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Transcription through the iab-7 cis-regulatory domain of the bithorax complex interferes with maintenance of Polycomb-mediated silencing

Department of Zoology and Animal Biology, University of Geneva, 30 quai E. Ansermet, 1211 Geneva-4, Switzerland

View larger version (87K): [in a new window]   Fig. 2. Homeotic transformations in promscs and scsprom. Wholemounts of abdominal cuticles (see Materials and Methods for description of the abdominal segments). (A) In homozygous promscs males, the presence of bristles on the sixth sternite (shown by arrows in wild type, left) indicates a homeotic transformation of A6 towards a more anterior segment. On the dorsal side, A5 and A6 tergites have a patchy pigmentation, indicating a transformation into a more anterior abdominal segment. This could reflect a transformation into A4. However, dissection of whole abdomen revealed that they contain only rudimentary gonads. As the somatic part of the gonads is derived from A3 (Bender and Hudson, 2000), rudimentary gonads reflect a transformation towards a more anterior segment. Taken altogether, these homeotic transformations indicate that A3, A4, A5 and A6 are transformed into a more anterior abdominal segment [a mixture of A2 and A3 (2-3)]. (B) A wild-type male has six abdominal segments. The seventh abdominal segment (A7), which is present in larvae, is suppressed during metamorphosis. In Fab-72, iab-7 (left) is ectopically activated in most cells of A6. As a consequence, A6 assumes A7 identity and most of the sixth tergite and sternite are absent. There are, however, cells of A6 in which ectopic activation of iab-7 does not take place. These cells, which are visible as a small tergite (shown by an arrow), adopt A5 identity, indicating that not only iab-7 is inactive, but also iab-6. In Fab-712 homozygotes (right), iab-7 is ectopically expressed in all cells of A6, giving rise to a fly with no apparent tissue in A6 (open arrow). In scsprom homozygotes, A6 is completely transformed into A7, as revealed by the complete absence of tergite or sternite tissue in A6 (open arrow). (C) The derepression of iab-8 in A6 and A7 (Fab-7 and Fab-8 phenotype). The A7 into A8 transformation is detectable in females where A7 develops. scsprom homozygous females show a phenotype reminiscent of an Fab-8 boundary deletion, as revealed by the strong reduction of the seventh tergite. Because in scsprom, the Fab-7 boundary function is also affected, the sixth tergite is also reduced in size.

View larger version (16K): [in a new window]   Fig. 1. Structure of the different P elements used to induce the swapping of Fab-7 by scs. The 3.1 kb fragment (containing the Fab-72 deletion) that serves as ectopic donor in the gene conversion experiment is drawn in dark blue at the scale indicated at the bottom of the figure. The iab-7PRE abutting the Fab-7 boundary is indicated. This Fab-7 fragment is inserted in front of a miniwhite gene within a P-element (the feet of the P element are indicated by black rectangles; the miniwhite gene in red is not drawn at scale). The structure of the SCS element that was inserted in the NsiI site just upstream from the Fab72 deletion endpoint is shown in green above the Fab-7 DNA line with a few relevant restriction sites (drawn at the same scale; B, BamH1; H, HpaI: M, MluI; P, PstI). The promoter driving transcription under the control of the iab cis- regulatory domains from within the BX-C is indicated in dark green. The promoter described by Avramova and Thikonov (Avramova and Thikonov, 1999) is shown in light green. The percentage of pairing sensitive lines from each construct is indicated with the numbers of lines scored (only homozygous viable lines are reported).

View larger version (19K): [in a new window]   Fig. 5. Summaries of the effects caused by the replacement of Fab-7 by promscs or scsprom. (A) The thin horizontal line represents the genomic DNA of the abdominal region of the BX-C marked off in kb according to Karch et al. (Karch et al., 1985). The structures of the abd-A and Abd-B transcription units are shown. The cis-regulatory interaction between the iab domains and their respective target promoters are shown by loops. While iab-2, iab-3 and iab-4 regulate abd-A in PS7, PS8 and PS9 respectively, iab-5, iab-6, iab-7 and iab-8 regulate Abd-B expression in PS10, PS11, PS12 and PS13. The red vertical bar represents the scsmin insulator that prevents Abd-B activation by iab-5 and iab-6. Upon isolation from Abd-B, iab-5 is able to interact with abd-A (Hogga et al., 2001). In promscs, the promoter on the left of the insulator would be activated by iab-3, iab-4 and iab-5, diverting these regulatory domains from the abd-A promoter. This results in the loss-of-function phenotype of iab-3, iab-4 and iab-5 (loss-of-function of iab-6 is due to the inability of iab-6 to regulate Abd-B properly because of the intervening scsmin insulator) (Hogga et al., 2001). (B) The posterior abdomen of a larva from A4/PS9 to A8/PS13 is shown on the left. On the right, the activity states of iab-4 to iab-8 cis-regulatory domains are shown in their respective segments/parasegments. For example, in A4/PS9, iab-4 is active (red), whereas the remaining iab-5,6,7 and iab-8 are kept inactive by the Pc-G-repressing complex (shown in black). In A5/PS10 the next adjacent cis-regulatory domain iab-5 is activated. In A6/PS11, iab-6 becomes active. As the scsmin insulator (red bar) does not insulate iab-6 fully from Abd-B, we envision that iab-6 activates the scs promoter across the insulator in A6/PS11, leading to transcription through iab-7 and iab-8 (shown by the thick arrow). As transcription would interfere with Pc-G complex silencing, ectopic activation of iab-7 and iab-8 occurs in A6/PS11, giving rise to the Fab-7/8 homeotic transformations.

View larger version (90K): [in a new window]   Fig. 3. Abd-B expression patterns in the central nervous system of homozygous scsmin, wild type and scsprom embryos. Central nervous systems (CNS) were dissected out from embryos stained with an antibody directed against ABDB as described elsewhere (Hogga et al., 2001). In wild type, the typical graded ABDB expression pattern from PS10 to PS14 is visible. In scsmin CNS, a partial block between iab-5/iab-6 and Abd-B is visible by the weaker signal in PS10 and PS11. In scsprom, the same impediment appears in PS10. Note however, that in PS11 and PS12, the ABDB expression patterns are similar (unlike in scsmin). The PS11 and PS12 levels and patterns of ABDB expression are not a reiteration of the PS12-specific pattern in wild type (as it would be expected in a Fab-7 embryo). They reach a level intermediate between the PS10 and PS11 from wild type. The regulatory output of the fused iab-6-iab-7 domain may be shared between the scsprom and Abd-B promoters (see text).

View larger version (51K): [in a new window]   Fig. 4. Transcripts arising from the scs promoter in the BX-C. Organization of the genomic DNA around Fab-7 in Fab-72 (left), promscs (middle) and scsprom (right) flies. The promoter active in promscs and scsprom is drawn. The A and B strand-specific probes are shown in red: probe A, bases 84,732 to 86,947 in the BX-C sequence (Martin et al., 1995); probe B, 82,554-84,732. The expression pattern of the transcripts in wild type (or Fab-72), promscs and scsprom embryos at 4 and 12 hours of development are shown below. Parasegments are indicated. In scsprom, we detected a similar expression pattern with a probe spanning the iab-8PRE (not shown; from 59,446 to 62,117). It should be noticed that the intensity of rightwards transcription is higher from scsprom than leftwards transcription originating from promscs. Although the signal usually appears after 1 hour of incubation with the alkaline substrates, with probe B, we had to wait more than twice as long as we did with probe A.