Identification in vivo of different rate-limiting steps associated with transcriptional activators in the presence and absence of a GAGA element

The GAGA element in Drosophila melanogaster is a paradigm for a class of DNA elements that can function to establish the transcriptional potential of genes without necessarily leading to gene expression. Another member of this class of elements could be the GC box recognized by Sp1, as Sp1 and its cognate binding site have been implicated in setting up polymerase II (Pol II) on the A20 promoter prior to activation by NF-κB (1). The existence of this class of regulatory elements is predicated on the identification of numerous genes that have Pol II associated with their promoters prior to transcriptional activation (1, 8, 14, 27, 28). Most of these elements remain to be identified, and their contributions to transcription are largely unknown. The impact of the GAGA element on Pol II-promoter interactions is probably best characterized for the heat shock genes in Drosophila. hsp70 is rapidly induced during heat shock by heat shock factor (HSF) (19). Prior to heat shock induction, RNA Pol II initiates transcription but stably pauses 20 to 40 nucleotides further downstream. In vivo protein-DNA cross-linking reveals that the GAGA factor, which binds the GAGA element, associates with the hsp70 promoter prior to heat shock induction (25, 44). Mutation of a GAGA element in hsp70 diminishes the level of paused Pol II (16), but the decrease is likely to be caused by a defect in initiation rather than pausing (18, 32). Biochemical studies indicate that the GAGA factor functions with the chromatin remodeling factor, NURF, to form a nuclease sensitive region over the hsp70 promoter in reconstituted chromatin (35, 36). Although the contribution of the GAGA elements to the nuclease sensitivity of the hsp70 promoter has not been rigorously evaluated in vivo, analyses of the hsp26 heat shock gene in Drosophila tissues reveal that the GAGA elements in this promoter contribute to formation of a nuclease sensitive chromatin structure prior to heat shock induction (17, 20, 21). Collectively, these data indicate that the GAGA element functions prior to heat shock induction to establish an open chromatin structure and to allow transcription to occur, resulting in a paused Pol II. In addition to the heat shock genes, GAGA factor has been implicated in regulation of numerous genes having a variety of cellular functions (39, 42). A genome-wide analysis of the distribution of GAGA factor in Drosophila cells identified approximately 250 potential target genes (38). The function of the GAGA factor at these genes is largely unknown. To learn more about how the GAGA element contributes to transcription, we developed a transgenic system that allowed us to compare the function of different activators on promoters that contain or lack a GAGA element. For the present study, we compared an activator containing amino acids 610 to 691 from HSF to an activator containing three copies of a 13-amino-acid array derived from VP16. Amino acids 610 to 691 from Drosophila HSF fused to the Gal4 DNA binding domain (DBD) had previously been shown to activate transcription of a promoter located on extrachromosomal DNA (43). This information has been used to understand the mechanism of activation by HSF (23, 26, 45), yet it is not clear whether this information has relevance to activation in the context of the GAGA element or a paused Pol II. The three tandem repeats of the N-terminal portion of the VP16 activation domain fused to the tetracycline repressor of Escherichia coli had previously been shown to be a potent activator of a chromosomal copy of gene in human cells (2). We wanted to know whether the GAGA element would have any impact on such a potent activation domain. Each activation domain was fused to a derivative of the DBD from the tetracycline repressor that could be induced to bind DNA upon addition of the tetracycline analog, doxycycline (37). This provided the opportunity to analyze the effect of the GAGA element on promoter activity under steady-state conditions and on the rate of transcriptional activation. The latter was of interest because one function of the GAGA element suggested by the heat shock genes is that it provides a means for rapidly inducing a gene.