TAD boundaries and gene activity are uncoupled
- National Cancer Institute, NIH, United States
- Systems Biology of Gene Expression, National Cancer Institute, NIH, United States
- High-throughput Imaging Facility, National Cancer Institute, NIH, United States
Figures
Figure 1
High-throughput DNA/RNA FISH.
(A) Schematic overview of the DNA/RNA high-throughput FISH imaging (HiFISH) pipeline used to simultaneously measure topologically associating domain (TAD) boundary distance and gene activity at the single-cell and single-allele levels. Step 1: Design of DNA FISH probes based on Micro-C profiling and detection of DNA and nascent RNA by HiFISH. Step 2: Measurement of center-to-center TAD boundary distances and RNA signal at individual alleles by image analysis using HiTIPS (Keikhosravi et al., 2024). Step 3: Quantitative comparison of TAD boundary distances with gene activity at each allele. (B) DNA/RNA HiFISH detection of 5’ (green) and 3’ (red) MYC TAD boundaries and nascent RNA (blue) in human bronchial epithelial cells (HBECs). Scale bar: 10 μm. (C) Quantification of MYC nascent RNA signals using DNA/RNA HiFISH in fixed HBECs or an MS2-tagged MYC reporter in living HBECs. Bars represent means ± SEM from two experiments. Dots indicate means from individual experiments. 166,953 cells were analyzed for MS2, and 30,137 cells for DNA/RNA HiFISH. Statistical significance was calculated using two-way ANOVA with Bonferroni correction: ns, not significant (p≥0.05).
© 2026, Guin. Panel A was created with BioRender and is published under a Creative Commons Attribution License. Further reproductions must adhere to the terms of this license.
Figure 2 with 2 supplements
Topologically associating domain (TAD) boundaries interact more frequently than non-TAD regions.
(A) Micro-C contact maps for EGFR and MYC TADs and adjacent regions in hTERT-HFFc6 fibroblast (HFF) cells, highlighting TAD boundaries and genomically equidistant non-TAD regions. Squares denote the probe positions used for 3’ (green), 5’ boundary (red), and equidistant non-TAD controls (purple). Interactions between the 5’ TAD boundaries and the 3’ TAD (yellow) or non-TAD boundaries (gray) are highlighted, and total Micro-C contacts between regions are quantified, emphasizing high TAD boundary contact frequency in both EGFR and MYC TADs, as well as weaker signals in the non-TAD regions. (B–C) Representative DNA high-throughput FISH imaging (HiFISH) images of EGFR and MYC TAD boundary and non-TAD regions in HFF cells (B) and human bronchial epithelial cells (HBECs) (C). Scale bar: 10 μm. (D–E) Measurement of boundary distances. Distance distributions of EGFR and MYC TAD boundaries vs. matched non-TAD regions in HFF cells (D) and HBECs (E). Dashed line indicates 250 nm threshold used to define physical interaction. Between 2,000 and 18,000 alleles were analyzed per sample. Values represent an individual dataset from a single experiment of multiple experiments. Mann-Whitney U test p-values are: ***p<1 × 10–100; ** 1×10–100≤p<1×10–20; * 1×10–20≤p<0.01.
© 2026, Guin. Panel A was created with BioRender and is published under a Creative Commons Attribution License. Further reproductions must adhere to the terms of this license.
Figure 2—figure supplement 1
Micro-C chromosome interaction maps and ChromHMM analysis of EGFR, MYC, ERRFI1, FKBP5, and VARS2 topologically associating domains (TADs) in HFFc6.
Diamonds denote probe interaction sites in Micro-C for both the non-TAD probes (gray) and the TAD boundary probes (yellow). The cytogenetic chromosome band track (black) indicates the chromosome location of the indicated loci. The gene reference track shows all coding and noncoding genes. The binding sites track shows CTCF (navy blue) and RAD21 (purple). ChromHMM chromatin states for two different foreskin fibroblast cell lines.
Figure 2—figure supplement 2
Sequence and location of DNA and RNA probes binding sites for DNA/RNA high-throughput FISH imaging (HiFISH).
Schematic representation of DNA and RNA probes target regions. RNA probes targeting sequences are indicated in blue.
Figure 3 with 1 supplement
Topologically associating domain (TAD) boundary proximity is not related to gene activity status.
(A) Representative DNA/RNA high-throughput FISH imaging (HiFISH) image of EGFR nascent RNA (blue) and its associated 5’ (red) and 3’ (green) TAD boundaries in HBECs, illustrating detection of active (RNA-positive) and inactive (RNA-negative) alleles. Scale bar: 10 μm. (B – E) Comparison of TAD boundary distances for EGFR and MYC alleles based on transcriptional activity status. Histograms of allele-specific distance distributions from a representative dataset from a single experiment; Mann-Whitney U test p-values are indicated as follows: ns, not significant (p≥0.05). Dot plots of the mean of median distances from multiple experiments (500–20,000 alleles per condition); error bars represent SEM, and statistical significance was calculated using two-way ANOVA with Bonferroni correction: ns, not significant (p≥0.05).
© 2026, Guin. Panel A was created with BioRender and is published under a Creative Commons Attribution License. Further reproductions must adhere to the terms of this license.
Figure 3—figure supplement 1
Topologically associating domain (TAD) boundary proximity is uncoupled from allelic gene activity in single nuclei in both 2D and 3D imaging.
(A) Comparison of TAD boundary distances for EGFR and MYC alleles in HCT116 based on transcriptional activity status. Histograms of allele-specific distance distributions from a representative dataset from a single experiment. Mann-Whitney U test p-values are indicated as follows: ****p<0.0001; ns, not significant (p≥0.05). (B) Comparative analysis of TAD boundary distances between active and inactive alleles within the same nucleus for EGFR and MYC loci in human bronchial epithelial cells (HBECs) and hTERT-HFFc6 fibroblast (HFF) cells. (C) Reciprocal analysis of MYC alleles in HBEC stratified by TAD boundary distance, shown for both 2D and 3D measurements. Alleles were grouped into TAD boundary-distance bins using a histogram bin width of 0.108 µm, corresponding to the xy pixel size of the imaging system, and transcriptional activity was then assessed across these distance categories. Histograms show data from a representative dataset of a single experiment. Mann-Whitney U test p-values are indicated as follows: ns, not significant (p≥0.05).
Figure 4 with 1 supplement
Global transcription inhibition does not alter topologically associating domain (TAD) boundary pairing.
(A) Representative DNA/RNA high-throughput FISH imaging (HiFISH) images of EGFR and MYC TAD boundary and nascent RNA in HBECs and HFF cells with and without 2 hr 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DRB) treatment. Scale bars: 10 μm. (B) Quantification of TAD boundary distances for EGFR and MYC in the presence or absence of DRB. Dot plots of the mean of median distances from multiple experiments (500–20,000 alleles per condition). Error bars represent SEM. Statistical significance was calculated using two-way ANOVA with Bonferroni correction: ns, not significant (p≥0.05).
Figure 4—figure supplement 1
Transcriptional inhibition does not affect the spatial organization of non-topologically associating domain (TAD) control regions.
(A) Comparison of TAD boundary and non-TAD distances for EGFR and MYC alleles in human bronchial epithelial cell (HBEC) and hTERT-HFFc6 fibroblast (HFF) based on transcriptional inhibition status. Histograms show allele-specific distance distributions from a representative dataset of a single experiment.
Figure 5 with 1 supplement
Transcription stimulation does not alter topologically associating domain (TAD) boundary interactions.
(A) Micro-C maps for the ERRFI1, FKBP5, and VARS2 TADs and neighboring regions, showing TAD boundaries (green, red) and equidistant non-TAD control regions (red, purple) in hTERT-HFFc6 fibroblast (HFF); corresponding probe positions are indicated. Interactions between the 5’ TAD boundaries and the 3’ TAD (yellow) or equidistant non-TAD control regions (gray) are highlighted, and total Micro-C contacts between regions are quantified, showing prominent contact frequency between ERRFI1, VARS2, and FKBP5 TAD boundaries, as well as the non-TAD region of ERRFI1. (B) Comparison of TAD boundary and non-TAD region distances for EGFR, MYC, ERRFI1, FKBP5, and VARS2 in human bronchial epithelial cells (HBECs) as measured by DNA HiFISH. Dot plots of the mean of median distances from two experiments (11,000–49,000 alleles per condition). Error bars represent SEM. Statistical significance was calculated using two-way ANOVA with Bonferroni correction: ****p<0.0001; **p<0.01; ns, not significant (p≥0.05). (C) Measurement of boundary distances. Distance distributions of ERRFI1, FKBP5, and VARS2 TADs in untreated and 2 hr dexamethasone (Dex)-treated HBEC. Between 2,500 and 6,000 alleles were analyzed per condition. Values represent an individual dataset from a single experiment representative of multiple experiments. Mann-Whitney U test p-values are indicated as follows: ns, not significant (p≥0.05).
Figure 5—figure supplement 1
RNA levels following dexamethasone (Dex) treatment.
(A) RNA-seq analysis of ERRFI1, VARS2, and FKBP5 RNA levels following Dex treatment for the indicated durations in human bronchial epithelial cells (HBECs). Values were calculated for RPKM fold-change (Dex/No_Dex) ratio. Data represent the mean of three independent experiments. (B) Histograms of the distribution of nascent RNA transcription sites per nucleus in HBECs upon Dex treatment. Data represent values from at least two independent experiments (diamonds and circles); diamonds (EtOH control) and circles (Dex 2 hr) represent the mean of means, and error bars indicate SD. p-Values from two-way ANOVA with Bonferroni correction are shown as: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant (p≥0.05).
Figure 6 with 1 supplement
Effects of RAD21 and CTCF depletion on topologically associating domain (TAD) boundary distances and gene expression.
(A) Representative DNA/RNA high-throughput FISH imaging (HiFISH) images of EGFR and MYC nascent RNA FISH (blue) and its 3’ (green) and 5’ (red) TAD boundaries DNA FISH in HCT116-RAD21-AID1 and HCT116-CTCF-AID2 cells, respectively, in control and auxin-treated conditions. Scale bar: 10 μm. (B) TAD boundary distances and non-TAD controls after RAD21 or CTCF depletion for 3 hr. Values represent an individual dataset from a single experiment representative of multiple experiments. Between 13,000 and 127,500 alleles were analyzed per condition. Mann-Whitney U test p-values are indicated as follows: ***p<1 × 10–100; ns, not significant (p≥0.05). (C) Fraction of silent (0), monoallelic (1), biallelic (2), and triallelic or more (≥3) expression of the indicated genes in individual cells after 3 hr or no auxin treatment in HCT116-RAD21-AID1 or HCT116-CTCF-AID2 cells. At least 20,000 cells were measured per experiment. Data represent values from at least two independent experiments (diamonds and circles); diamonds (DMSO control) and circles (RAD21 or CTCF-depleted) represent the mean of means, and error bars indicate SD. p-Values from two-way ANOVA with Bonferroni correction are shown as: ****p<0.0001; **p<0.01; *p<0.05; ns, not significant (p≥0.05).
Figure 6—figure supplement 1
Depletion of RAD21 and CTCF.
(A) Loss of RAD21 or CTCF in HCT116-RAD21-AID1 or HCT116-CTCF-AID2 cells, respectively, following DMSO (control) or auxin treatment. RAD21 and CTCF degradation were assessed using mClover fluorescence (green). Scale bar: 20 μm. (B–C) Fraction of silent (0), monoallelic (1), biallelic (2), and triallelic or more (≥3) expression of the indicated genes in individual cells after 3 hr or no auxin treatment in HCT116-RAD21-AID1 (B) or HCT116-CTCF-AID2 (C) cells. Data represent values from at least two independent experiments (diamonds and circles); diamonds (DMSO control) and circles (RAD21 or CTCF-depleted) represent the mean of means, and error bars indicate SD. p-Values from two-way ANOVA with Bonferroni correction are shown as: *p<0.05; ns, not significant (p≥0.05).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | Cell line HBEC3-KT (HBEC) | Ramirez et al., 2004 | RRID:CVCL_X491 | Human bronchial epithelial cells immortalized with hTERT and CDK4 |
| Cell line (Homo sapiens) | Cell line HFF-hTERT clone 6 (HFFc6; HFF) | Cellosaurus | RRID:CVCL_VC41 | hTERT-immortalized human foreskin fibroblasts (clone 6) |
| Cell line (Homo sapiens) | Cell line HCT 116 | Cellosaurus | RRID:CVCL_0291 | Human colorectal carcinoma line; used as parent for degron derivatives |
| Cell line (Homo sapiens) | Cell line HCT-116 RAD21-mAID-mClover (RAD21-mAC) | Natsume et al., 2016 | Human colorectal carcinoma line (HCT116) derivative used for AID1-mediated RAD21 depletion | |
| Cell line (Homo sapiens) | Cell line HCT116-CTCF-AID2 | Yesbolatova et al., 2020 | HCT116 derivative used for AID2-mediated CTCF depletion | |
| Commercial assay or kit | Airway Epithelial Cell Basal Medium | ATCC | ATCC:PCS-300-030 | |
| Commercial assay or kit | Bronchial Epithelial Cell Growth Kit | ATCC | ATCC:PCS-300-040 | |
| Chemical compound, drug | DRB | Sigma-Aldrich | Sigma:D1916 | Transcription inhibition |
| Chemical compound, drug | Dexamethasone (Dex) | Sigma-Aldrich | Sigma:D4902 | Glucocorticoid receptor agonist |
| Chemical compound, drug | Auxin | Sigma-Aldrich | Sigma:I3750 | Used for RAD21 depletion in AID1 degron system |
| Chemical compound, drug | 5-Ph-IAA | GLPBio | GLPBio:GC46061 | AID2 ligand used in AID2 degron system |
| Recombinant DNA reagent | BAC probe RP11-112A3 (EGFR upstream control) | BACPAC Resources Center | BACPAC:RP11-112A3 | |
| Recombinant DNA reagent | BAC probe RP11-117I14 (EGFR 5′ TAD) | BACPAC Resources Center | BACPAC:RP11-117I14 | |
| Recombinant DNA reagent | BAC probe RP11-98C17 (EGFR 3′ TAD) | BACPAC Resources Center | BACPAC:RP11-98C17 | |
| recombinant DNA reagent | BAC probe RP11-788I22 (MYC upstream control) | BACPAC Resources Center | BACPAC:RP11-788I22 | |
| Recombinant DNA reagent | BAC probe RP11-765K23 (MYC 5′ TAD) | BACPAC Resources Center | BACPAC:RP11-765K23 | |
| Recombinant DNA reagent | BAC probe RP11-717D13 (MYC 3′ TAD) | BACPAC Resources Center | BACPAC:RP11-717D13 | – |
| Recombinant DNA reagent | BAC probe RP11-279H6 (ERRFI1 upstream control) | BACPAC Resources Center | BACPAC:RP11-279H6 | |
| Recombinant DNA reagent | BAC probe RP11-338N10 (ERRFI1 5′ TAD) | BACPAC Resources Center | BACPAC:RP11-338N10 | |
| Recombinant DNA reagent | BAC probe RP11-366K21 (ERRFI1 3′ TAD) | BACPAC Resources Center | BACPAC:RP11-366K21 | |
| Recombinant DNA reagent | BAC probe RP11-192H11 (VARS2 upstream control) | BACPAC Resources Center | BACPAC:RP11-192H11 | |
| Recombinant DNA reagent | BAC probe RP11-159K11 (VARS2 5′ TAD) | BACPAC Resources Center | BACPAC:RP11-159K11 | |
| Recombinant DNA reagent | BAC probe RP11-803D22 (VARS2 3′ TAD) | BACPAC Resources Center | BACPAC:RP11-803D22 | |
| Recombinant DNA reagent | BAC probe RP11-107C8 (FKBP5 upstream control) | BACPAC Resources Center | BACPAC:RP11-107C8 | |
| Recombinant DNA reagent | BAC probe RP11-78C20 (FKBP5 5′ TAD) | BACPAC Resources Center | BACPAC:RP11-78C20 | |
| Recombinant DNA reagent | BAC probe RP11-828B18 (FKBP5 3′ TAD) | BACPAC Resources Center | BACPAC:RP11-828B18 | |
| Sequence-based reagent | Stellaris RNA probe set: EGFR (Atto647N) | LGC Biosearch Technologies | ||
| Sequence-based reagent | Stellaris RNA probe set: MYC (Atto647N) | LGC Biosearch Technologies | ||
| Sequence-based reagent | Stellaris RNA probe set: ERRFI1 (Quasar 670) | LGC Biosearch Technologies | ||
| Sequence-based reagent | Stellaris RNA probe set: VARS2 (Atto647N) | LGC Biosearch Technologies | ||
| Sequence-based reagent | Stellaris RNA probe set: FKBP5 (Atto647N) | LGC Biosearch Technologies | LGC:ISMF-2059-5 | |
| Software, algorithm | HiTIPS | Keikhosravi et al., 2024; Keikhosravi, 2025 | https://github.com/CBIIT/HiTIPS | High-throughput segmentation/detection and quantification pipeline |
| Software, algorithm | CellPose | PMID:33318659 | RRID:SCR_021716 | Nucleus segmentation within HiTIPS workflow |
| Software, algorithm | DNA/RNA registration (cross-correlation) | Keikhosravi, 2024: Almansour et al., 2024 | https://github.com/CBIIT/DNA_RNA_registration | Used for sequential DNA/RNA image registration; implemented in Python 3.8 |
| Software, algorithm | Analysis scripts for DNA/RNA HiFISH quantification | Other | https://github.com/CBIIT/mistelilab-tad-ge | R scripts used for boundary-distance and single-cell gene-expression calculations |
| Software, algorithm | UCSC Genome Browser | Nassar et al., 2023 | RRID:SCR_005780 | |
| Software, algorithm | 4DN Data Portal | Dekker et al., 2017 | RRID:SCR_016925 | |
| Other | Charcoal-stripped fetal bovine serum | R&D Systems | R&D:S11650H | |
| Other | 384-Well imaging plates (PhenoPlate) | Revvity | Revvity:6057500 | High-throughput imaging format |
| Other | DY549P1-dUTP | Dyomics | Used for BAC probe labeling by nick translation | |
| Other | DY488-dUTP | Dyomics | Used for BAC probe labeling by nick translation | |
| Other | THE RNA Storage Solution | Thermo Fisher Scientific | Thermo Fisher:AM7001 | Included in hybridization buffer |
| Other | Yokogawa CV8000 spinning-disk confocal microscope | Yokogawa | 60× water objective (NA 1.2); 4 laser lines (405/488/561/640 nm) |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/110197/elife-110197-mdarchecklist1-v1.pdf
-
Supplementary file 1
RNA probe sequences.
- https://cdn.elifesciences.org/articles/110197/elife-110197-supp1-v1.xlsx
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TAD boundaries and gene activity are uncoupled
eLife 15:RP110197.
https://doi.org/10.7554/eLife.110197.3
