Functional characterization of the sciarid BhC4-1 core promoter in transgenic Drosophila

Abstract

Background: Core promoters are cis-regulatory modules to which bind the basal transcriptional machinery and which participate in the regulation of transcription initiation. Although core promoters have not been extensively investigated through functional assays in a chromosomal context, the available data suggested that the response of a given core promoter might vary depending on the promoter context. Previous studies suggest that a (-57/+40) fragment constitutes the core promoter of the BhC4-1 gene which is located in DNA puff C4 of the sciarid fly Bradysia hygida. Here we tested this (-57/+40) fragment in distinct regulatory contexts in order to verify if promoter context affects its core promoter activity.

Results: Consistent with the activity of a core promoter, we showed that in the absence of upstream regulatory sequences the (-57/+40) fragment drives low levels of reporter gene mRNA expression throughout development in transgenic Drosophila. By assaying the (-57/+40) fragment in two distinct regulatory contexts, either downstream of the previously characterized Fbp1 enhancer or downstream of the UAS element, we showed that the BhC4-1 core promoter drives regulated transcription in both the germline and in various tissues throughout development. Furthermore, the use of the BhC4-1 core promoter in a UAS construct significantly reduced salivary gland ectopic expression in third instar larvae, which was previously described to occur in the context of the GAL4/UAS system.

Conclusions: Our results from functional analysis in transgenic Drosophila show that the BhC4-1 core promoter drives gene expression regardless of the promoter context that was assayed. New insights into the functioning of the GAL4/UAS system in Drosophila were obtained, indicating that the presence of the SV40 sequence in the 3' UTR of a UAS construct does not preclude expression in the germline. Furthermore, our analysis indicated that ectopic salivary gland expression in the GAL4/UAS system does not depend only on sequences present in the GAL4 construct, but can also be affected by the core promoter sequences in the UAS construct. In this context, we propose that the sciarid BhC4-1 core promoter constitutes a valuable core promoter which can be employed in functional assays in insects.

Figure 1

The BhC4-1 core promoter and diagrams of the analyzed constructs. The sequence of the 97 bp (-57/+40) BhC4-1 core promoter region [GenBank:U13892] is shown in (A). The TATA motif and the initiator element (Inr) are underlined and in italic, respectively. The experimentally defined transcription start site (+1) is the first T (underlined, italic) in the Inr element [13]. The sequences similar to Motif 8 and Motif 10 (MTE) [7,29] are shown in bold and bold/italic, respectively. (B) The upper diagram shows the core promoter elements identified in the BhC4-1 core promoter. The numbers below the diagram correspond to the position of each element, defined in relation to the experimentally mapped transcription start site (+1) [13]. The lower diagram, drawn for comparison, consists of a hypothetical insect core promoter containing a TATA motif, and Inr and an MTE. The numbers below the diagram are defined in relation to the transcription start site (+1), and indicate the position in which these elements are more commonly found in insect core promoters. The core promoter elements drawn in the lower diagram consist of the subset of core promoter elements that have been identified. A particular core promoter might contain some, all or none of these elements [2,4]. The motif 8 element (Motif 8 E) is not shown in the bottom diagram because its preferential location in insect core promoters has not been defined. (C) Diagrams of the transgenes analyzed in this work. The arrow above the BhC4-1 core promoter diagram indicates the transcription start site and the direction of transcription.

Figure 2

BhC4-1-lacZ mRNA expression in a (-57/+40) transgenic line. Thirty micrograms of total RNA extracted from embryos (E), first and second instar larvae (L1,2), third instar larvae (L3), prepupae at 0 h, 3 h, 6 h, 9 h and 12 h, 24 h pupae, adult males (M) and adult females (F) were analyzed by the Ribonuclease Protection Assay using a mixture of two radiolabeled probes. The protected BhC4-1-lacZ RNA mRNA is 617 nt long (BhC4-1-lacZ arrow). The protected Actin mRNA is about 260 nt long (Actin arrow). In lane (y,w), 30 μg of total RNA extracted from embryos of the parental strain y,w were hybridized with both probes. In lanes (3 h prepupae/lacZ) and (3 h prepupae/Actin), 30 μg of total RNA extracted from 3 h prepupae of the (-57/+40) transgenic line were hybridized either with the lacZ probe or the Actin probe, as indicated. The migration of the RNA size markers is indicated on the right.

Figure 3

β-galactosidase expression in the fat body of larvae and prepupae of the Fbp1-97bp transgenic line. (A) 96 h third instar larvae, (B) 100 h third instar larvae and (C) 0 h prepupae. Reporter gene expression was detected after 1 h incubation in the presence of X-gal. The arrows indicate the trachea (t), proventriculus (p), imaginal discs (id), salivary glands (sg), brain (b), ventral ganglion (g), midintestine (mi), fat body (fb) and ring gland (rg), respectively. The blue staining detected in the imaginal discs (id) and in the midintestine (mi) corresponds to endogenous β-galactosidase activity [56].

Figure 4

Patterns of GFP expression driven by different GAL4 lines. (A) UAS-97 bp/GAL4-twi.2xPE 3-4 h old embryo; (B) whole ovary dissected from an UAS-97 bp; GAL4::VP16-nos.UTR female. The arrows indicate the germarium; (C) whole ovary from an UAS-97 bp female, note the absence of GFP expression at the tip of the ovary where the germarium is located (arrows); (D) dissected ovariole from an UAS-97 bp; GAL4::VP16-nos.UTR female; (E) dissected UAS-97 bp; Lsp2-GAL4 third instar larvae showing GFP expression in the larval fat body; (F, G) dissected fat body and associated organs from an UAS-97 bp; Lsp2-GAL4 early prepupae, note that GFP expression is only verified in the fat body; (H) dissected UAS-97 bp/GAL4-GMR larva, the inset shows a dissected antenna-eye imaginal disc from a larva of the same genotype, note that GFP expression occurs only in the eye imaginal disc; (I) dissected UAS-GFP.S65T/GAL4-GMR larva showing GFP expression both in the eye disc and in the salivary gland; (J) dissected UAS-97 bp; pPTGAL26 larva, note the presence of a low frequency of GFP positive cells in the salivary glands; (K) dissected UAS-GFP.S65T; pPTGAL26 larva, note GFP expression in the salivary glands. (A) confocal microscopy; (A') bright field image of the same embryo as shown in (A), (B, C, E, F, G, H, I, J, K) fluorescence stereoscopy; (D) overlay between confocal microscopy and bright field. (rg) ring gland, (b) brain, (fb) fat body, (mi) midintestine, (sg) salivary gland, (gc) gastric caeca, (ed) eye imaginal disc, (as) anterior spiracle, (p) proventriculus, (g) ventral ganglion.