Fig 7. JAK/STAT activates expression of Abd-B through a feedback loop.
(A) Genomic region contained in [CH321-91P18] BAC-Abd-B-gfp and location of the VT42855 enhancer sequence. Asterisks represent 3N (red) and 4N (blue) STAT sites present in the VT42855, green boxes and arrows in the scale line mark the position of the control and experimental oligos used in the ChIP experiment below. (B) Endogenous Abd-B protein expression. (C) GFP-tagged Abd-B expression from the BAC-Abd-B-gfp is indistinguishable in the A8 and A9 abdominal segments from the endogenous protein. (D) Ectopic Abd-Bm expression in the embryonic ectoderm using 69B-Gal4 induces ectopic BAC-Abd-B-gfp activation anterior to A8. (E) In Df(1)os1A embryos lacking JAK/STAT ligands ectopic Abd-Bm expression cannot activate high levels of BAC-Abd-B-gfp anterior to A8. (F) Ectopic Upd expression using 69B-Gal4 induces ectopic BAC-Abd-B-gfp activation. Note in D and F that the expression observed in A8 and A9 is stronger than the ectopic expression in anterior segments. This is likely due to the BAC element containing several A8-A9 enhancers not all of which will respond to the feedback and become activated ectopically. (G) Posterior spiracle gal4 RNA expression (arrowhead) driven by the 2.2kb Abd-B genomic fragment in VT42855-Gal4 line. (H) Posterior spiracle expression in VT42855-Gal4 disappears in Df(1)os1A embryos lacking JAK/STAT ligands. Note that central nervous expression is unaffected. (I-K) VT42855-Gal4/+, UAS-lacZ/+ embryos double stained with anti-βGal (green in I, white in K) and anti-Ct (red in I white in J) to prove the posterior spiracle expression (inset) of VT42855. (L) STAT92E-GFP Chromatin immunoprecipitation in S2 cells shows STAT binding to the VT42855 region of the Abd-B cis regulatory element compared with the negative control region devoid of STAT sites indicated in A (green boxes). Cells were transfected with pAC-Gal4 to activate the expression of UAS constructs, pAC-HopTum-l expressing an activated JAK kinase to induce STAT92E activation by phosphorylation and either with pUAST-STAT92E-GFP or empty pUAST as a control. ChIP of STAT92E was performed using an anti-GFP antibody. The position of oligos used to amplify the VT42855 region is indicated in (A) by an arrow and their sequences indicated in Materials and Methods. (M) The crb518 posterior spiracle enhancer is composed of a specificity module sufficient to drive posterior spiracles expression, a STAT binding module and a repressor module. Early Abd-B expression activates the JAK/STAT Unpaired ligand and other primary posterior spiracle targets, but fails to activate crb518 CRM as a result of the repressor module activity. The JAK/STAT pathway plays a key role in the activation of crb by, first, reinforcing Abd-B expression in A8 through a feedback loop mechanism; second, inducing additional activator factors, which together with ABD-B act over the specificity module and finally by direct binding of STAT92E to crb518 to counteract the repressor element. Arrowheads in G and H indicate the position of the posterior spiracle primordium; arrows in D and F indicate some of the segments that exhibit ectopic activation of the BAC-Abd-B-gfp.