Chromosome Structure I: Loop extrusion or boundary:boundary pairing?

  1. Xinyang Bing
  2. Wenfan Ke
  3. Miki Fujioka
  4. Amina Kurbidaeva
  5. Sarah Levitt
  6. Michael Levine
  7. Paul Schedl
  8. James B Jaynes

  • Curated by eLife

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    eLife assessment

    This valuable work presents elegant experimental data from the Drosophila embryo supporting the notion that interactions among specific loci, called boundary elements, contribute to topologically associated domain (TAD) formation and gene regulation. Although the evidence supporting boundary elements as determinants of 3D structures is compelling, the evidence rejecting loop extrusion is incomplete. This study will be of interest to the nuclear structure community, particularly those using Drosophila as a model.

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Abstract

Two different models have been proposed to explain how the endpoints of chromatin looped domains (TADs) in eukaryotic chromosomes are determined. In the first, a cohesin complex extrudes a loop until it encounters a boundary element roadblock, generating a stem-loop. In this model, boundaries are functionally autonomous: they have an intrinsic ability to halt the movement of incoming cohesin complexes that is independent of the properties of neighboring boundaries. In the second, loops are generated by boundary:boundary pairing. In this model, boundaries are functionally non-autonomous, and their ability to form a loop depends upon how well they match with their neighbors. Moreover, unlike the loop-extrusion model, pairing interactions can generate both stem-loops and circle-loops. We have used a combination of MicroC to analyze how TADs are organized, and experimental manipulations of the even skipped TAD boundary, homie, to test the predictions of the loop-extrusion and the boundary-pairing models. Our findings are incompatible with the loop-extrusion model and instead suggest that the endpoints of TADs in flies are determined by a mechanism in which boundary elements physically pair with their partners, either head-to-head or head-to-tail, with varying degrees of specificity. Although our experiments do not address how partners find each other, the mechanism is unlikely to require loop extrusion.

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  1. Version published to 10.1101/2023.11.17.567501v2 on bioRxiv
  2. Version published to 10.7554/elife.94070 on eLife
  3. Version published to 10.7554/elife.94070.1 on eLife
  4. eLife

    eLife assessment

    This valuable work presents elegant experimental data from the Drosophila embryo supporting the notion that interactions among specific loci, called boundary elements, contribute to topologically associated domain (TAD) formation and gene regulation. Although the evidence supporting boundary elements as determinants of 3D structures is compelling, the evidence rejecting loop extrusion is incomplete. This study will be of interest to the nuclear structure community, particularly those using Drosophila as a model.

  5. eLife

    Reviewer #1 (Public Review):

    Summary:
    The authors addressed how long-range interactions between boundary elements are established and influence their function in enhancer specificity. Briefly, the authors placed two different reporters separated by a boundary element. They inserted this construct ectopically ~140 kb away from an endogenous locus that contains the same boundary element. The authors used expression patterns driven by nearby enhancers as an output to determine which enhancers the reporters interact with. They complemented this analysis with 3D DNA contact mapping. The authors found that the orientation of the boundary element determined which enhancers each reporter interacted with. They proposed that the 3D interaction topology, whether being circular or stem configuration, distinguished whether the interaction was cohesin mediated or through an independent mechanism termed pairing.

    Strengths:
    The transgene expression assays are built upon prior knowledge of the enhancer activities. The 3D DNA contacts confirm that transgene expression correlates with the contacts. Using 4 different orientations covers all combinations of the reporter genes and the boundary placement.

    Weaknesses:
    The interpretation of the data as a refusal of loop extrusion playing a role in TAD formation is not warranted, as the authors did not deplete the loop extruders to show that what they measure is independent. As the authors show, the single long DNA loop mediated by cohesin loop extrusion connecting the ectopic and endogenous boundary is clearly inconsistent with the results, therefore the main conclusion of the paper that the 3D topology of the boundary elements a consequence of pairing is strong. However, the loop extrusion and pairing are not mutually exclusive models for the formation of TADs. Loop-extruding cohesin complexes need not make a 140 kb loop, multiple smaller loops could bring together the two boundary elements, which are then held together by pairing proteins that can make circular topologies.

  6. eLife

    Reviewer #2 (Public Review):

    In Bing et al, the authors analyze micro-C data from NC14 fly embryos, focusing on the eve locus, to assess different models of chromatin looping. They conclude that fly TADs are less consistent with conventional cohesin-based loop extrusion models and instead rely more heavily on boundary-boundary pairings in an orientation-dependent manner.

    Overall, I found the manuscript to be interesting and thought-provoking. However, this paper reads much more like a perspective than a research article. I strongly suggest the authors spend some time editing their introduction to the most salient points as well as organizing their results section in a more conventional way with conclusion-based titles. It was very difficult to follow the authors' logic throughout the manuscript as written. It was also not clear as written which experiments were performed as part of this study and which were reanalyzed but published elsewhere. This should be made clearer throughout.

    It has been shown several times that Drosophila Hi-C maps do not contain all of the features (frequent corner peaks, stripes, etc.) observed when compared to mammalian cells. Considering these features are thought to be products of extrusion events, it is not an entirely new concept that Drosophila domains form via mechanisms other than extrusion. That being said, the authors' analyses do not distinguish between the formation and the maintenance of domains. It is not clear to this reviewer why a single mechanism should explain the formation of the complex structures observed in static Hi-C heatmaps from a population of cells at a single developmental time point. For example, how can the authors rule out that extrusion initially provides the necessary proximity and possibly the cis preference of contacts required for boundary-boundary pairing whereas the latter may more reflect the structures observed at maintenance? Future work aimed at analyzing micro-C data in cohesin-depleted cells might shed additional light on this.

    Additional mechanisms at play include compartment-level interactions driven by chromatin states. Indeed, in mammalian cells, these interactions often manifest as a "plume" on Hi-C maps similar to what the authors attribute to boundary interactions in this manuscript. How do the chromatin states in the neighboring domains of the eve locus impact the model if at all?

    How does intrachromosomal homolog pairing impact the models proposed in this manuscript (Abed et al. 2019; Erceg et al., 2019). Several papers recently have shown that somatic homolog pairing is not uniform and shows significant variation across the genome with evidence for both tight pairing regions and loose pairing regions. Might loose pairing interactions have the capacity to alter the cis configuration of the eve locus?
    In summary, the transgenic experiments are extensive and elegant and fully support the authors' models. However, in my opinion, they do not completely rule out additional models at play, including extrusion-based mechanisms. Indeed, my major issue is the limited conceptual advance in this manuscript. The authors essentially repeat many of their previous work and analyses. The authors make no attempt to dissect the mechanism of this process by modifying extrusion components directly. Some discussion of Rollins et al., 1999 on the discovery of Nipped-B and its role in enhancer-promoter communication should also be made to reconcile their conclusions in the proposed absence of extrusion events.

  7. eLife

    Reviewer #3 (Public Review):

    Bing et al. attempt to address fundamental mechanisms of TAD formation in Drosophila by analyzing gene expression and 3D conformation within the vicinity of the eve TAD after insertion of a transgene harboring a Homie insulator sequence 142 kb away in different orientations. These transgenes along with spatial gene expression analysis were previously published in Fujioka et al. 2016, and the underlying interpretations regarding resulting DNA configuration in this genomic region were also previously published. This manuscript repeats the expression analysis using smFISH probes in order to achieve more quantitative analysis, but the main results are the same as previously published. The only new data are the Micro-C and an additional modeling/analysis of what they refer to as the 'Z3' orientation of the transgenes. The rest of the manuscript merely synthesizes further interpretation with the goal of addressing whether loop extrusion may be occurring or if boundary:boundary pairing without loop extrusion is responsible for TAD formation. The authors conclude that their results are more consistent with boundary:boundary pairing and not loop extrusion; however, most of this imaging data seems to support both loop extrusion and the boundary:boundary models. This manuscript lacks support, especially new data, for its conclusions. Furthermore, there are many parts of the manuscript that are difficult to follow. There are some minor errors in the labelling of the figures that if fixed would help elevate understanding. Lastly, there are several major points that if elaborated on, would potentially be helpful for the clarity of the manuscript.

    Major Points:
    1. The authors suggest and attempt to visualize in the supplemental figures, that loop extrusion mechanisms would appear during crosslinking and show as vertical stripes in the micro-C data. In order to see stripes, a majority of the nuclei would need to undergo loop extrusion at the same rate, starting from exactly the same spots, and the loops would also have to be released and restarted at the same rate. If these patterns truly result from loop extrusion, the authors should provide experimental evidence from another organism undergoing loop extrusion.
    2. On lines 311-314, the authors discuss that stem-loops generated by cohesin extrusion would possibly be expected to have more next-next-door neighbor contacts than next-door neighbor contacts and site their models in Figure 1. Based on the boundary:boundary pairing models in the same figure would the stem-loops created by head-to-tail pairing also have the same phenotype? Making possible enrichment of next-next-door neighbor contacts possible in both situations? The concepts in the text are not clear, and the diagrams are not well-labeled relative to the two models.
    3. The authors appear to cite Chen et al., 2018 as a reference for the location of these transgenes being 700nM away in a majority of the nuclei. However, the exact transgenes in this manuscript do not appear to have been measured for distance. The authors could do this experiment and include expression measurements.
    4. The authors discuss the possible importance of CTCF orientation in forming the roadblock to cohesin extrusion and discuss that Homie orientation in the transgene may impact Homie function as an effective roadblock. However, the Homie region inserted in the transgene does not contain the CTCF motif. Can the authors elaborate on why they feel the orientation of Homie is important in its ability to function as a roadblock if the CTCF motif is not present? Trans-acting factors responsible for Homie function have not been identified and this point is not discussed in the manuscript.
    5. The imaging results seem to be consistent with both boundary:boundary interaction and loop extrusion stem looping.
    6. The authors suggest that the eveMa TAD could only be formed by extrusion after the breakthrough of Nhomie and several other roadblocks. Additionally, the overall long-range interactions with Nhomie appear to be less than the interactions with endogenous Homie (Figures 7, 8, and supplemental 5). Is it possible that in some cases boundary:boundary pairing is occurring between only the transgenic Homie and endogenous Homie and not including Nhomie?
    7. In Figure 4E, the GFP hebe expression shown in the LhomieG Z5 transgenic embryo does not appear in the same locations as the LlambdaG Z5 control. Is this actually hebe expression or just a background signal?
    8. Figure 6- The LhomieG Z3 late-stage embryo appears to be showing the ventral orientation of the embryo rather than the lateral side of the embryo as was shown in the previous figure. Is this for a reason? Additionally, there are no statistics shown for the Z3 transgenic images. Were these images analyzed in the same way as the Z5 line images?
    9. Do the Micro-C data align with the developmental time points used in the smFISH probe assays?

  8. Version published to 10.1101/2023.11.17.567501v1 on bioRxiv