LexA chimeras reveal the function of Drosophila Fos as a context-dependent transcriptional activator

Abstract

The transcriptional activation potential of proteins can be assayed in chimeras containing a heterologous DNA-binding domain that mediates their recruitment to reporter genes. This approach has been widely used in yeast and in transient mammalian cell assays. Here, we applied it to assay the transactivation potential of proteins in transgenic Drosophila embryos. We found that a chimera between the DNA-binding bacterial LexA protein and the transactivation domain from yeast GAL4 behaved as a potent synthetic activator in all embryonic tissues. In contrast, a LexA chimera containing Drosophila Fos (Dfos) required an unexpected degree of context to function as a transcriptional activator. We provide evidence to suggest that this context is provided by Djun and Mad (a Drosophila Smad), and that these partner factors need to be activated by signaling from Jun N-terminal kinase and decapentaplegic, respectively. Because Dfos behaves as an autonomous transcriptional activator in more artificial assays systems, our data suggest that context-dependence of transcription factors may be more prevalent than previously thought.

Figure 1

Maps and sequences of constructs and of the signal-responsive module of the Ubx midgut enhancer. (A) Layout of the LexA chimeras. The LexA coding sequence (white) is flanked by translation initiation and nuclear translocation signals (left-hand black box) and flexible linker (right-hand black box). Dark gray top line, GAL4 activation domain (GAD); dark gray underneath, DNA-binding and dimerization domain of Dfos. (B) Sequences in the LL and MadL reporters; four tandem copies were inserted upstream of a canonical TATA box. The LexA binding site is underlined. The context sequence in MadL is derived from the Dpp/Ras responsive element in the Ubx midgut enhancer (bracketed in C). (C) The signal responsive module of the Ubx midgut enhancer contains a Wingless response sequence (binding site for TCF) and an adjacent bipartite Dpp/Ras response sequence (CRE, Dpp/Ras target; tandem Mad binding sites, Dpp target). Note that the Mad binding sites are also the target for a Wingless induced repressor (see text).

Figure 2

LexGAD is a potent autonomous activator in transgenic embryos. Side views of ≈14-h-old embryos, bearing lacZ reporter and expressing various LexA chimeras, stained with α-lacZ antibody. (A) LL reporter only. (B) LL with ubiquitous LexGAD; LexGAD activates LL throughout the embryo. (C) MadL reporter only. (D) MadL with mesodermal LexGAD; LexGAD cannot activate MadL in regions of the visceral mesoderm that do not experience Dpp stimulation (open triangles). (E) LL with mesodermal LexGAD; LexGAD activates LL throughout the visceral mesoderm (arrows). (F) MadL with mesodermal LexGAD and Dpp; in the presence of Dpp, LexGAD activates MadL throughout the mesoderm (arrows). Anterior to the left, dorsal up in all panels.

Figure 3

LexFos is a context-dependent transcriptional activator. Side views of ≈14-h-old embryos bearing MadL, stained with α-lacZ antibody (A and B, surface views; C–F, focused on midgut). (A) MadL reporter only. (B) MadL with ubiquitous LexFos; note lacZ staining in the dorsal leading edge cells (arrows) because of LexFos. (C) MadL only; endodermal background staining is indicated by arrowhead. (D) MadL with ubiquitous LexFos; the background staining because of MadL is suppressed by LexFos (▵). (E) MadL with ubiquitous Jun*. (F) MadL with ubiquitous LexFos and Jun*; Jun* potently synergizes with LexFos in various embryonic tissues.

Figure 4

LexFos synergizes with JNK and Dpp signaling. Side views of ≈14-h-old embryos bearing MadL, stained with α-lacZ antibody. (A) MadL with mesodermal Drac*. (B) MadL with mesodermal LexFos and Drac*; Drac* potently synergizes with LexFos in various embryonic tissues. (C) MadL with ubiquitous Dpp. (D) MadL with ubiquitous LexFos and Dpp; Dpp synergizes with LexFos in the anterior endoderm (arrows; lacZ staining in the salivary glands, arrowheads in C and D depends on Dpp, but not on LexFos).