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

Adriana C Garcia 0 Daniel LG Gita Fernanda C Humann Maria L Pa-Larson Nadia Monesi 0 0 Departamento de Analises Clinicas, Toxicologicas e Bromatologicas, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Universidade de Sao Paulo , Ribeirao Preto, SP, Brazil, 14040-903 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. - Background One of the most important events in the control of gene expression is the regulation of transcription initiation. Transcription of eukaryotic protein-coding genes by RNA polymerase II involves a tightly regulated sequence of steps including decondensation of the locus, nucleosome remodeling, histone modifications, binding of transcriptional activators and coactivators to enhancers and promoters that culminates in the recruitment of the basal transcription machinery to the core promoter [1-3]. The basal transcription machinery comprises a set of factors, including RNA polymerase II, that are minimally essential for in vitro transcription from an isolated core promoter, whereas the RNA polymerase II core promoter has been defined as a cis-regulatory module whose function is to direct the initiation of transcription [2]. Recent studies have revealed that the structure and function of core promoters is more diverse than previously thought. The majority of the eukaryotic core promoters are classified as focused core promoters [1,4]. In a focused core promoter transcription starts at a single nucleotide or within a distinct cluster of start sites in a short region of several nucleotides. Studies in vertebrates have also revealed a second type of core promoter, named dispersed core promoters, in which several weak transcription start sites are distributed over a broad region ranging from 50 to 100 nucleotides [1,4]. Even though there are apparently no universal core promoter elements, molecular studies on focused core promoters have identified and characterized sequence motifs that are characteristic of core promoters such as the Inr (Initiator), TATA box, BRE (TFIIB Recognition Element), DPE (Downstream core Promoter Element), MTE (Motif Ten Element) and DCE (Downstream Core Element) [1,2,4]. Central to the transcription initiation process is the binding of TBP [TATA binding protein, a subunit of TFIID (Transcription Factor IID)] to DNA at a specific distance from the transcription start site, regardless of the presence or absence of a TATA box. The core promoter motifs usually work in cooperation and biochemical studies have demonstrated synergism between different pairs of core promoter motifs [2-4]. These studies of core promoter motifs have been further extended by the use of computational approaches [5-7]. Furthermore, core promoter function is not restricted to the binding of general transcription factors. Several studies have shown the existence of specificity between enhancers and core promoters demonstrating that core promoter motifs constitute cisregulatory elements that participate in transcriptional regulation [8-10] and are an important component in transcriptional networks [11]. Focused core promoters have also been identified in the regulatory region of sciarid DNA puff genes [12-16]. In addition to RNA puffs, which are of general occurrence in Diptera, the polytene chromosomes of sciarid flies also present DNA puffs that are formed at discrete sites in the salivary gland polytene chromosomes at the end the last larval instar. The molecular characterization of DNA puffs revealed that they constitute sites of developmentally regulated gene amplification and transcription [17-19]. These three processes, gene amplification, gene expression and DNA puff formation are all induced in the salivary glands as a late response to the increased ecdysone levels that trigger metamorphosis [20,21]. Earlier studies have shown that the BhC4-1 DNA puff gene of the sciarid Bradysia hygida is amplified 21-fold and is abundantly transcribed in the salivary gland when DNA puff C4 is formed at the end of the fourth larval instar [13,22]. The characterization of the mechanisms that regulate BhC4-1 expression have been extended through functional studies performed in transgenic Drosophila, and have resulted in the identification of cisregulatory elements in the BhC4-1 promoter region [23-25]. Similar to events in the sciarid, in transgenic Drosophila BhC4-1 expression in the salivary gland is induced as a late response to the increase in ecdysone levels [20]. In addition, genetic interaction experiments have shown that the BhC4-1 cis-regulatory elements are recognized by trans-activating factors of D. melanogaster, indicating that the regulatory mechanisms of BhC41 transcription in the salivary gland are highly conserved in D. melan (...truncated)