Abstract
Using a Drosophila transgenic system we investigated the ability of GAGA factor, a putative anti-repressor, to modulate transcription-related events in the absence or presence of a bona fide activator, the Adf-1 transcription factor. In contrast to previous in vitro and in vivo data linking the binding of GAGA factor to the acquisition of DNase hypersensitivity at heat shock promoters, we observed that inserting multiple GAGA binding motifs adjacent to a minimal alcohol dehydrogenase (Adh) promoter led to strongly elevated embryonic transcription without creation of a promoter-associated DNase-hypersensitive (DH) site. Establishment of DNase hypersensitivity required the presence of both GAGA and Adf-1 binding sites and was accompanied by a further, synergistic increase in transcription. Because Adf-1 is capable neither of establishing a DH site nor of promoting efficient transcription by itself in embryos, it is likely that DH site formation depends on a GAGA factor-mediated binding of Adf-1 to chromatin, perhaps facilitated by a locally remodeled downstream promoter region. More generally we suggest that GAGA factor-binding sequences may operate in a promoter-specific context, with transcriptional activation, polymerase pausing, and/or DH site formation critically dependent on the nature of the sequences (and their binding partners) linked in cis.
Highlights
Control of eukaryotic transcription is a complex, tightly regulated process requiring the action of many distinct proteins, including chromatin-interacting/modifying proteins, transcriptional activators, and general transcription factors (GTFs
Controlled in vitro experiments showed that GAGA factor had no overt transcription activating ability on naked DNA templates [13, 14] but could relieve the repression of basal transcription at a linked promoter assembled into chromatin when the remodeling factor nucleosome remodeling factor (NURF) was present [12]
With GAGA factor characterized purely as an anti-repressor, we were intrigued to find that the placement of high affinity GAGA-binding sites adjacent to a minimal alcohol dehydrogenase (Adh) TATA promoter was sufficient for robust stimulation of transcription in an in vivo chromatin context (Fig. 2 and Table I)
Summary
Construction of the P Element Constructs and Transgenic Lines—All recombinant DNA procedures were performed using standard techniques [22]; full details can be provided upon request. A minimal derivative of the Drosophila distal Adh promoter (Ϫ41 to ϩ20) driving a luciferase reporter gene was isolated from p-41ADHLUC The su(Hw)-binding element of the gyspy retrotransposon was isolated by SalI digestion of pGBaBx [23] and inserted at the XhoI site of pSK:L to generate pSK:LS. A bacterial lacZ gene flanked by promoter and terminator sequences for T7 RNA polymerase was cloned into the BamHI site of pSK:LS, in a divergent orientation relative to the Adh-luciferase transcription unit, to generate pSK:ZLS. For the GAGA construct, single-stranded synthetic oligonucleotides comprising the proximal GAGA element of Drosophila hsp (Ϫ125 to Ϫ80) (along with appropriate flanking restriction sites) were annealed and inserted between the PstI and SmaI sites of pSK:ZLS, i.e. in the region between the T7 RNA polymerase and Adh promoters, to generate pSK:ZGLS. Luciferase activity for each sample was normalized for protein concentration and adjusted for background luminescence by subtracting the signal obtained from samples derived from the nontransgenic parental line
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