Activation by eve enhancers of nhomie-containing transgenes inserted at -142 kb from eve.

A. Map of eve locus, a subset of the intervening genes and the attP site in the first exon of the hebe gene. The three different transgene inserts oriented so that the LacZ reporter is closest to eve are shown: lambda DNA (G-lambda-L), nhomie in the same (forward) orientation as the eve nhomie (G-nhomie-L), and nhomie in the opposite (reverse) orientation as the eve nhomie (G-eimohn-L). B) The panels in each row show DAPI staining, eve mRNA, GFP mRNA, and LacZ mRNA for G-lambda-L, G-nhomie-L and G-eimohn-L blastoderm stage embryos. C) The panels in each row show DAPI staining, eve mRNA, GFP mRNA, and LacZ mRNA for G-lambda-L, G-nhomie-L and G-eimohn-L in stage 13-14 embryos. D) Quantitation of transgene expression (LacZ or gfp) for G-lambda-L, G-nhomie-L, and G-eimohn-L in stage 13-14 embryos.

nhomie-containing transgenes physically interact with the eve TAD.

A) Top panel shows the MicroC contact profile in the wild-type chromosomal segment that contains both the attP transgene insertion site (in the hebe gene) and the eve TAD. Bottom panels show blowups of the MicroC contact profile for the vicinity of the attP site in the hebe gene and the eve TAD. Note that the MicroC contact profile for the eve TAD has the characteristic signature of a stem-loop TAD—a plume above the volcano triangle. B) and C) MicroC contact profile and blow up for the G-lambda-L transgene. D) and E) MicroC contact profile and blowup for the G-nhomie-L transgene. Blue and green arrowheads indicate interactions between the GFP reporter and the TADs flanking the eve TAD. F) and G) MicroC contact profile and blow up for the G-eimohn-L transgene. Blue and green arrowheads indicate interactions between the LacZ reporter and the TADs flanking the eve TAD.

MicroC viewpoints from the G-nhomie-L and G-eimohn-L reporters.

Viewpoints for the G-nhomie-L transgene for A) GFP and B) LacZ reporters. Viewpoints for the G-eimohn-L transgene for C) GFP and D) LacZ reporters. E) The average contact count per bin for the GFP and LacZ reporters, as indicated for the three transgene inserts G-lambda-L, G-nhomie-L, and G-eimohn-L. F) Models for chromatin organization of G-nhomie-L and G-eimohn-L.

Mutation of nhomie Su(Hw) binding site disrupts activation of transgene reporter expression by the eve enhancers.

A) The panels in each row show DAPI staining, eve mRNA, GFP mRNA, and LacZ mRNA for G-lambda-L, G-nhomie-L, and G-nhomieΔSu(Hw)-L in blastoderm stage embryos. B) The panels in each row show DAPI staining, eve mRNA, GFP mRNA, and LacZ mRNA for G-lambda-L, G-nhomie-L, and G-nhomieΔsu(Hw)-L in stage13-14 embryos. C) Normalized maximum intensity projections of GFP and LacZ mRNA staining in the APR of stage13-14 embryos for each transgene.

Digoxigenin in situ hybridization in wild type and the Su(Hw) binding site mutants in nhomie and homie.

Expression of the LacZ reporter by A) G-nhomie-L, B) G-nhomieΔSu(Hw)-L, C) G-eimoh-L, D) G-eimohΔSu(Hw)-L, and E) G-lambda-L transgene inserts are shown. The transgene constructs are illustrated on the top. Embryonic stages 5, 7, 11, 13 are shown. Ventral views of stage 13 (13v) are shown in the bottom row. Arrowheads indicate the following: blue: APR, yellow: eve-expressing neuronal cells, green: eve-expressing mesodermal cells, and red: hebe-expressing midline cell clusters. Scale bar: 50μm.

Mutation of the homie Su(Hw) binding site disrupts activation of transgene reporter expression by eve enhancers.

A) The panels in each row show DAPI staining, eve mRNA, GFP mRNA, and LacZ mRNA for G-lambda-L, G-eimoh-L, and G-eimohΔSu(Hw)-L in blastoderm stage embryos. B) The panels in each row show DAPI staining, eve mRNA, GFP mRNA, and LacZ mRNA for G-lambda-L, G-eimoh-L, and G-eimohΔSu(Hw)-L in stage 13-14 embryos. C) Normalized maximum intensity projections of GFP and LacZ mRNA staining in the APR of stage 13-14 embryos for each transgene.

Mutation of the nhomie Su(Hw) binding site disrupts physical interactions between the transgene and sequences in the eve TAD.

The MicroC contact profile and blow up for A) G-nhomie-L and B) G-nhomieΔSu(Hw)-L transgenes. Viewpoints from the GFP and LacZ reporters in C) G-nhomie-L and D) G-nhomieΔSu(Hw)-L transgenes. E) Average contact count per bin for the GFP and LacZ reporters, as indicated for the two transgene inserts G-nhomie-L and G-nhomieΔSu(Hw)-L. F) Insulation score for the transgene boundaries nhomie and nhomieΔSu(Hw).

Mutation of the homie Su(Hw) binding site disrupts physical interactions between the transgene and sequences in the eve TAD.

MicroC contact profile and blow up for A) G-eimoh-L and B) G-eimohΔSu(Hw)-L transgenes. Viewpoints from the GFP and LacZ reporters in C) G-eimoh-L and D) G-eimohΔSu(Hw)-L transgenes. E) Average contact count per bin for the GFP and LacZ reporters, as indicated for the two transgene inserts G-eimoh-L and G-eimohΔSu(Hw)-L. F) Insulation score for the transgene boundaries eimoh and eimohΔSu(Hw).

Viewpoints from the transgene boundaries.

A) nhomie, B) nhomieΔSu(Hw), C) eimoh, and D) eimohΔSu(Hw). Note the reduction in contacts between the Su(Hw) site mutant boundaries in the transgene and both nhomie and homie (at the ends of the eve TAD).

homie-dependent transvection is weakened by loss of the Su(Hw) binding site.

Digoxigenin in situ hybridization showing LacZ expression in the transvection assay. The two transgene constructs used to assay transvection are illustrated at the top. The different transgene constructs used for each cross are indicated on the left as: reporter x enhancer. The negative control has a 500 bp lambda DNA fragment. The 367 bp homie (homieCDEF), 271 bp homie (homieDEF), and the Su(Hw) site-mutated homieDEF (DΔSuEF) were tested. Stages 11, 12, and 13 are shown. Arrowheads indicate the following: blue: APR and green: mesoderm. Scale bar: 50μm.

The su(Hw) gypsy insulator supports transvection with homie.

Digoxigenin in situ hybridization showing LacZ expression in the transvection assay. The two transgene constructs for each experiment are shown on the top. The transgene combinations used in each case are indicated as reporter x enhancer. The negative control has a 500 bp lambda DNA fragment. The 349 bp gypsy fragment, the 367 bp homie (homieCDEF and homieFEDC) fragments and the 329 bp Fab-8 fragment (Kyrchanova et al., 2016) were tested. Note that the orientation of homie in homieFEDC is inverted in the transgene to test whether gypsy interaction with homie is orientation-dependent. Stages 11, 12, and 13 are shown. Arrowheads indicate the following: blue: APR and green: mesoderm. Scale bar: 50μm.