Diagram of the possible loop topologies generated by loop-extrusion and boundary:boundary pairing.

A) Cohesin embraces a loop at a loading site somewhere within the TAD-to-be and then extrudes a loop at an equal rate on both strands. Extrusion continues until cohesin encounters boundary roadblocks on both strands. B) In one model, the orientation of the roadblock is important. As a consequence, the chromatin fiber will be organized into a series of stem-loops separated from each other by unanchored loops. C) If the presence of a boundary roadblock, but not its orientation, is sufficient to halt extrusion, the chromatin fiber will be organized into a series of linked stem-loops. The main axis of the chromosome will be defined by the cohesin:CTCF roadblocks. D) The pairing of two boundaries head-to-tail generates a stem-loop. E) If boundary pairing interactions are strictly pairwise, head-to-tail pairing will generate a series of stem-loops separated from each other by unanchored loops. F) If boundaries can engage in multiple head-to-tail pairing interaction, the chromosome will be organized into a series of linked stem-loops. The main axis of the chromosome will be defined by a series paired boundaries. G) The pairing of two boundaries head-to-head generates a circle-loop. H) If boundary pairing interactions are strictly pairwise there will be a series of circle-loops separated from each other by unanchored loops. I) If boundaries can engage in multiple head-to-head pairing interactions, the chromatin fiber will be organized into a series of circle-loos connected to each other at their base. Pairing interactions between boundaries #1 and #2 need not be in register with pairing of boundaries #2 and #3. In this case, the main axis of the chromosome may bend and twist and this could impact the relative orientation of the circle-loops.

TAD organization of the even-skipped locus and neighboring sequences.

A) The eve skipped TAD is a volcano with a plume that is anchored by nhomie and homie. ChIP-seq data below the MicroC map indicate many of the known fly chromosomal architectural proteins proteins are associated with the two eve boundary elements in vivo (Cuartero et al., 2014; Duan et al., 2021; Gaskill et al., 2021; Li et al., 2015; Sun et al., 2015; Ueberschär et al., 2019; Zolotarev et al., 2016). B) The eve locus forms a stem-loop structure. In this illustration nhomie pairs with homie head-to-tail, and this configuration forms a stem-loop which brings sequences upstream nhomie and downstream of homie in contact as is observed in insulator bypass assays (Kyrchanova et al., 2008a). The eve locus is shown assembled into a coiled “30 nM” chromatin fiber. C) MicroC contact pattern for the chromosomal region spanning the attP site at –142 kb and the eve locus. Like the eve volcano, this contact pattern was generated using aggregated previously published NC14 data (Batut et al., 2022; Levo et al., 2022). A black arrow indicates the –142kb locus where transgenes are integrated into the genome. A blue arrow indicates position of the Etf-QO gene. Note the numerous TADs separating the –142kb hebe locus from the eve “volcano TAD”. Individual TADs are labeled TA—TM. Black boxes indicate positions of sequences that, based on ChIP experiments, are bound by one or more known insulator proteins in vivo.

Schematics of boundary pairing and loop extrusion.

A) LhomieG Z5 and GhomieL Z5 transgenes and the eve locus on a linear map. The same color codes are used throughout. In the eve locus, nhomie (blue arrow) and homie (red arrow) are oriented, by convention, so that they are pointing towards the right. This convention is maintained in the two transgenes. In LhomieG Z5 homie (top) is in the opposite orientation from homie in the eve locus is pointing away from the eve locus. In GhomieL Z5 homie (bottom) homie is in the same orientation as the homie in the eve locus and is pointing towards the eve locus. B) Predicted boundary pairing interactions between LhomieG Z5 and the eve locus. homie in the transgene pairs with homie in the eve locus head-to-head. Since homie in the transgene is pointing in the opposite orientation from homie in the eve locus, a stem-loop will be generated. If homie in the transgene also pairs with nhomie in the eve locus head-to-tail, a loop structure like that shown in B will be generated. In this topology, eve-lacZ is in close proximity to eve enhancers and eve-gfp is in contact with the hebe enhancer. C) Predicted boundary pairing between GhomieL Z5 and the eve locus. The GhomieL Z5 transgene is inserted in the same chromosomal orientation as LhomieG Z5; however, the homie boundary is inverted so that it is pointing towards the eve in the same orientation as the homie in the eve locus. Homie in the transgene will pair with homie in the eve locus head-to-head, and this generates a circle-loop. If homie in the transgene also pairs with nhomie in the eve loucs a loop structure like that shown in C will be generated. In this topology eve-gfp will be activated by both the eve and hebe enhancers. D) Loop extrusion model for LhomieG Z5. Transgene homie and endogenous homie determine the endpoints of the extruded eveMammoth (eveMa) loop. In this topology, eve-lacZ is in close proximity to the eve enhancers, while eve-gfp is close to the hebe enhancers. E) Loop extrusion model for GhomieL Z5. The cohesin complex bypasses transgenic homie and is stopped at an upstream boundary X to generate a novel eveGarantuan (eveGa) loop. Both eve-gfp and eve-lacZ are located within the same eveGa TAD, and thus should interact with eve. Since the hebe enhancers are within this TAD as well, they would also be able to activate both reporters.

The lacZ reporter is activated by eve enhancers in the LhomieG Z5 transgene.

A) Expression patterns of endogenous eve in early (stage 4-5) and late (stage 14-16) embryos by smFISH. eve (Atto 633) probes were used to hybridize with eve mRNA. eve is shown in yellow and DAPI in blue. N>3. B) Schematics of transgenes. C) Expression patterns of transgenic reporters in early stages embryos. Top: LlambdaG Z5. Bottom: LhomieG Z5. GFP is in green, lacZ is in orange, and DAPI is in blue, here and in E. D) Quantification of normalized stripe signals for transgenes shown in C. The reporter smFISH intensity (signal to background), here and in F, was measured, normalized, and plotted as described in Methods. N>3. n=27 for LlambdaG Z5 and n=42 for LhomieG Z5. E) Expression patterns of transgenic reporters in late stages embryos. Top: LlambdaG Z5. Bottom: LhomieG Z5. F) Quantification of normalized stripe signals for transgenes shown in E. N>3. n=27 for LlambdaG Z5 and n=56 for LhomieG Z5. Scale bars = 200μm. N = # independent biological replicates. n = # animals. The paired two-tailed t-test were used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant. Raw measurements are available in the Source Data files.

The GFP reporter is activated by both eve and hebe enhancers in the LhomieG Z5 transgene.

A) Schematics of transgenes. B) Expression patterns of transgenic reporters in early stage embryos. Top: LlambdaG Z5. Bottom: GhomieL Z5. GFP is in green, lacZ is in orange, and DAPI is in blue, here and in D. C) Quantification of normalized stripes signal as represented in B. The reporter smFISH intensity (signal to background) was measured, normalized, and plotted as described in Methods, here and in E. N>3. n=27 for LlambdaG Z5, and n=46 for LhomieG Z5. D) Expression patterns of transgenic reporters in late stage embryos. Top: LlambdaG Z5. Bottom: GhomieL Z5. E) Quantification of normalized stripe signals for transgenes shown in D. N>3. n=27 for LlambdaG Z5, and n=59 for LhomieG Z5. Scale bars = 200μm. N = # independent biological replicates. n = # animals. The paired two-tailed t-test were used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant. Raw measurements are available in the Source Data files.

Expression of reporters in Z3 transgenes.

A) Schematics of Z3 transgenes. B) Boundary pairing for GhomieL Z3 transgene long-range interactions. In this topology, GFP is in close proximity to eve enhancers, and lacZ is in contact with the hebe enhancer region. C) Boundary pairing for the LhomieG Z3 transgene. In this topology, lacZ is in close proximity to both the eve enhancers and the hebe enhancers. D) Loop extrusion model for GhomieL Z3. Transgenic homie and endogenous homie flank the eveMammoth loop. In this topology, GFP is in close proximity to eve enhancers, and lacZ is close to the hebe enhancers. E) Loop extrusion model for the LhomieG Z3 transgene. The cohesin complex bypasses transgenic homie and is stopped at an upstream boundary X to create the eveGarantuan loop. Both GFP and lacZ are at a similar physical distance from eve enhancers and hebe enhancers in this topology. F) Expression patterns of transgenic reporters in early stage embryos. Top: GhomieL Z3. Bottom: LhomieG Z3. GFP is in green, lacZ is in orange, and DAPI is in blue. N=3, n=24 for both GhomieL Z3 and LhomieG Z3. G) Expression patterns of transgenic reporters in late stage embryos. Top: GhomieL Z3. Bottom: LhomieG Z3. N=3, n=24 for both GhomieL Z3 and LhomieG Z3. Scale bars = 200μm, N = # independent biological replicates, n = # animals.

homie in Z5 transgene LhomieG and GhomieL Z3 mediates long-range interactions with the eve locus

Small dark blue arrows – insulators upstream and downstream of hebe that appear important to demarcate interaction domains between –142kb and eve. Large blue arrow – endogenous nhomie. Large red arrow – homie, either endogenous or in the transgene. The direction of arrows follow established convention on nhomie/homie, and do not reflect orientation of insulator protein binding motifs per se. Gray boxes – enhancers. A) MicroC map of the control line LlambdaG Z5. Scaled cartoons of the two loci of interest are shown directly below the MicroC map, and an unscaled blowup of the elements of interest at each locus is provided. Within the MicroC map of the entire locus, a zoom-in of the off-diagonal interaction between the –142kb insertion cassette and the endogenous eve locus is shown. Note a slight increase in interaction frequency (compare to Figure 1A). B, C) MicroC map of LhomieG Z5 and GhomieL Z5, respectively. The only difference between the two lines is the orientation of transgenic homie, as indicate below each MicroC map. Blow up of the off-diagonal interaction between –142kb and eve, including scaled cartoons denoting features of interest in the top right corner. Note the changed pattern of interaction with the endogenous eve locus due to the orientation switch. D) “Virtual 4C” maps obtained from MicroC maps of “B” (top panel), and “C” (bottom panel). Viewpoints are shown in both panels from either the lacZ gene (orange) or the GFP gene (green). Note in each cassette the altered interaction of either gene with endogenous eve, depending on the change of orientation of homie.

homie in the transgene interacts with eve homie and nhomie. “Virtual 4C” maps of the LhomieG (A) or GhomieL (B) transgenes, for the Z5 (blue) and Z3 (orange) orientations. Viewpoints are taken from the transgenic homie sequence. Importantly, note that transgenic homie preferentially interacts with endogenous homie no matter the orientation. Intriguingly, the orientation switch of transgenic homie leads to an apparent change in its degree of preference for endogenous homie over nhomie.