Isolation of cDNAs encoding the Drosophila GAGA transcription factor.

Abstract

To investigate the mechanisms involved in expression of the Drosophila melanogaster engrailed gene, we purified GAGA protein, one of several putative transcriptional activator proteins that binds to the proximal region of the engrailed promoter. Antibodies raised against GAGA protein were used to demonstrate that the protein is present in all nuclei of young embryos. We isolated cDNA clones encoding GAGA protein in which a putative 519-codon open reading frame contains general sequence motifs characteristic of other transcription factors. These include stretches of polyglutamine, a 60-amino-acid region with 18 (30%o) lysine or arginine residues, and a single putative zinc finger motif. In addition, a 120-residue N-terminal region shares significant sequence homology with several other known Drosophila transcription factors, including those encoded by Broad Complex and tramtrack. Up to 35-fold GAGA protein-dependent stimulation of transcription in Schneider line 2 tissue culture cells was observed after transfection of GAGA protein-encoding sequences. The GAGA gene is present in one copy in the Drosophila genome, at cytological location 70EF, and it encodes RNAs which vary in size between 2.4 and 4.4 kb.Many of the gene products that organize the Drosophila melanogaster embryo are expressed in restricted domains, and their accurate deployment is critical to normal development. Among these is engrailed, a homeodomain-containing protein that is expressed in a subset of the ectoderm and neuroectoderm. In gastrulating embryos, cells containing engrailed protein are in 15 concentric, single-cell-wide rings (15,19,29), and the presence of functional engrailed protein in these cells and in the posterior compartments that are populated by their descendants contributes to processes that form and maintain the segments and compartments.Genetic analysis has implicated the pair-rule segmentation genes even-skipped (eve) and fushi tarazu (ftz) in activating engrailed transcription in the gastrulating embryo (reviewed by Lawrence [31]). Genes that have been implicated in its subsequent regulation include engrailed itself (16), Polycomb (9,39), and wingless (3,16,38). It is not known whether the action of the protein products of any of these genes on engrailed regulation is direct, although logic might suggest that eve and ftz proteins, both of which are homeodomain-containing transcription factors, directly activate engrailed transcription in the gastrulating embryo. However, there is little direct evidence to suggest that these or other transcription factors act directly on the engrailed promoter. In vitro studies have identified sequences in the engrailed 5' upstream region that bind eve and ftz proteins (23), and engrailed is activated in a pattern that corresponds precisely with the anterior borders of the eve and ftz stripes (32). However, functional studies of the engrailed promoter failed to identify the eve and ftz binding sites in the engrailed upstream region as important sequences for regulation (10), and the respons...

Identification of engrailed promoter elements essential for interactions with a stripe enhancer in Drosophila embryos

“…1b). A footprinting analysis indicated that they are binding sites for non-RNA-polymerase II proteins, most probably GAGA factors involved in transcriptional activation in cultured cells (Soeller et al 1988(Soeller et al , 1993; there is no evidence for GAGA factor binding to the fourth CTCTC at À36/±32. The ®rst three CTCTCs are surrounded or associated with CX (X an arbitrary base) or GX repeats, and these repeats represent about 50% of the sequence upstream of RNA starts of D-168 (for the en sequence, see Kassis et al 1989).…”

ATP-dependent nucleosome disruption at a heat-shock promoter mediated by binding of GAGA transcription factor

“…and C.w., manuscript in preparation). POlyclonal antibodies against GAGA were prepared in rabbits from protein expressed in E. cofi 10 and purified to >95% homogeneity on heparin-Sepharose CL6B and by preparative SDS-PAGE. Preimmune and immune sera (5 111 -100 +1 +100…”

Biological functions of the ISWI chromatin remodeling complex NURF

“…Four genes we show to be dependent on nurf301 for expression-Ubx, en, hsp26, and hsp70-all contain multiple binding sites for the GAGA factor, which is genetically required for their correct expression (Soeller et al 1993;Farkas et al 1994;Bhat et al 1996). On the Drosophila hsp70 and hsp26 promoters (GA.CT) n cognate elements to which the GAGA factor binds are required for HSF-binding.…”