Shared binding sites for the chromosomal architectural protein Su(Hw) mediate physical interactions between Drosophila TAD boundaries

Reviewer #1 (Public review):

Summary:

This study extends the authors' prior work on transgenic nhomie/homie boundary pairing (Fujioka et al. 2016 PLoS Genetics), which showed that these elements - corresponding to the eve TAD's left and right boundaries - can pair with endogenous copies over large genomic distances (142 kb here), bridging a linked reporter gene to endogenous eve enhancers for long-range gene activation (shown again here in Figure 1). Physical pairing was previously confirmed (Chen et al. 2018 Nat Genet) and further resolved by Micro-C (Bing et al. 2024 eLife), supporting the hypothesized "stem-loop" or "circle loop" topologies used to explain homie/nhomie directional pairing (shown here for nhomie in Figures 2-3). The authors recently showed that a Su(Hw) binding site is required for homie-mediated reporter gene activation by eve enhancers (Fujioka et al. 2025 Genetics); here, they extend this finding to nhomie (Figures 4-6), further showing that Su(Hw) motifs are required for Micro-C-detectable looping between transgenic and endogenous eve boundaries (Figures 7-9). Finally, they show that while cis-pairing over 142 kb is highly specific to homie/nhomie elements, transvection between homologous transgene insertions is more permissive (functioning with the Su(Hw)-bound gypsy insulator) but still shows some specificity (failing with the CTCF-bound Fab8 boundary, Figures 10-11).

Strengths:

The question of how pairs of loci can specifically physically pair over relatively long genomic distances is an interesting fundamental question. The study's strengths are the clarity and meticulous interpretation of the results, and the authors' conclusions are compelling.

Weaknesses:

A major weakness is that some figures reproduce previously published findings; in some cases it is unclear whether the same fly lines were used, and in others, the lines differ only slightly from those used previously (e.g., a shorter version of the Homie transgene than the one used previously). Most conclusions in this manuscript have already been published elsewhere. As a result, the paper does not report a genuine new discovery, and only incrementally advances our understanding of boundary pairing.

Reviewer #2 (Public review):

The results in Ke et al., build on 15 years of work focused on dissecting the pairing properties of the Drosophila Homie insulator. Here, the authors use similar methods to those shown in Fujioka et al., 2016, Ke et al., 2024, and Fujioka et al., 2025, but with a focus on nHomie pairing and the role of Su(Hw) in both Homie and nHomie long-range interactions. The main question the authors hope to address is what the mechanisms are behind the physical interactions involved in boundary:boundary pairing. They attempt to answer this question through mutating the Su(Hw) binding sites located within the nHomie and Homie transgenic sequences and observing how pairing is altered.

The work presented is thorough and thought out; however, some of the conclusions that the authors focus on are not what makes the work interesting and could be reprioritized. For example, the authors spend several paragraphs in the discussion (lines 531-595) addressing how the data presented does not support an argument for cohesion-mediated loop extrusion. While the interactions shown throughout the manuscript do not support cohesion-mediated loop extrusion occurring at the Homie locus, the authors have already made this point in both Bing et al., 2024 and Ke et al., 2024 and thus do not need to expound on this point.

Instead, the authors have a more compelling story in their specificity vs promiscuity arguments. Homie is a unique insulator in Drosophila and even when located 142kb away will still find its unique pairing partners (itself and nHomie). The authors have shown this several times prior, yet here they show that some level of this long-distance homing interaction is dependent upon the Su(Hw) binding site. Additionally, the authors show in this study that addition of gypsy sequence, in a less demanding assay, is sufficient for transvection pairing with Homie. This transvection result is a novel finding, as gypsy was previously shown to be insufficient for long-distance pairing with Homie based on the authors' prior studies. It is likely different architectural proteins that bind within the Homie sequence and allow it to pair specifically with itself, regardless of assay type, and these elements are likely absent from the gypsy sequence, leading to pairing that is more situational (see point 8 in recommendations).

Finally, to no fault of the authors, the art of visualizing complex 3D pairing configurations is difficult. Unfortunately, that can at times mask the ultimate points that the authors are trying to make about pairing early in the manuscript.

Overall, the work mainly supports the authors' claims, and the findings are a useful addition to the insulator and Drosophila 3D genome organization field.

Reviewer #3 (Public review):

Summary:

This manuscript investigates the function of Su(Hw) binding sites found in two boundaries/insulators, homie and nhomie, in TAD formation that encompasses the eve gene. They tested the hypothesis that Su(Hw) binds homie and nhomie, thereby forming a stem-loop TAD. The authors used transgene reporters with various mutations, and the results support the hypothesis strongly.

Strengths:

They combine reporter assays (GFP and LacZ expression) with MicroC contact profiling to robustly support their conclusion. Overall, they propose how Su(Hw) mediates physical interaction between boundary elements (homie and nhomie).

Weaknesses:

The writing is quite dense and not easily accessible to outside readers.