Inserting the Ftz homeodomain into engrailed creates a dominant transcriptional repressor that specifically turns off Ftz target genes in vivo

Abstract

The Engrailed homeodomain protein is an 'active' or dominant transcriptional repressor in cultured cells. In contrast, the Fushi Tarazu homeodomain protein is an activator, both in cultured cells and in Drosophila embryos, where it activates several known target genes, including its own gene. This auto-activation has been shown to depend on targeting to a fushi tarazu enhancer by the Fushi Tarazu homeodomain. We combined Fushi Tarazu targeting and Engrailed active repression in a chimeric regulator, EFE. When EFE is ubiquitously expressed, it overrides endogenous Fushi Tarazu and causes a fushi tarazu mutant phenotype. Normal Fushi Tarazu target genes are affected as they are in fushi tarazu mutants. One such target gene is repressed by EFE even where Fushi Tarazu is not expressed, suggesting that the repression is active. This is confirmed by showing that the in vivo activity of EFE depends on a domain that is required for active repression in culture. A derivative that lacks this domain, while it cannot repress the endogenous fushi tarazu gene, can still reduce the activity of the fushi tarazu autoregulatory enhancer, suggesting that it competes with endogenous Fushi Tarazu for binding sites in vivo. However, this passive repression is much less effective than active repression.

Fig. 1

(A–D) Cuticular pattern deletions caused by EFE and its parental proteins. Cuticle patterns in (A) wild-type embryos, or 20 hours after transient induction of either (B) Ftz, (C) EFE, or (D) En from heat-inducible transgenes. Embryos were subjected to a 15 minute heat pulse at 37°C (see Materials and Methods) beginning 2.5 hours after the end of a 30 minute egg collection period. Ftz caused deletion of the even-numbered abdominal denticle bands, as well as the first and third thoracic bands, while EFE-induced deletions were essentially complementary. The positions of the A1 (arrow) and A8 (arrowhead) denticle bands are indicated. The EFE pattern is very similar to that of ftz mutants (Wakimoto et al. 1984). EFE pair-rule embryos retain ventral pits posterior to the T3 denticle band but usually lose the Keilin’s organs, which lie just posterior, suggesting that the register of the deletions is very similar, if not identical, to that of ftz mutants (Struhl, 1985). More than 70% of EFE embryos had preferential deletions of ftz-dependent parts of the ventral denticle bands, including the following categories: pair-rule deletions of ftz-dependent bands, 20–30%; deletions of a subset of the ftz-dependent bands, 20–30%; and deletions of all of the ftz-dependent bands plus some additional bands, 15–30%. 10–20% of these cuticles showed normal patterns, most likely due to embryos that were partially developed at the time of egg laying, and therefore escaped the effects of EFE (see part E). In contrast, En caused extensive disorganization of repeating pattern, including polarity reversals. All embryos in all figures are oriented ventral down and anterior to the left. (E) Ectopically expressed EFE causes defects within a time window similar to that of Ftz, but different from that of En. Hatching rates were determined (after 40 hours) following a 15 minute heat pulse beginning at the indicated time after the end of a 30 minute egg collection. The indicated protein was ectopically expressed from a heat-inducible transgene. At least 3 independently heat-shocked plates with at least 100 eggs each were counted (both unhatched eggs and empty egg casings) for each data point. The graph indicates the average and range of the data obtained. Normal embryos hatch after about 24 hours.

Fig. 2

ftz gene expression is repressed strongly by EFE, but not by either parental protein. The pattern of ftz RNA expression was monitored in embryos at two times after induction (as in Fig. 1) of (A) no transgene, (B) hs-Ftz, (C) hs-EFE or (D) hs-En. Embryos were fixed either 10 minutes (column 1) or 40 minutes (column 2) after induction, and stained by in situ hybridization (as described in Materials and Methods) using a probe to ftz RNA sequences outside the HD. The apparent background staining in B, column 1, is due at least in part to cross-reaction of the probe with ftz RNA sequences expressed from the Ftz transgene.

Fig. 3

EFE represses both even- and odd-numbered en stripes. The patterns of en RNA were detected (as in Fig. 2, except using an en probe) in embryos 40 minutes following induction (as in Fig. 1) of either (A) no transgene, (B) hs-Ftz, (C) hs-EFE, or (D) hs-En. Ubiquitous expression from both the En- and EFE-producing transgenes was observed at earlier times after induction, with RNA from the En transgene persisting somewhat longer (not shown). Note that both the even- and odd-numbered en stripes are repressed by EFE.

Fig. 4

eve expression is repressed by ectopic En, but not by EFE. The patterns of eve RNA were detected (as in Fig. 2, except using an eve probe) in embryos 20 minutes after heat induction (as in Fig. 1) of either (A) no transgene, (B) hs-Ftz, (C) hs-EFE, or (D) hs-En. Ftz and EFE had little or no effect. En repressed all eve stripes, with stripes 5, 6, and 1 being least affected. Thus En repression activity is re-targeted away from the eve gene by the HD swap.

Fig. 5

The effect of ectopic EFE on wg expression is distinct from that of En and Ftz. The patterns of wg RNA were detected (as in Fig. 2, except using a wg probe) in embryos 40 minutes after induction (as in Fig. 1) of either (A) no transgene, (B) hs-Ftz, (C) hs-EFE, or (D) hs-En. wg is repressed by Ftz (in odd-numbered parasegments) and by En, while it is strongly de-repressed by EFE in the Ftz-dependent (even-numbered) parasegments, as it is in ftz-mutant embryos.

Fig. 6

Repression of ftz requires an active repression domain from En. In situ hybridization to ftz RNA in EFEΔ embryos was carried out as in Fig. 2 following a 30 minute heat shock beginning between 2.5 and 3 hours of development (30 minute collections). Embryos were fixed either (A) 10 minutes or (B) 40 minutes after the end of heat shock. EFEΔ embryos showed a slight repression initially (compare to Fig. 2A), followed by full recovery. (C) EFEΔ is innocuous in vivo. Hatching rates were determined for the wild-type recipient strain, and for transgenic derivatives carrying either an EFE- or an EFEΔ -producing transgene either without heat shock, or following a 15 minute heat shock beginning 2.5 hours after the end of a 30 minute egg collection.

Fig. 7

(A–D) Both EFE and EFEΔ are localized to nuclei, and EFEΔ is more stable. Embryos were heat shocked for 15 minutes to induce transgene expression, then fixed and stained with α-En (affinity purified using the N-terminal 150 aa of En, present in both EFE and EFEΔ ) and with alkaline phosphatase (AP) coupled 2° antibodies. AP activity was first quantified using the soluble-product-producing substrate PNP (Manoukian and Krause, 1992) (results shown in E), then the embryos were stained as in Fig. 2 (final step). (A) EFE expression 10 minutes after induction, (B) EFEΔ expression at the same time, (C) staining of EFE transformants 55 minutes after induction, leaving only a trace of endogenous en stripes (consistent with the RNA analysis of Fig. 3C), (D) EFEΔ staining 55 minutes after induction. In spite of its persistence, EFEΔ is unable to repress ftz expression or disrupt normal embryogenesis. (E) EFEΔ is expressed at the same level as EFE, and is more stable. α -En antibody staining was quantified as described in A-D. Both proteins were initially expressed at equal levels, but EFEΔ was more stable. A background value (amounting to 7% at the first time point and 10% at the second and third) was subtracted from the numbers plotted based on parallel staining of control embryos.

Fig. 8

EFEΔ can compete for Ftz-binding sites in cultured cells, but cannot actively repress transcription. Drosophila S2 cells were co-transfected with a CAT (chloramphenicol acetyltransferase) reporter plasmid, which contains binding sites for both the glucocorticoid receptor (GR) and the Ftz HD, separated by 40 bp, upstream of a basal promoter, and a plasmid that expresses either Ftz or GR (see Materials and Methods for details). Each of the latter activate reporter expression 50- to 100-fold above the basal level. The ability of either EFE or EFEΔ to repress this activated transcription was determined by co-transfection of an appropriate expression plasmid. The non-repressed level was determined by co-transfection of the same quantity of ‘empty’ parental expression plasmid, which is a P-element transformation vector (see Materials and Methods). CAT activity was determined and normalized to the activity of a co-transfected reference gene (see Materials and Methods for details). The graph represents the average and range of at least 4 independent transfections with 2 different plasmid preparations, in at least 2 separate experiments.

Fig. 9

(A,B) EFEΔ inhibits the activity of a ftz enhancer in vivo. Embryos carrying a ftz-lacZ transgene, and either (A) lacking or (B) containing the EFEΔ transgene, were double-stained for lacZ RNA (black), as in Fig. 2 except using a lacZ probe, and endogenous Ftz protein (brown), using an α-Ftz monoclonal antibody (kindly provided by Ian Duncan) that does not bind the Ftz HD and therefore does not cross-react with EFEΔ. Embryos were fixed 30 minutes after a 30 minute heat shock beginning between 2.5 and 3 hours of development (30 minute collections). Although the ftz-lacZ transgene gives a range of staining intensities in different embryos, there was a clear shift in the entire range due to repression by EFEΔ, most clearly discernible as a lack of embryos with strong, mostly complete stripes like that shown in A. (C) Quantitation of ftz enhancer activity. Embryos were probed for lacZ RNA expressed from the ftz enhancer, or, as a control, from an eve enhancer (8.0 eve-lacZ of Harding et al., 1989), as in A and B (fixed either 15 or 30 minutes after heat shock, as indicated), and the signal was quantified using a soluble chromogenic substrate, as described in Materials and Methods. A background from non-lacZ RNA-producing embryos processed in parallel was subtracted from each of the values presented. The graph represents the average and range of two separate experiments for the heat-shock-induced values. For the non-heat-shocked values, 2 sets of embryos were collected for each transgenic line at each of the two stages used for the heat-shocked values, and processed in parallel; the values shown are the average and range of these 4 determinations.