DNA methylation insulates genic regions from CTCF loops near nuclear speckles

Shelby A Roseman
Allison P Siegenfeld
Ceejay Lee
Nicholas Z Lue
Amanda L Waterbury
Brian B Liau author has email address

Harvard University Department of Chemistry and Chemical Biology, Cambridge, United States
Broad Institute of MIT and Harvard, Cambridge, United States
Dana Farber Cancer Institute, Boston, United States
Harvard Medical School, Boston, MA 02215, United States
University of California, Berkeley, Berkeley, United States

https://doi.org/10.7554/eLife.102930.2

Open access
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Figures and data

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 grey 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.

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. IGV representation of highly-looping (yellow) and normal-looping CTCF peaks (grey) 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 (grey). 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, 20kb 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.

DNMT1i-induced gene activation and speckle association depend on CTCF
A. Schematic showing the CTCF degradation experimental workflow. Following pre-treatment with HaloPROTAC3 for 8 hours to degrade HaloTag-CTCF, cells are co-treated with HaloPROTAC3 and DNMT1i for 24 hours. 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. Number of genes per cluster are 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 wildtype 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.

Acute disruption of nuclear speckles alters gene expression without disrupting CTCF
A. Schematic illustrating 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 hours 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 hours or DMSO for 12 hours.
C. Replicate-averaged input normalized tags between CTCF ChIP-seq in 6-hour dTAG-13 treatment (xaxis) 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 hours 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, 20kb bins).
E. Model for methylation-mediated insulation of genes from regulatory elements near nuclear speckles 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.

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