hi-C data. Median distance is shown with a solid black line. K562 LIMe-Hi-C data from Siegenfeld et al., 2022. (E) Proportion of all, DNMT1i-specific, and highly looping CTCF K562 DNMT1i peaks in each published subcompartment (y-axis) assigned by SNIPER for wild-type K562 cells. (F) Boxplot showing replicate-averaged SON Cut&Tag signal (RPKM, 20 kb bins, y-axis) after DNMT1i treatment in the respective cell type over DNMT1i-specific peaks vs. all other CTCF peaks called in DNMT1i treatment for K562 and HCT116 cells. Same as Figure 2G, but not broken into looping categories. (G) Boxplots showing SON TSA-seq normalized counts (y-axis) for DNMT1i-specific vs. non-DNMT1i-specific CTCF DNMT1i peaks for K562 and HCT116 (left) broken down by whether the CTCF peak is in a loop anchor. (H) Boxplots showing SON TSA-seq normalized counts (y-axis) for highly looping vs. normal-looping CTCF DNMT1i peaks for K562 and HCT116 (right). SON TSA-seq normalized counts were analyzed from public data (4DNFIVZSO9RI.bw, 4DNFIBY8G6RZ.bw) in the untreated, wild-type respective cell type (see Methods). (I) Density plot showing the correlation between SON Cut&Tag signal (RPKM, 20 kb bins) over a given CTCF peak (y-axis) vs. the number of different loops (partners) assigned to that CTCF peak (x-axis) for K562 DNMT1i treatment. In (A, C, F, G, and H), the interquartile range (IQR) is depicted by the box with the median represented by the center line. Outliers are excluded. P-Values were calculated by a Mann-Whitney test and are annotated as follows: ns: not significant; *: 0.01<p≤0.05; **: 0.001<p≤0.01; ***: 0.0001<p≤0.001; ****: p≤0.0001.
Figure 3 with 1 supplement
DNMT1i-induced gene activation and speckle association depend on CTCF.
(A) Schematic showing the CTCF degradation experimental workflow. Following pretreatment with HaloPROTAC3 for 8 hr to degrade HaloTag-CTCF, cells are co-treated with HaloPROTAC3 and DNMT1i for 24 hr. DMSO controls without CTCF depletion and/or DNMT1 inhibition were also included. (B) Western blot showing the depletion of CTCF in HaloTag-CTCF knock-in cell line in a HaloPROTAC3-dependent and DNMT1i-independent manner (330 nM HaloPROTAC3, 10 µM DNMT1i). (C) Aggregate profile of replicate-averaged SON Cut&Tag signal (RPKM) over 1-day DNMT1i-specific CTCF peaks in HaloTag-CTCF HCT116 cells with and without CTCF degradation through HaloPROTAC3. (D) Left: Heatmap showing Z-score normalized r-log transformed counts for genes that are differentially expressed in DNMT1i vs. DMSO non-HaloPROTAC3 conditions (p-adj<0.05, |log2(fold-change)|>0.5) clustered by K-means. The number of genes per cluster is as follows: Cluster 1: 33, Cluster 2: 48, Cluster 3: 6, Cluster 4: 54. Right: Average log2(fold-change) in CTCF binding in DNMT1i vs. DMSO for all CTCF peaks on genes within the four gene clusters. (E) Boxplot showing normalized SON TSA-seq signal (20 kb bins, y-axis, 4D Nucleome 4DNFIBY8G6RZ) in wild-type HCT116 cells over genes within the different clusters identified in D. In (E), the interquartile range (IQR) is depicted by the box with the median represented by the center line. Outliers are excluded. P-Values were calculated by a Mann-Whitney test and are annotated as follows: ns: not significant; *: 0.01<p≤0.05; **: 0.001<p≤0.01; ***: 0.0001<p≤0.001; ****: p≤0.0001.
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Figure 3—source data 1
PDF file containing original western blots for Figure 3B, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/102930/elife-102930-fig3-data1-v1.pdf
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Figure 3—source data 2
Original files for western blot analysis displayed in Figure 3B.
- https://cdn.elifesciences.org/articles/102930/elife-102930-fig3-data2-v1.zip
Figure 3—figure supplement 1
DNMT1i-induced gene activation and speckle association depend on CTCF.
(A) Western blot for CTCF levels over a dose/time course of HaloPROTAC3 in HaloTag-CTCF knock-in HCT116 cells. GAPDH is shown as a loading control. 330 nM was chosen as the final dose. (B) Volcano plot showing –log10(p-adj) (y-axis) vs. log2(fold-change) in gene expression (x-axis) upon 24 hr DNMT1i treatment (left) or 32 hr HaloPROTAC3 treatment (right) in HaloTag-CTCF HCT116 cells. Upregulated genes are highlighted in red, and downregulated genes are highlighted in blue (p-adj<0.05, |log2(fold-change)|>0.5). <5 points per plot are omitted for visualization. (C) Aggregate profile plot of replicate-averaged RPKM normalized CTCF binding in DMSO over the transcription start sites of differential HaloPROTAC3 genes. (D) Boxplot showing expression levels of genes within clusters from Figure 3D in fragments per kilobase per million mapped fragments (FPKM). Expression in cells treated with vehicle (-HaloPROTAC3, - DNMT1i) is shown. (E) Heatmap of per-CTCF peak log2(fold-change CTCF binding) after 1 day of DNMT1i treatment vs. DMSO control centered over all CTCF peaks overlapping genes in the clusters from Figure 3D. Each gene could have multiple CTCF peaks on it. (F) Boxplot showing log2(fold-change SON Cut&Tag signal DNMT1i vs. DMSO, y-axis) over gene bodies for genes in the different clusters from Figure 3D. log2(fold-change) Cut&Tag signal with and without HaloPROTAC3 treatment are shown for each gene cluster. The same genes are shown for the + and - HaloPROTAC3 conditions for comparison. In (D and F), the interquartile range (IQR) is depicted by the box with the median represented by the center line. Outliers are excluded. P-Values were calculated by a Mann-Whitney test and are annotated as follows: ns: not significant; *: 0.01<p≤0.05; **: 0.001<p≤0.01; ***: 0.0001<p≤0.001; ****: p≤0.0001.
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Figure 3—figure supplement 1—source data 1
PDF file containing original western blots for Figure 3—figure supplement 1A, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/102930/elife-102930-fig3-figsupp1-data1-v1.pdf
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Figure 3—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 3—figure supplement 1A.
- https://cdn.elifesciences.org/articles/102930/elife-102930-fig3-figsupp1-data2-v1.zip
Figure 4 with 1 supplement
Acute disruption of nuclear speckles alters gene expression without disrupting CTCF.
(A) Schematic illustrating the use of the dTAG system to acutely deplete SON and SRRM2, thus disrupting nuclear speckles. (B) Immunofluorescence for SON and SRRM2 in K562 speckle knock-in cells treated with dTAG-13 for 6 or 12 hr to deplete SON and SRRM2. Right: per-nucleus mean fluorescence following immunofluorescence for SON/SRRM2 double dTAG knock-in K562 cells treated with dTAG-13 for 6 or 12 hr or DMSO for 12 hr. (C) Replicate-averaged input normalized tags between CTCF ChIP-seq in 6 hr dTAG-13 treatment (x-axis) vs. DMSO (y-axis) for K562 speckle dTAG knock-in cells. (D) Boxplot showing log2(fold-change) in loop strength for CTCF HiChIP loops in speckle knock-in K562 cells treated with dTAG-13 vs. DMSO (y-axis) for 6 hr relative to SON Cut&Tag signal (x-axis). Loops are segregated into equally sized deciles by Cut&Tag signal with 10 representing the decile closest to speckles (RPKM, 20 kb bins). (E) Model for methylation-mediated insulation of genes from regulatory elements near nuclear speckle. In (B and D), the interquartile range (IQR) is depicted by the box with the median represented by the center line. Outliers are excluded. P-Values were calculated by a Mann-Whitney test and are annotated as follows: ns: not significant; *: 0.01<p≤0.05; **: 0.001<p≤0.01; ***: 0.0001<p≤0.001; ****: p≤0.0001.
Figure 4—figure supplement 1
Acute disruption of nuclear speckles alters gene expression without disrupting CTCF.
(A) Separate western blots for SON and SRRM2 in speckle dTAG knock-in K562 cells ±6 hr of 500 nM dTAG-13 treatment. Vinculin was used as a loading control. (B) Volcano plot showing –log10(p-adj) (y-axis) vs. log2(fold-change) in gene expression (x-axis) upon speckle degradation with 6 hr dTAG-13 treatment. Upregulated genes are highlighted in red, and downregulated genes are highlighted in blue (p-adj<0.05, |log2(fold-change)|>0.25). (C) Proportion (y-axis) of all nonzero (left) and all 6 hr dTAG-13 downregulated (right, p-adj<0.05) genes in each SON TSA-seq decile (x-axis) from wild-type cells. 10 is the decile closest to speckles. (D) Density plot showing SON Cut&Tag signal in wild-type K562 cells treated with DMSO (RPKM 20 kb bins, y-axis) vs. the change in gene expression (x-axis) for genes that decrease in expression upon dTAG-13 treatment (Spearman R, –0.43). (E) Aggregate profile plot of CTCF ChIP-seq signal (RPKM) in speckle knock-in K562 cells with and without 6 hr speckle degradation (y-axis) over all CTCF peaks (x-axis). (F) Boxplot showing log2(fold-change) loop strength for dTAG-13 treated vs. DMSO-treated speckle dTAG knock-in cells relative to TSA-seq decile after 6 or 12 hr of dTAG-13 treatment with matched DMSO controls. Speckle refers to the decile closest to speckles, and non-speckle refers to the deciles 1–9 that are furthest from speckles. (G) Aggregate profile plot of CTCF ChIP-seq signal (RPKM) in speckle dTAG knock-in K562 cells with dTAG-13 vs. DMSO treatment (y-axis) across expressed genes (top) and genes that are downregulated with dTAG-13 treatment (bottom) (x-axis). (H) Boxplot showing log2(fold-change) loop strength for 6 hr dTAG-13 treated vs. DMSO-treated speckle dTAG knock-in cells (y-axis) for loops with anchors that do not overlap any genes, overlap-only genes that do not change expression, and overlap genes that increase or decrease in expression for at least one anchor (x-axis). (I) Representative SON and SRRM2 immunofluorescence images (green, Alexa Fluor 488) in HaloTag-CTCF HCT116 cells with 24 hr of 330 nM HaloPROTAC3 treatment to deplete CTCF or vehicle control. Overlaid on DAPI in blue. In (F and H), the interquartile range (IQR) is depicted by the box with the median represented by the center line. Outliers are excluded. P-Values were calculated by a Mann-Whitney test and are annotated as follows: ns: not significant; *: 0.01<p≤0.05; **: 0.001<p≤0.01; ***: 0.0001<p≤0.001; ****: p≤0.0001.
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Figure 4—figure supplement 1—source data 1
PDF file containing original western blots for Figure 4—figure supplement 1A, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/102930/elife-102930-fig4-figsupp1-data1-v1.pdf
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Figure 4—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 4—figure supplement 1A.
- https://cdn.elifesciences.org/articles/102930/elife-102930-fig4-figsupp1-data2-v1.zip
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Additional files
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Supplementary file 1
HiChIP looping information.
- https://cdn.elifesciences.org/articles/102930/elife-102930-supp1-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/102930/elife-102930-mdarchecklist1-v1.pdf
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DNA methylation insulates genic regions from CTCF loops near nuclear speckles
eLife 13:RP102930.
https://doi.org/10.7554/eLife.102930.3
