Boundary Bypass Activity in the Abdominal-B Region of the Drosophila Bithorax Complex is Position Dependent and Regulated

Abstract

Expression of Abdominal-B ( Abd-B ) in abdominal segments A5 - A8 is controlled by four regulatory domains, iab-5 - iab-8 . Each domain has an initiator element (which sets the activity state), elements that maintain this state and tissue-specific enhancers. To ensure their functional autonomy, each domain is bracketed by boundary elements ( Mcp , Fab-7 , Fab-7 and Fab-8 ). In addition to blocking crosstalk between adjacent regulatory domains, the Fab boundaries must also have bypass activity so the relevant regulatory domains can "jump over" intervening boundaries and activate the Abd-B promoter. In the studies reported here we have investigated the parameters governing bypass activity. We find that the bypass elements in the Fab-7 and Fab-8 boundaries must be located in the regulatory domain that is responsible for driving Abd-B expression. We suggest that bypass activity may also be subject to regulation.

Summary statement: Boundaries separating Abd-B regulatory domains block crosstalk between domains and mediate their interactions with Abd-B . The latter function is location but not orientation dependent.

Fig. 1.. A) Map of the Ubx, abd-A and Abd-B regions of the Drosophila melanogaster BX-C.

The Ubx gene is regulated by abx/bx and bxd/pbx domains (marked with yellow and orange) in parasegments PS5 and PS6 respectively (which correspond approximately to segments T3 and A1 in adults). Three regulatory domains, iab-2, iab-3 and iab-4 (shades of blue), control abd-A expression in PS7(A2), PS8(A3), PS4(A4) respectively. Abd-B expression in PS10(A5), PS11(A6), PS12(A7) and PS13(A8) is controlled by iab-5, iab-6, iab-7 and iab-8 (shades of green) respectively. The embryo and adult segments are indicated using the same color code as the iab domain that is required for their specification. The black lines with colored circles mark chromatin boundaries: Fab-1, Fub, Fab-3, Fab-4, Mcp, Fab-6, Fab-7, and Fab-8. The red circles indicate the number of CTCF binding sites in each boundary, and the blue – the number of Pita sites. LBC – as cyan. (B) Deletion of the Fab-7 boundary. When the Fab-7 boundary is deleted, iab-6 and iab-7 fuse into one domain. As a result, PS11/A6 is transformed into a copy of PS12/A7. The Fab-7^attP50 platform, in which four hypersensitive regions, HS*, HS1, HS2, and HS3 (marked with gray boxes) are deleted, is indicated by broken black lines. Maps of the (C) Fab-7 and (D) Fab8 fragments. Maps of fragments that were used for replacements. The Fab-8 fragments are indicated as follows: the bypass element is indicated by the light green line, the insulator element by the dark green line. The coordinates are according to the complete sequence of BX-C in the SEQ89E numbering [71]. The Fab-7 fragments are shown as a light blue line.

Fig. 2.. The Fab-8 bypass element (F8¹⁶⁵) must be located adjacent to iab-6 in order to overcome the blocking activity of the Fab-8 insulator (F8²⁰⁹).

(A) Scheme of the Abd-B regulatory domains and fragments used for Fab-7 replacements. All other designation as in Fig. 1. (B) Morphology of the male abdominal segments (numbered) in wild type (wt) and in F8, F8^R, F8²⁰⁹, F8^165R+F8^209R, F8²⁰⁹+F8¹⁶⁵. Trichomes on the A5 and A6 tergites are shown in dark field. In wt the A6 sternite has a banana shape and is devoid of bristles, while the A5 sternite has a quadrilateral shape and is covered in bristles. The A6 tergite has trichomes along the anterior and ventral edges, while the entire A5 tergite is typically covered in trichomes with small internal patch that lack trichomes. The filled red arrowheads show morphological features indicative of GOF transformations, the empty arrowheads – LOF transformations. (C) Abd-B expression in the CNS of wt and the Fab-7 replacement lines. Each panel shows an image of the CNS of stage 14 embryos stained with antibodies to Abd-B (red). White horizontal bars delimit parasegment boundaries. Parasegments are numbered from 9 to 14 on the right side of the panels; approximate positions of segments are shown on the left side of the wt panel and marked 4 to 8. The wt expression pattern of Abd-B in the embryonic CNS is characterized by a stepwise gradient of increasing protein level from PS10 to PS14. Staining for Engrailed was used to define the boundaries of the parasegments (Fig S1).

Fig. 3.. Testing the ability of F8²⁰⁹ to block the Fab-7 dependent bypass.

(A) Map of the AbdB regulatory region and F8²⁰⁹+F7 fragment used for Fab-7 replacements. All designation as in Fig. 1 (B) Bright field (top) and dark field (bottom) images of cuticles prepared from wt, F8²⁰⁹, F8²⁰⁹+F7, F8²⁰⁹+F7+GE24185, GE24185, where GE24185 is null dCTCF allele in GE24185/GE24185 flies[60]. The empty red arrowheads point to signs of LOF transformations, which are correlated with the loss/lack of bypass functions of the tested DNA fragments. The filled red arrowheads show morphological features indicative of GOF transformations.

Fig. 4.. The gypsy (Su(Hw)) and CTCF^×4 insulators block iab-6 enhancers from regulating Abd-B when placed next to the iab-6 domain.

(A) Map of the Abd-B regulatory region and fragments used for Fab-7 replacements. All designation as in Fig. 1 and 2. gy is shown as 12 orange circles reflecting 12 binding sites for Su(Hw) in insulator from the gypsy transposon. (B) Bright and darkfield images of cuticles prepared from the different replacements: gy, F7+gy, gy+F7, gy+F7 su(Hw)^-, CTCF^×4, F7+CTCF^×4, CTCF^×4+F7 male flies. F7 replacement correspond to wt. (C) Abd-B expression in the CNS of stage 14 embryos. Staining with Engrailed to mark parasegment borders is shown in Fig. S1. All other designations are as in Fig. 1 and 2.

Fig. 5.. Multimerized Pita sites disrupt the bypass activity of Fab-7 dHS1

(A) Schematic presentation of Fab-7 substitutions with different combinations of the Pita^×5 and F7^dHS1. (B) Images of cuticles prepared from Pita^×5, F7^dHS1+Pita^×5 (phenotype similar to wt), Pita^×5+F7^dHS1, and Pita^×5+F7^dHS1+Pita^×5 male flies. (C) Abd-B expression in the CNS of stage 14 embryos. (D) Images of cuticles prepared from wt, , Pita^×5, F7^dHS1+Pita^×5 (phenotype similar to wt), Pita^×5+F7^dHS1, and Pita^×5+F7^dHS1+Pita^×5 male flies.. All designations are as in Fig. 1 and 2.

Fig. 6.. Multimerized Pita sites disrupt Fab-8 bypass activity.

A) Schematic presentation of Fab-7 substitutions. B) Images of cuticles prepared from wt, F8 (phenotype similar to wt), F8+Pita^×5, and Pita^×5+ F8 male flies. All designations are as in Fig. 2 and 3. (C) Abd-B expression in the CNS of stage 14 embryos. (D) Images of cuticles prepared from wt, F8 (phenotype similar to wt), F8+Pita^×5, and Pita^×5+ F8 male flies. All designations are as in Fig. 1 and 2.

Fig. 7.. Model of the functional role of the iab-6 activation in the regulation of distance interaction between the Fab-7 boundary and the Abd-B promoter region.

(A) In wt in A4 segment Abd-B regulatory region is inactive, initiators are repressed and the Fab-boundaries does not interact with the Abd-B promoter region. Activation of the iab-6 domain in PS11/A6 results in the stimulation of the Fab-7 bypass module. The interaction between the Fab-7 boundary and AbdB promoter region facilitates enhancer-promoter communication. (B) An insulator inserted between the iab-6 enhancers and the bypass element (proximal side) blocks interaction with the Abd-B promoter region. (C) The insulator inserted on the distal side of the Fab-7 boundary does not interfere with correct stimulation of the bypass element.