Figures and data
Diagram of the possible loop topologies generated by head-to-head and head-to-tail pairing.
A) Head-to-tail boundary (arrows) pairing generates a series of stem-loops linked together by unanchored loop. In this case, the main axis of the chromosome would correspond to the unanchored loops connecting different stem-loops. B) If boundaries pair with both neighbors (head-to-tail) the stem-loops would be linked to each other by the paired boundaries. In this case the main axis of the chromosome will correspond to the paired boundaries. C) Head-to-head boundary pairing generates a series of circle-loops linked together by an unanchored loop. The unanchored loop will be the main axis of the chromosome. D) If boundaries pair with both neighbors (head-to-head), the chromatin fiber will be organized into a series of circle-loops connected to each other at their base and these paired boundaries will define the chromosomal axis. In both B) and D), the pairing interactions between the blue and red boundaries need not be in register with the pairing of the red boundary to the next-door green boundary. In this case, the main axis of the chromosome may bend and twist, and this could impact the relative orientation of the stem-loops/circle-loops. More complex structures would be generated by mixtures of stem-loops and circle-loops.
Stem-loops and circle-loops.
“Once in a while you get shown the light in the strangest of places if you look at it right.” MicroC contact profile for Drosophila wild-type (yw) stage 14 embryos. A) eve and neighboring TADs (TI, TJ, TK TL, TM and TN). The eve TAD is a volcano with a plume that is anchored by nhomie (nh) and homie (h). The plume is generated by crosslinking of sequences in neighboring TADs. At the bottom of the plume, TL sequences are linked to sequences in TM close to eve including ter94. At the next level, sequences in TK are linked to TM. In addition, sequences in LM are linked to sequences in TM located beyond the TER94 gene. At the next level, sequences in TJ are linked to sequences in TM. Note that interactions between sequences in TL and TJ and sequences in TM close to the eve TAD are somewhat less frequent than those farther away from the eve TAD. Sequences in the neighboring TAD also interact with each other as indicated. For example, sequences in TK and TJ interact with each other (J-K) and also with interact with sequences in TI (I-K and I-J). B) The Bithorax Abd-B gene and the parasegment (PS) specific regulatory domains, iab-3, iab-4, iab-5, iab-6, iab-7, iab-8. iab-4 regulates the abd-A gene in PS9. iab-5 regulates Abd-B in PS10, iab-6 regulates Abd-B in PS11, iab-7 regulates Abd-B in PS12 and iab-8 regulates Abd-B in PS13. The domains are separated from each other by the boundary elements Fab-4, Mcp, Fab-6, Fab-7, and Fab-8 as indicated. The AB-I boundary is located upstream of the Abd-B promoter. Each regulatory domain corresponds to a TAD. Though insulated from each other, each TAD interacts with its immediate neighbor. For example, iab-5 interacts with its immediate neighbor iab-4 and iab-6 to give 4-5 and 5-6 respectively. It also interacts with the next-next door neighbor iab-7 (5-7) and even its next-next-next door neighbor iab-8 (5-8). (x: Scarlet Begonias GD: 1974).
TAD organization of the nhomie forward and λDNA replacements.
A) MicroC contact profile of stage 14 nhomie forward embryos. In this replacement nhomie is inserted in the same orientation as the endogenous nhomie. N(replicates)=2. Resolution = 200bp. L-M: Interactions between TADs TL and TM flanking the eve locus. Asterisk: sub-TAD linking the eve promoter to the eve PRE and homie. Dark blue arrow: sub-TAD linking the eve promoter to nhomie. Light blue arrowhead: nhomie. Red arrowhead: homie. Green arrowhead: sub-TAD boundary formed by the CG12134 promoter region. Dark blue arrowhead: sub-TAD boundary formed by eIF3j promoter region. Diagram: Map of eve locus and surrounding genes. B) Virtual 4C with viewpoint from homie (black arrow) towards nhomie forward. C) Diagram of the eve stem-loop TAD. D) MicroC contact profile of stage 14 λ DNA embryos. In this replacement, λ DNA is inserted in place nhomie. N(replicates)=2. Resolution = 200bp. Asterisks: sub-TAD linking the eve promoter to the eve PRE and homie. Purple arrowhead: sub-TAD linking CG12134 promoter region to eve promoter. The eIF3j sub-TAD (demarked by the blue and green arrowheads) is still present. E) Virtual 4C with viewpoint from homie reading toward eIF3j. F) Diagram of the “unanchored” eve TAD. Arrows show novel interactions.
nhomie deletion (lambda DNA replacement) exposes eIF3j to the eve enhancers.
nh forward: nhomie replacement with nhomie in the forward (WT) orientation. λ DNA: nhomie replacement with λ DNA. At the syncytial blastoderm stage, a series of stripe-specific enhancers upstream (stripes 1, 2, 3, 7) and downstream (stripes 1, 4, 5, 6) of the eve gene drive eve expression. During cellularization of the blastoderm and gastrulation, a single enhancer located upstream of eve drives expression of all seven stripes. DAPI: DNA stained with DAPI (blue). eIF3j: Embryo hybridized with probe complementary to eIF3j mRNA. eve: Embryo hybridized with probe complementary to eve mRNA. TER94: Embryo hybridized with probe complementary to TER94. Yellow carets: eve-enhancer-driven eIF3j stripes. Control non-specific probes for each channel indicates autofluorescence background for each channel in the top panel.
Manipulating the nhomie boundary impacts the regulatory landscape.
N=# of independent replicate, n= # of embryos. Two-way ANOVA with Tukey’s multiple comparisons test for each pair of groups was used to determine the statistical significance. *p≤ 0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001. A) Quantitation of numbers of embryos showing stripe patterns in HCR-FISH for eIF3j and TER94, as shown in Fig. 4 and Fig 7. N=3. n=45 for each group. B) Quantitation of the number of missing ventral denticle bands in larvae from a cross of BSC/CyO,hb-lacZ deficiency females to males of the indicated genotypes (N=6): wild-type control (yw) n = 767. The nhomie forward replacement n = 1099. The λDNA replacement n = 1175. The nhomie reverse replacement: n = 1083. The homie forward replacement: n = 1137.
TAD organization of the nhomie reverse and homie forward replacements.
A) MicroC contact profile of stage 14 nhomie reverse embryos. In this replacement nhomie is inserted in the reverse orientation compared to WT nhomie. N(replicates)=2. Resolution = 200bp. B) Virtual 4C with viewpoint from homie (black arrow) towards nhomie reverse. C) MicroC contact profile of stage 14 homie forward embryos. In this replacement homie is inserted in the forward orientation (the same as the endogenous homie). N(replicates)=2. Resolution = 200bp. D) Virtual 4C with viewpoint from homie (black arrow) towards homie forward. A) and C) Note that interactions between the TADs flanking the eve locus (purple double arrow) are suppressed compared to nhomie forward (see Fig. 3), while interactions between eve and TL, TM are enhanced (L-ev and ev-M). Asterisks: sub-TAD linking the eve promoter to the eve PRE and homie. Dark blue arrow: sub-TAD linking the eve promoter to nhomie reverse. Light blue bar: nhomie reverse. Red bar: homie. Green arrowhead: sub-TAD boundary formed by the CG12134 promoter region. Dark blue arrowhead: sub-TAD boundary formed by the eIF3j promoter region. Diagram: Map of eve locus and surrounding genes.
eve enhancers activate neighboring genes when the eve TAD is a circle-loop.
hcrFISH hybridization to mRNA expressed by eIF3j, eve and TER94 at the blastoderm stage (embryonic stage 5). nh forward: nhomie replacement with nhomie in forward (normal) orientation (same as in Fig. 3). nh reverse: nhomie replacement with nhomie in reverse orientation. h forward: nhomie replacement with homie in forward orientation. Yellow arrowhead: position of stripes. DAPI (blue): DNA stained with DAPI. eIF3j (green): Embryo hybridized with probe complementary to eIF3j mRNA. eve (orange): Embryo hybridized with probe complementary to eve mRNA. TER94 (red): Embryo hybridized with probe complementary to TER94. Control non-specific probes for each channel indicates autofluorescence background for each channel in the top panel.
