© 2000 Oxford University Press Nucleic Acids Research, 2000, Vol. 28, No. 6 1439–1446 The initiator element of the Drosophila β2 tubulin gene core promoter contributes to gene expression in vivo but is not required for male germ-cell specific expression Ansgar Santel*, Jörg Kaufmann 1 , Ruth Hyland and Renate Renkawitz-Pohl Zoologie-Entwicklungsbiologie am Fachbereich Biologie, Philipps-Universität Marburg, Karl-von-Frisch-Straße, D-35032 Marburg, Germany and 1 Chiron Corporation, Chiron Technologies, 4560 Horton Street, Emeryville, CA 94608, USA Received November 8, 1999; Revised and Accepted January 28, 2000 ABSTRACT The tissue-specific expression of the Drosophila β2 tubulin gene (B2t) is accomplished by the action of a 14-bp activator element (β2UE1) in combination with certain regulatory elements of the TATA-less, Inr- containing B2t core promoter. We performed an in vivo analysis of the Inr element function in the B2t core promoter using a transgenic approach. Our experiments demonstrate that the Inr element acts as a functional cis-regulatory element in vivo and quan- titatively regulates tissue-specific reporter expres- sion in transgenic animals. However, our mutational analysis of the Inr element demonstrates no essen- tial role of the Inr in mediating tissue specificity of the B2t promoter. In addition, a downstream element seems to affect promoter activity in combination with the Inr. In summary, our data show for the first time the functionality of the Inr element in an in vivo back- ground situation in Drosophila. INTRODUCTION Tissue-specific gene expression in higher eukaryotes can be accomplished by the recruitment of cell type-specific tran- scription factors (activators) to distinct promoter/enhancer sequences. The binding of specific activators allows for the interaction with the general transcription initiation machinery at the core promoter and leads to transcriptional activation (1– 3). The analysis of core promoter function within transcrip- tional activation and initiation has led to the identification of relevant cis-acting promoter sequences and their trans-acting counterparts (4–6). The core promoter of genes transcribed by RNA-polymerase II is defined as the minimal sequence surrounding the transcription start site that is capable of initi- ating accurate basal transcription in vitro. The best character- ized core promoter elements are the TATA box at the –25 region and the initiator (Inr) sequence (6–8) encompassing the transcriptional initiation site (9). In addition to core promoters containing one or both of these elements, promoters that lack both elements have been identified. Recently, in Drosophila, an additional 7-bp core promoter element was found ~30 bp downstream of the transcription start site in TATA-less, but Inr-containing, core promoters, termed the DPE (downstream promoter element; 10,11). Functional analysis of these core promoter elements revealed their role in recruiting the TFIID complex for transcriptional start site selection by the binding of different components of the multisubunit TFIID complex to each of these elements. While the TATA box is recognized by the TATA box binding protein (TBP; 12), the Inr and the DPE serve as binding sites for certain TAFs (TBP associated factors; 11,13). In particular, the Inr is thought to function either as a TATA box analog in TATA-less core promoters (6) or as an enhancer of TATA box function in TATA box- containing core promoters (14). It has also been shown that the Inr element serves as a selective determinant for directing promoter accessibility and usage, resulting in differential gene expression (e.g. temporal versus spatial gene expression; 15– 18). Additionally, the observation of TAF-mediated direct TFIID recognition of the Inr and sequences located further downstream of the transcription start site (10,17,19–22) stresses the importance of TFIID recruitment to the core promoter as a crucial step for transcription initiation. Activated gene expression is conferred by the core promoter in conjunction with appropriate transcriptional activator element(s). Interestingly, concerning the mechanism of acti- vated gene expression, it has been shown that TFIID (i.e. certain TAFs) plays a fundamental role in mediating activation of transcription (23,24). In addition, efforts to analyze the regulatory activities of core promoter structure in vivo have been undertaken (25–28). However, currently less is known about the role of core promoter function in mediating tissue- dependent promoter specificity in vivo. While intensive studies address the question of promoter specificity due to different core promoter structures in combination with different activa- tors in vitro (29,30), we examined the properties and depend- ence of a distinct core promoter architecture for controlling tissue-specific gene expression using the Drosophila β2 tubulin gene promoter in vivo. *To whom correspondence should be addressed at present address: Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA. Tel: +1 650 725 7625; Fax: +1 650 725 7739; Email: santel@cmgm.stanford.edu