TAD boundary architecture and gene activity are uncoupled

Reviewer #1 (Public review):

Summary:

This is an important study that employs high-throughput single-cell imaging to directly investigate the relationship between topologically associating domain (TAD) boundaries and gene regulation. The authors rigorously test the prevailing model that TAD boundaries functionally regulate gene activity by modulating chromatin interactions. Their core finding is that, under their specific experimental conditions, the physical distance between TAD boundaries shows no consistent correlation with the transcriptional bursting activity of a gene within the TAD. However, the authors' leap from this specific observation to the broad conclusion that "TAD boundary architecture and gene activity are uncoupled" risks conceptual overgeneralization and may lead to misinterpretation, as it seemingly contradicts substantial prior evidence supporting the regulatory role of TAD structures.

Strengths:

The major strength of this work lies in its innovative high-throughput, multi-colour imaging platform, which enables the simultaneous detection of spatial distances between specific DNA elements (TAD boundaries) and transcriptional activity at the same genomic locus in single cells and single alleles. The high-throughput nature makes the results convincing. A second key strength is the incorporation of perturbations, including global transcriptional inhibition, cell-type comparison, and degradation of key architectural proteins (CTCF, cohesin). This provides a comprehensive methodological framework to examine the relationship between boundary proximity and gene activity from multiple angles under defined conditions.

Weaknesses:

(1) Conceptual framing and interpretation:

The central conclusion may require more precise framing to avoid potential overreach. The authors' interpretation equating "physical distance between TAD boundaries" with overall "TAD boundary architecture," and "transcriptional bursting events" with broader "gene activity," could benefit from clarification. This framing may not fully capture the temporal dynamics of transcription or the regulatory complexity within TADs. Furthermore, the broad conclusion of an uncoupled relationship appears to challenge extensive prior evidence from perturbation studies showing that disrupting TAD boundaries can alter gene expression. The authors' own observation of reduced gene activity upon RAD21 degradation suggests that global TAD disruption can affect transcription. A more precise and limited conclusion, acknowledging that their data demonstrate a lack of detectable correlation between boundary distance and bursting activity in their system, would be more accurate and help reconcile these findings with the existing literature.

(2) Technical methods and data presentation:

(2.1) Accuracy and dimensionality of distance measurements: The manuscript does not clearly state whether distances are measured in 2D or 3D, nor does it sufficiently address precision limits. The stated Z-step size (1 µm) may be inadequate for accurately measuring sub-micron chromatin distances in 3D.

(2.2) Probe design and systematic error: The genomic coverage size of the BAC probes used for DNA FISH is not explicitly stated. Large probe coverage could inherently blur the precise spatial location of adjacent DNA loci. The reported average distance (~300 nm) may be influenced by the physical size of the probes, as well as systematic expansion or distortion introduced by sample fixation and FISH processing. Although such technical limitations are currently unavoidable, the authors should clarify how these factors might affect their ability to detect subtle distance changes.

(2.3) Data Visualization: The manuscript would benefit from including representative, zoomed-in regions of interest from the raw imaging data. This would allow readers to visually assess measured distance differences against background noise.

(2.4) Potential impact of resolution limits: In Figure 5, the micro-C data reveal a clear difference in interaction patterns inside versus outside the VARS2 locus TAD, yet the imaging data show no corresponding distance difference. This strongly suggests that the current imaging system, limited by optical resolution, probe size, and localisation accuracy, may be unable to resolve finer-scale spatial reorganizations associated with specific chromatin conformations (e.g., enhancer-promoter loops). The authors should explicitly discuss that their conclusion of "no coupling observed" may be constrained by the resolution and sensitivity of their method and does not preclude the possibility of detecting such associations with higher-precision measurements or in live-cell dynamics.

In summary, this study provides a valuable single-cell perspective. However, the authors should more cautiously define the scope of their findings in the manuscript and provide a more balanced discussion situating their work within the broader field.

Reviewer #2 (Public review):

Summary:

Almansour et al. investigate whether the proximity of TAD boundaries is directly linked to gene activity. The authors use high-throughput imaging to simultaneously measure the gene activity and physical distances between boundary regions in an allele-specific manner. Using transcriptional inhibitors, expression induction, and acute depletion of CTCF and cohesin, they test whether proximity of boundaries affects, or is affected by, gene activity.

Strengths:

The combined use of DNA and RNA imaging enabled simultaneous measurement of boundary proximity and transcriptional status at individual alleles. This allows single-allele correlation between boundary proximity and gene activity at multiple loci across thousands of alleles.

The use of both transcription inhibitors and transcription stimulation provides compelling and consistent evidence that boundary proximity can be disconnected from a gene's activity. The data convincingly support the conclusion that stable proximity between boundary regions is not required for ongoing transcription at the loci and timescales examined.

This work strengthens the emerging view that genome organization at the level of domain boundaries does not impose a deterministic control over transcription.

Weaknesses:

In untreated cells, the distribution of distance measurements between boundary probes is exceptionally narrow. While depletion of RAD21 clearly demonstrates an ability to detect changes in this distribution, this tight baseline distribution may limit sensitivity to more subtle changes (like those one might expect from transcriptional influences). In addition, the correlation analysis is asymmetric, primarily stratifying by transcriptional status and then comparing boundary distances. Given the central claim that boundary architecture does not influence gene activity, the analysis should be done from the opposite perspective (stratifying by boundary distance).

Strong disruption of boundary distances is only observed upon depletion of cohesin. Notably, this corresponds with the largest changes in gene activity. In contrast, depletion of CTCF actually had minimal impact on boundary distances and also had minimal impact on gene activity. This makes sense in light of previous work, where live cell imaging demonstrated that cohesin is more important for domain-structure, whereas CTCF is only important for blocking cohesin from continuing on, such that the fully formed loop occurs in a very small percentage of cells. Therefore, the fact that disruption of cohesin (more important for internal domain structure) affects gene activity while disruption of CTCF does not is exceptionally interesting but is lacking from the discussion.

On a related note, this approach primarily tests the role of boundary interactions rather than domain organization as a whole, and it should be acknowledged that internal domain structures are not directly assessed.

The comparison to work in other organisms (particularly the comparisons made to Drosophila) should be handled with care. The mechanisms underlying domain formation differ substantially across these systems, particularly regarding the differences in CTCF's role.