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Genomic context modulates insulator activity through promoter competition

Department of Cellular Biology, University of Georgia, Athens, GA 30602, USA

View larger version (9K): [in a new window]   Fig. 1. Two mechanisms that specify enhancer and promoter interaction in transgenic Drosophila. Schematic diagram of transcriptional interactions between AE1 and IAB5 enhancers and promoters in transgenic Drosophila. Transgenes are shown to contain AE1 or IAB5 (yellow rectangle) between divergently pointed promoter-reporter fusion genes (red and blue boxes). Direction of transcription and the position of start site are indicated by the arrows. Activating interactions between promoter and the enhancers are represented by arched arrows. Core promoter elements of eve and white are indicated underneath each promoter. The size and distance of the DNA elements are not to scale. (A) AE1 and IAB5 preferentially activate transcription from the evenskipped (eve) promoter which contains TATA, but not from the white promoter which contain initiator (INI) and DPE, but not TATA sequences. (B) AE1 and IAB5 can activate the white gene if the TATA region in the eve promoter is changed to the corresponding region from the white promoter. (C) AE1-eve interaction is completely blocked by the suHw insulator (red oval), with a redirection of transcription activation to the white promoter.

View larger version (80K): [in a new window]   Fig. 2. The ftz-AE1 interaction overcomes the suHw insulator. Transgenic Drosophila embryos containing the AE1 enhancer between divergently transcribed promoter-reporter fusion genes were hybridized with antisense probes for reporter genes. The transgene is diagrammed below each pair of embryos with the reporter gene directly below the embryo showing the corresponding expression. Orientation and location of the promoters in the diagrams are indicated by arrows. The size and distance of the DNA elements are not to scale. Germband extension stage embryos are shown anterior to the left and dorsal up. (A,B) AE1 specifically activates ftz/CAT expression (A, CAT expression) but not the Scr/lacZ expression (B, lacZ expression). (C,D) AE1-ftz/CAT interaction overcomes the intervening the suHw insulator (C, CAT expression) and no AE1 specific expression is detected from the Scr/lacZ fusion gene (D, lacZ expression). The inset in each panel shows the endogenous Scr expression at or near the same stage as an internal control of the in situ staining (see Results, and Materials and Methods for details).

View larger version (41K): [in a new window]   Fig. 3. Competing promoters can influence the suHw-mediated blockage of the ftz-AE1 interaction. Reporter gene expression are monitored in transgenic embryos containing the AE1 enhancer placed between two competing promoter-reporter fusion genes. Transgenes are diagrammed beneath each image pair (see Fig. 2 legend for general descriptions). (A-D) Transgenic embryos containing ftz-white promoter/reporters. (A,B) AE1 specifically activates ftz/lacZ expression (B, anti-lacZ probe) but not the white gene expression (A, white expression) in transgenic embryos. (C,D) AE1-ftz/lacZ interaction is partially attenuated by the intervening suHw insulator (D, lacZ expression) without concomitant restoration of AE1-white interaction (C, white expression). (E-H) Transgenic embryos containing ftz-eve promoter/reporters. (E,F) AE1 simultaneously activates ftz/lacZ (F, lacZ expression) and the eve/CAT expression (E, CAT expression) in transgenic embryos. (G,H) AE1-ftz/lacZ interaction can be completely blocked by the intervening suHw insulator (H, lacZ expression) with a concomitant increase in the level of AE1-eve/CAT interaction (G, CAT expression). The inset in each panel shows the internal control Scr expression (see Fig. 2 legend, and Materials and Methods for details).

View larger version (79K): [in a new window]   Fig. 4. Modulation of the suHw function by the regulatory context is independent of interacting promoters. Transgenic embryos containing AE1 between divergently transcribed eve-Scr promoter/reporter genes are hybridized with anti-reporter probes (A-D, see Fig. 2 legend for general descriptions). (A,B) AE1 activates eve/CAT expression (a, CAT expression) but not the Scr/lacZ expression (B, lacZ expression) in transgenic embryos. (C,D) AE1-eve/CAT interaction can only be partially blocked by the intervening suHw insulator (C, CAT expression) without concomitant restoration of AE1-Scr/lacZ interaction (D, lacZ expression). The insets show the internal control Scr expression (see Fig. 2 legend, and Methods for details).

View larger version (77K): [in a new window]   Fig. 5. AE1 specificity between ftz and Scr may be determined by promoter competition. AE1 can activate Scr/reporter expression in transgenic embryos containing divergently transcribed white-Scr promoter/reporter pairs (A-D, see Fig. 2 legend for general descriptions). AE1 activates white (A, white expression) but not the Scr/lacZ expression (B, lacZ expression). AE1-white interaction can be completely blocked by the intervening suHw insulator (C, white expression) with a moderate concomitant increase in the level of AE1-Scr interaction (D, lacZ expression). The insets show the internal control Scr expression (see Fig. 2 legend, and Materials and Methods for details).

View larger version (15K): [in a new window]   Fig. 6. Models for regulatory interdependence among closely linked genes. Regulatory interdependence ‘relays’ along neighboring genes and imposes organizational rigidity to closely linked genes or regions of chromosomes. Three closely linked neighboring genes (P2-P4) with their regulatory enhancers (E2-E3) are diagrammed. The specificity between the enhancers and their cognate promoters depends on the neighboring regulatory elements (E, enhancers; P, promoters; In, insulators), owing to the sharing and competing nature of the interactions between these elements (arrows). Changes in the relative positioning or regulatory capacity of any one component element, e.g. change of P4, a non-competitive promoter for E2 to P1, a highly competitive promoter for E2 (see purple arrow), will influence the regulatory outcome of neighboring interactions and in turn interactions further away thereby linking the entire genomic region into one regulatory, organizational and evolutionary unit.

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