An in vivo allelic series at the mouse Sox9–Kcnj2 locus showed that deleting every major CTCF site at a TAD boundary and inside the TAD fuses neighboring domains yet leaves gene expression and development largely intact, whereas inversions that reposition boundaries redirect enhancer activity and cause disease — TAD architecture is thus nonessential but instructive. The problem left open by this allelic series is to identify the molecular determinants that make individual CTCF-binding sites strong or weak insulators, establish the mechanism(s) by which enhancers contact and activate their promoters in the absence of CTCF-defined loops, determine how these principles generalize across loci and the genome, define how promoter-level competition shapes novel enhancer–promoter contacts, and derive rules predicting which structural variants redirect regulatory activity in a pathogenic manner.

Background

In mammalian genomes, regions of roughly a hundred kilobases to a megabase contact each other far more frequently than they contact their neighbors; these self-interacting units are called topologically associating domains (TADs), and they were discovered with Hi-C, a method that cross-links and sequences physically touching DNA fragments to map genome-wide contact frequencies (Dixon et al. 2012, Nature 485:376–380, doi:10.1038/nature11082). Most TAD boundaries are marked by clusters of binding sites for CTCF, a DNA-binding protein that, together with the ring-shaped cohesin complex, is thought to build domain structure through loop extrusion: cohesin progressively enlarges a loop until it stalls at a CTCF site, so that pairs of properly oriented CTCF sites anchor loops and clusters of them form boundaries. Two observations created a long-standing tension. First, acute degradation of CTCF or cohesin dissolves loops and domain structure, yet transcription of most genes changes only modestly (Nora et al. 2017, Cell 169:930–944.e22, doi:10.1016/j.cell.2017.05.004; Rao et al. 2017, Cell 171:305–320.e24, doi:10.1016/j.cell.2017.09.026). Second, genomic rearrangements that disrupt TADs can cause Mendelian disease by enhancer hijacking — repositioning a distal enhancer so that it activates the wrong gene — as in limb malformation syndromes caused by rearrangements near the SOX9 regulatory landscape (Lupiáñez et al. 2015, Cell 161:1012–1025, doi:10.1016/j.cell.2015.04.004). How can structure be dispensable for most genes yet its rearrangement be catastrophic?

Despang et al. (2019, Nature Genetics 51:1263–1271, doi:10.1038/s41588-019-0466-z) addressed this with an allelic series — a set of engineered mouse alleles, each carrying a different deletion, inversion, or CTCF-site rearrangement — at the Sox9–Kcnj2 locus in embryonic limb buds, assayed by capture Hi-C (cHi-C, a targeted Hi-C variant that enriches for a chosen locus), RNA profiling and skeletal phenotyping. Sox9 is a transcription-factor gene essential for skeleton development whose loss is lethal, and Kcnj2 is an adjacent potassium-channel gene in a neighboring TAD. Deleting the TAD boundary alone did not fuse the TADs; only removing the boundary together with four internal CTCF sites (C1–C4) achieved fusion, and even then gene expression changed only mildly — the enhancers still found and regulated their cognate promoters. Inverting the TAD or repositioning the boundary, however, redirected regulatory activity, producing loss of Sox9, gain of Kcnj2, and disease phenotypes. The authors concluded that CTCF-defined architecture provides redundancy, precision and robustness but is not essential, and that pathogenic gene activation requires active CTCF-dependent redirection of enhancer–promoter contacts, not mere loss of insulation. In the Discussion they state the questions this leaves open as (i)–(iv), and the findings additionally imply (v): (i) the analysis is of a single locus, and the structure–function relationship might differ at other TADs, while a comparable allelic series is not feasible genome-wide; (ii) individual internal CTCF sites contributed unequally to insulation (the blocking of regulatory interactions across a boundary) — deleting C1 with the boundary affected contacts more than other sites — yet no correlation with CTCF ChIP-seq (chromatin immunoprecipitation followed by sequencing, which measures CTCF occupancy) or motif orientation was found, so the molecular determinants of a site's insulating potency are unknown; (iii) the mechanism allowing enhancer–promoter communication without CTCF-defined loops is unestablished — candidate explanations include cohesin remaining on chromatin independently of CTCF, intrinsic enhancer–promoter specificity (promoter type, histone modifications, chromatin openness), homotypic interactions of transcription factors bound at enhancers and promoters, and transcriptional condensates (membraneless aggregates of transcription machinery); (iv) promoter competition between Sox9 and Kcnj2 for shared enhancers could not be tested directly because Sox9 promoter deletions are lethal; and (v) the findings imply, but do not deliver, predictive rules for interpreting which structural variants will redirect regulatory activity pathogenically in the clinic.

Problem Statement

The problem left open by the functional dissection of the Sox9–Kcnj2 locus is to replace its locus-specific conclusions with a mechanistic, locus-general account of how CTCF-defined chromatin architecture relates to gene regulation, composed of five scientifically linked questions: (1) Determinants of insulating potency — identify the molecular properties (such as sequence context, occupancy dynamics, motif orientation within clusters, boundary composition and width, transcriptional activity at the site) that make individual CTCF-binding sites strong or weak insulators, explaining why at Sox9–Kcnj2 the internal site C1 dominates insulation while potency does not correlate with CTCF ChIP-seq strength or motif orientation. (2) Mechanism of CTCF-independent enhancer–promoter communication — establish how enhancers contact and activate their cognate promoters when all CTCF-defined loops are removed, discriminating among candidate mechanisms such as residual cohesin-mediated contacts, homotypic interactions of transcription factors bound at enhancers and promoters, transcriptional condensates, and promoter-intrinsic specificity (promoter type, chromatin state, histone modifications). (3) Generalization — determine how the principles observed at Sox9–Kcnj2 (redundancy of CTCF sites; architecture providing precision and robustness but not necessity; pathogenicity requiring active redirection rather than mere loss of insulation) distribute across loci with different enhancer densities, promoter architectures and tissue contexts, given that outcomes at loci dissected so far range from negligible to fully penetrant lethality. (4) Promoter-level shaping of novel contacts — define how promoter competition and promoter-intrinsic properties determine the transcriptional outcome when a rearrangement places a gene in contact with foreign enhancers, including cases where direct promoter deletion is lethal. (5) Prediction — derive from (1)–(4) rules that predict which structural variants will be tolerated and which will redirect regulatory activity in a pathogenic manner, the capability that clinical interpretation of noncoding structural variants currently lacks. These five questions are jointly mandatory: the task is resolved only by a complete causal account from CTCF-site composition to insulation strength to enhancer–promoter communication to transcriptional and phenotypic outcome, validated across loci and applied to rearranged genomes.

References

  1. Despang A, Schöpflin R, Franke M, Ali S, Jerković I, Paliou C, Chan W, Timmermann B, Wittler L, Vingron M, Mundlos S, Ibrahim DM (2019) Functional dissection of the Sox9–Kcnj2 locus identifies nonessential and instructive roles of TAD architecture. Nature Genetics 51:1263–1271. doi:10.1038/s41588-019-0466-z. https://doi.org/10.1038/s41588-019-0466-z
  2. Dixon JR, Selvaraj S, Yue F, Kim A, Li Y, Shen Y, Hu M, Liu JS, Ren B (2012) Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485:376–380. doi:10.1038/nature11082. https://doi.org/10.1038/nature11082
  3. Nora EP, Goloborodko A, Valton A, Gibcus JH, Uebersohn A, Abdennur N, Dekker J, Mirny LA, Bruneau BG (2017) Targeted degradation of CTCF decouples local insulation of chromosome domains from genomic compartmentalization. Cell 169:930–944.e22. doi:10.1016/j.cell.2017.05.004. https://doi.org/10.1016/j.cell.2017.05.004
  4. Rao SSP, Huang S, Glenn St Hilaire B, Engreitz JM, Perez EM, Kieffer-Kwon K, Sanborn AL, Johnstone SE, Bascom GD, Bochkov ID, Huang X, Shamim MS, Shin J, Turner D, Ye Z, Omer AD, Robinson JT, Schlick T, Bernstein BE, Casellas R, Lander ES, Aiden EL (2017) Cohesin loss eliminates all loop domains. Cell 171:305–320.e24. doi:10.1016/j.cell.2017.09.026. https://doi.org/10.1016/j.cell.2017.09.026
  5. Lupiáñez DG, Kraft K, Heinrich V, Krawitz P, Brancati F, Klopocki E, Horn D, Kayserili H, Opitz JM, Laxova R, Santos-Simarro F, Gilbert-Dussardier B, Wittler L, Borschiwer M, Haas SA, Osterwalder M, Franke M, Timmermann B, Hecht J, Spielmann M, Visel A, Mundlos S (2015) Disruptions of topological chromatin domains cause pathogenic rewiring of gene-enhancer interactions. Cell 161:1012–1025. doi:10.1016/j.cell.2015.04.004. https://doi.org/10.1016/j.cell.2015.04.004
  6. Huang H, Zhu Q, Jussila A, Han Y, Bintu B, Kern C, Conte M, Zhang Y, Bianco S, Chiariello AM, Yu M, Hu R, Tastemel T, Juric I, Hu M, Nicodemi M, Zhuang X, Ren B (2021) CTCF mediates dosage- and sequence-context-dependent transcriptional insulation by forming local chromatin domains. Nature Genetics 53:1064–1074. doi:10.1038/s41588-021-00863-6. https://doi.org/10.1038/s41588-021-00863-6
  7. Anania C, Acemel RD, Jedamzick J, Bolondi A, Cova G, Brieske N, Kühn R, Wittler L, Real FM, Lupiáñez DG (2022) In vivo dissection of a clustered-CTCF domain boundary reveals developmental principles of regulatory insulation. Nature Genetics 54:1026–1036. doi:10.1038/s41588-022-01117-9. https://doi.org/10.1038/s41588-022-01117-9
  8. Hsieh TS, Cattoglio C, Slobodyanyuk E, Hansen AS, Darzacq X, Tjian R (2022) Enhancer–promoter interactions and transcription are largely maintained upon acute loss of CTCF, cohesin, WAPL or YY1. Nature Genetics 54:1919–1932. doi:10.1038/s41588-022-01223-8. https://doi.org/10.1038/s41588-022-01223-8
  9. Zuin J, Roth G, Zhan Y, Cramard J, Redolfi J, Piskadlo E, Mach P, Kryzhanovska M, Tihanyi G, Kohler H, Eder M, Leemans C, van Steensel B, Meister P, Smallwood S, Giorgetti L (2022) Nonlinear control of transcription through enhancer–promoter interactions. Nature 604:571–577. doi:10.1038/s41586-022-04570-y. https://doi.org/10.1038/s41586-022-04570-y
  10. Chakraborty S, Kopitchinski N, Zuo Z, Eraso A, Awasthi P, Chari R, Mitra A, Tobias IC, Moorthy SD, Dale RK, Mitchell JA, Petros TJ, Rocha PP (2023) Enhancer–promoter interactions can bypass CTCF-mediated boundaries and contribute to phenotypic robustness. Nature Genetics 55:280–290. doi:10.1038/s41588-022-01295-6. https://doi.org/10.1038/s41588-022-01295-6
  11. Chang L, Ghosh S, Papale A, Luppino JM, Miranda M, Piras V, Degrouard J, Edouard J, Poncelet M, Lecouvreur N, Bloyer S, Leforestier A, Joyce EF, Holcman D, Noordermeer D (2023) Multi-feature clustering of CTCF binding creates robustness for loop extrusion blocking and topologically associating domain boundaries. Nature Communications 14:5615. doi:10.1038/s41467-023-41265-y. https://doi.org/10.1038/s41467-023-41265-y
  12. Ge X, Huang H, Han K, Xu W, Wang Z, Wu Q (2023) Outward-oriented sites within clustered CTCF boundaries are key for intra-TAD chromatin interactions and gene regulation. Nature Communications 14:8101. doi:10.1038/s41467-023-43849-0. https://doi.org/10.1038/s41467-023-43849-0
  13. Islam Z, Saravanan B, Walavalkar K, Farooq U, Singh AK, Radhakrishnan S, Thakur J, Pandit A, Henikoff S, Notani D (2023) Active enhancers strengthen insulation by RNA-mediated CTCF binding at chromatin domain boundaries. Genome Research 33:1–17. doi:10.1101/gr.276643.122. https://doi.org/10.1101/gr.276643.122
  14. Ealo T, Sanchez-Gaya V, Respuela P, Muñoz-San Martín M, Martin-Batista E, Haro E, Rada-Iglesias A (2024) Cooperative insulation of regulatory domains by CTCF-dependent physical insulation and promoter competition. Nature Communications 15:7258. doi:10.1038/s41467-024-51602-4. https://doi.org/10.1038/s41467-024-51602-4
  15. Baudic M, Murata H, Bosada FM, Melo US, Aizawa T, Lindenbaum P, van der Maarel LE, Guedon A, Baron E, Fremy E, Foucal A, Ishikawa T, Ushinohama H, Jurgens SJ, Choi SH, Kyndt F, Le Scouarnec S, Wakker V, Thollet A, Rajalu A, Takaki T, Ohno S, Shimizu W, Horie M, Kimura T, Ellinor PT, Petit F, Dulac Y, Bru P, Boland A, Deleuze J, Redon R, Le Marec H, Le Tourneau T, Gourraud J, Yoshida Y, Makita N, Vieyres C, Makiyama T, Mundlos S, Christoffels VM, Probst V, Schott J, Barc J (2024) TAD boundary deletion causes PITX2-related cardiac electrical and structural defects. Nature Communications 15:3380. doi:10.1038/s41467-024-47739-x. https://doi.org/10.1038/s41467-024-47739-x
  16. Chakraborty S, Wenzlitschke N, Anderson MJ, Eraso A, Baudic M, Thompson JJ, Evans AA, Shatford-Adams LM, Chari R, Awasthi P, Dale RK, Lewandoski M, Petros TJ, Rocha PP (2025) Deletion of a single CTCF motif at the boundary of a chromatin domain with three FGF genes disrupts gene expression and embryonic development. Developmental Cell 60:1838–1853.e9. doi:10.1016/j.devcel.2025.02.002. https://doi.org/10.1016/j.devcel.2025.02.002
  17. Papale A, Segueni J, El Maroufi H, Noordermeer D, Holcman D (2025) Insulation between adjacent TADs is controlled by the width of their boundaries through distinct mechanisms. Proceedings of the National Academy of Sciences of the United States of America 122:e2413112122. doi:10.1073/pnas.2413112122. https://doi.org/10.1073/pnas.2413112122
  18. Salnikov P, Belokopytova P, Yan A, Viesná E, Korablev A, Serova I, Lukyanchikova V, Stepanchuk Y, Torgunakov N, Tikhomirov S, Fishman V (2025) Direction and modality of transcription changes caused by TAD boundary disruption in Slc29a3/Unc5b locus depends on tissue-specific epigenetic context. Epigenetics & Chromatin 18:55. doi:10.1186/s13072-025-00618-1. https://doi.org/10.1186/s13072-025-00618-1
  19. Fudenberg G, Kelley DR, Pollard KS (2020) Predicting 3D genome folding from DNA sequence with Akita. Nature Methods 17:1111–1117. doi:10.1038/s41592-020-0958-x. https://doi.org/10.1038/s41592-020-0958-x
  20. Smaruj PN, Kamulegeya F, Kelley DR, Fudenberg G (2025) Interpreting the CTCF-mediated sequence grammar of genome folding with AkitaV2. PLOS Computational Biology 21:e1012824. doi:10.1371/journal.pcbi.1012824. https://doi.org/10.1371/journal.pcbi.1012824
  21. Senapati S, Irshad IU, Sharma AK, Kumar H (2025) Predicting gene expression changes from chromatin structure modification. npj Systems Biology and Applications 11:34. doi:10.1038/s41540-025-00510-4. https://doi.org/10.1038/s41540-025-00510-4
  22. Sreenivasan VKA, Yumiceba V, Spielmann M (2025) Structural variants in the 3D genome as drivers of disease. Nature Reviews Genetics 26:742–760. doi:10.1038/s41576-025-00862-x. https://doi.org/10.1038/s41576-025-00862-x
  23. Koska M, Nagano M, Swigut T, Boettiger AN, Hansen AS, Wysocka J (2026) Promoter strength and position govern promoter competition through transcript-dependent insulation. Nature Genetics 58:1928–1940. doi:10.1038/s41588-026-02691-y. https://doi.org/10.1038/s41588-026-02691-y
  24. Trivellin G, Sánchez-Gaya V, Grasso A, Pasińska M, Stratakis CA, Milnes D, Kirk EP, Beckers A, Lania AG, Pétrossians P, Rada-Iglesias A, Franke M, Daly AF (2026) Distinguishing benign from pathogenic duplications involving GPR101 and VGLL1-adjacent enhancers in the clinical setting with the bioinformatic tool POSTRE. npj Genomic Medicine 11:12. doi:10.1038/s41525-025-00548-7. https://doi.org/10.1038/s41525-025-00548-7