Fractionation of a nuclear extract derived from Drosophila tissue culture cells reveals the presence of multiple components involved in accurate transcription of both distal and proximal promoters of the alcohol dehydrogenase (Adh) gene. Transcription of deletion mutants indicates that a region between -24 and -85 upstream of the distal start site contains sequences required for RNA synthesis in vitro. Moreover, sequences that overlap this same upstream control region are specifically bound and protected from DNAase digestion by a promoter-specific transcription factor, Adf-1. Analysis of proximal promoter mutants has identified multiple upstream elements that influence transcription, and DNAase footprint analysis has detected three specific binding regions. Adf-1 binds at least one of these proximal promoter regions but interaction at this site is not specifically required for transcription. These results suggest that multiple sequence-specific DNA binding proteins interact differentially with the proximal and distal promoters of Adh to activate transcription (Heberlein, 1985).
The sibling species Drosophila melanogaster and D. orena show similar patterns of alcohol dehydrogenase expression, both spatially and temporally. These two species diverged from a common ancestor 6 million to 15 million years ago, and the DNA sequences of the promoter regions of their Adh genes show a mosaic pattern of conservation and change. By interspecific transformation of D. orena sequences into D. melanogaster, a functional equivalence between these sequences has been demonstrated. Using both D. melanogaster embryo extracts and purified transcription factor Adf-1, the protection of these promoter sequences from nuclease has been compared, demonstrating considerable conservation (Moses, 1990).
Adh distal factor-1 (Adf-1) is a sequence-specific DNA-binding activity originally identified in Drosophila tissue culture cells and embryos. Adf-1 binds to upstream recognition elements in each of the two promoters of the Drosophila alcohol dehydrogenase gene (Adh), and binding of Adf-1 to the Adh distal promoter site activates transcription. A mutational analysis of the Adh distal promoter has been carried out using both an in vitro transcription assay and a transient transfection assay in Drosophila tissue culture cells, and in both cases deletion of sequences required for Adf-1 binding leads to a 3-4-fold drop in transcription. Purified Adf-1 activates Adh distal promoter transcription in vitro in a binding site-dependent manner. DNase I footprint analysis shows that the purified protein binds not only to the two previously characterized sites in Adh but also to transcriptional regulatory elements in the dopa decarboxylase and Antennapedia P1 promoters. Thus, it appears that Adf-1 may play an important role not only in the regulation of Adh expression but also in the transcription of other Drosophila genes as well (England, 1990).
The distal promoter of Adh is differentially expressed in Drosophila tissue culture cell lines. After transfection with an exogenous Adh gene, there was a specific increase in distal alcohol dehydrogenase (ADH) transcripts in ADH-expressing (ADH+) cells above the levels observed in transfected ADH-nonexpressing (ADH-) cells. Deletion mutations and a comparative transient-expression assay have been used to identify the cis-acting elements responsible for enhanced Adh distal transcription in ADH+ cells. DNA sequences controlling high levels of distal transcription have been localized to a 15-base-pair (bp) region nearly 500 bp upstream of the distal RNA start site. In addition, a 61-bp negative cis-acting element was found upstream from and adjacent to the enhancer. When this silencer element is deleted, distal transcription increases only in the ADH+ cell line. These distant upstream elements must interact with the promoter elements, the Adf-1-binding site and the TATA box, since they only influence transcription when at least one of these two positive distal promoter elements is present. Internal deletions targeted to the Adf-1-binding site or the TATA box reduce transcription in both cell types but do not affect the transcription initiation site. Distal transcription in transfected ADH- cells appears to be controlled primarily through these promoter elements and does not involve the upstream regulatory elements. Evolutionary conservation in distantly related Drosophila species suggests the importance of these upstream elements in correct developmental and tissue-specific expression of ADH (Ayer, 1990).
Sequence-specific transcription factors need to gain access to regulatory sequences in chromatin. Previous studies utilizing model systems have suggested many mechanisms involved in this process. It is unclear however how these findings relate to natural promoters. The Drosophila alcohol dehydrogenase gene distal promoter is organized into an ordered nucleosome array before multiple transcription factors recognize their sites within this nucleosomal context and activate transcription. A purified in vitro system has been used to study the binding of the ubiquitous Drosophila transcription factors Adf-1 and GAGA factor to the Adh distal promoter in chromatin. Several nucleosome core particles are assembled on 150 bp DNA fragments containing the Adh distal cis-acting elements in the natural promoter context but different DNA-histone environments. The Adh distal promoter regulatory sequences can position nucleosomes in the same rotational setting as observed in vivo. In one particular nucleosome position, the wrapping of the Adf-1 and adjacent GAGA factor binding sites around the histone octamer creates a unique local DNA conformation. Therefore high-affinity but non-cooperative nucleosome binding of Adf-1 and GAGA factor occurs, in contrast to the inhibition of Adf-1 and GAGA factor binding in other nucleosome positions. Thus, local histone-DNA sequence contacts giving rise to a specific asymmetric nucleosome structure may play important roles in modulating the affinities of transcription factors for their nucleosomal sites (Gao, 1998).
The Drosophila homeobox gene fushi tarazu is expressed in a highly dynamic striped pattern in early embryos. A key regulatory element that controls the ftz pattern is the ftz proximal enhancer, which mediates positive autoregulation via multiple binding sites for the Ftz protein. In addition, the enhancer is necessary for stripe establishment prior to the onset of autoregulation. Nine binding sites for multiple Drosophila nuclear proteins have been identified in a core 323-bp region of the enhancer. Three of these nine sites interact with the same cohort of nuclear proteins in vitro. The nuclear receptor Ftz-F1 interacts with this repeated module. Additional proteins interacting with this module have been purified from Drosophila nuclear extracts. Peptide sequences of the zinc finger protein Ttk and the transcription factor Adf-1 were obtained. While Ttk is thought to be a repressor of ftz stripes, both Adf-1 and Ftz-F1 activate transcription in a binding site-dependent fashion. These two proteins are expressed ubiquitously at the time ftz is expressed in stripes, suggesting that either may activate striped expression alone or in combination with the Ftz protein. The roles of the nine nuclear factor binding sites were tested in vivo, by site-directed mutagenesis of individual and multiple sites. The three Ftz-F1-Adf-1-Ttk binding sites were found to be functionally redundant and essential for stripe expression in transgenic embryos. Thus, a biochemical analysis identified cis-acting regulatory modules that are required for gene expression in vivo. The finding of repeated binding sites for multiple nuclear proteins underscores the high degree of redundancy built into embryonic gene regulatory networks (Han, 1998).
Using a Drosophila transgenic system an examination was performed of 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, it was observed that inserting multiple GAGA binding motifs adjacent to a minimal alcohol dehydrogenase (Adh) promoter, normally a target of Adf-1, leads to strongly elevated embryonic transcription without creation of a promoter-associated DNase-hypersensitive (DH) site. Establishment of DNase hypersensitivity requires the presence of both GAGA and Adf-1 binding sites and is 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. Furthermore, it is suggested 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 (Pile, 2000).
Mitochondrial biogenesis is a complex and highly regulated process that requires the controlled expression of hundreds of genes encoded in two separated genomes, namely the nuclear and mitochondrial genomes. To identify regulatory proteins involved in the transcriptional control of key nuclear-encoded mitochondrial genes, a detailed analysis was performed of the promoter region of the alpha subunit of the Drosophila F1F0 ATP synthase complex. Using transient transfection assays, a 56 bp cis-acting proximal regulatory region was identified that contains binding sites for the GAGA factor and the alcohol dehydrogenase distal factor 1. In vitro mutagenesis revealed that both sites are functional, and phylogenetic footprinting showed that they are conserved in other Drosophila species and in Anopheles gambiae. The 56 bp region has regulatory enhancer properties and strongly activates heterologous promoters in an orientation-independent manner. In addition, Northern blot and RT-PCR analysis identified two alpha-F1-ATPase mRNAs that differ in the length of the 3' untranslated region due to the selection of alternative polyadenylation sites (Talamillo, 2004).
Many transcriptional activators have been shown to interact with the TAF subunits of the TFIID complex, and this interaction has been correlated with transactivation in several cases. Tests were carried out to discover whether TAFs are important in Adf-1-directed transcription and, if so, whether there is any correlation between activation and potential Adf-1-TAF interactions. An Adf-1-dependent in vitro transcription system was depleted of TFIID by using antibodies against both TAFII250 and TBP. In the absence of an exogenous source of TFIID, this transcription system is inactive. The addition of either purified TFIID or purified recombinant TBP to the depleted transcription system restores basal transcription levels. The addition of purified Adf-1 to these reaction mixtures leads to a high level of activation when TFIID is present but has no effect on the reaction mixture containing only TBP. These results suggest that Adf-1, like many Drosophila activators, requires the presence of TAFs in order to activate transcription in vitro (Cutler, 1998).
To determine if the transcriptional requirement for TAFs is due to direct interactions between one or more of these proteins and Adf-1, in vitro binding experiments were conducted. Beads containing anti-TAFII250 monoclonal antibodies were used to immunoprecipitate TFIID from partially purified TFIID fractions. Silver staining and Western blotting using anti-TAFII80 antibodies has confirmed that an intact holo-TFIID complex is immunoprecipitated. Purified wild-type or mutant Adf-1 protein was incubated with these TFIID beads or with negative-control beads. Anti-Adf-1 antibodies were used to visualize the amounts of bound and input Adf-1 proteins on Western blots. Wild-type Adf-1 binds strongly to beads containing TFIID. In contrast, the two carboxy-terminal transcriptionally inactive mutants, 5A214 and N228, failed to bind the TFIID beads, suggesting that the carboxy terminus of Adf-1 is necessary for TFIID binding (Cutler, 1998).
The Drosophila TFIID complex includes TBP and approximately eight major TAFs. To determine which of these are bound by Adf-1, each of the eight TAFs and TBP were produced by in vitro transcription and translation in the presence of 35S-labeled methionine. The lysates were then incubated with glutathione beads bound to either GST fused to the carboxy terminus of Adf-1 (C92; amino acids 162 to 253) or to GST alone. The C92 fragment was used since the TFIID-binding data suggested that TAF-binding activity may reside in the carboxy terminus of Adf-1. After extensive washing, bound and input TAFs were visualized by autoradiography. Of the nine proteins, only TAFII110 and TAFII250 binds at significant levels to Adf-1. The similar amounts of TAF binding observed with full-length Adf-1 and with proteins having amino-terminal deletions of Adf-1 further confirm that the carboxy terminus of Adf-1 is the major TAF-binding determinant in the protein. Smaller fragments of TAFII110 and hTAFII250 were also tested for their ability to bind Adf-1. The carboxy-terminal 240 amino acids of TAFII110 are sufficient for binding Adf-1, while binding with regions at both the amino and carboxy termini of hTAFII250 is seen. The central portion of hTAFII250 also binds Adf-1, though to a lesser extent. This binding also demonstrates that the carboxy-terminal region of Adf-1 is sufficient for TAF binding (Cutler, 1998).
To verify that the in vitro binding observed reflects events that can occur inside of a cell, the interaction between Adf-1 and TAFII110 and hTAFII250 was tested in S. cerevisiae. Several portions of both TAFs were fused to the GAL4 DBD, and the carboxy terminus of Adf-1 was fused to the GAL4 AAD. These vectors were cotransformed into S. cerevisiae containing GAL4 sites upstream of the gene for beta-galactosidase. The regions of TAFII110 and hTAFII250 that bind Adf-1 in vitro also interact efficiently in this 'in vivo' two-hybrid assay (Cutler, 1998).
Several of the mutations in the carboxy-terminal region of Adf-1 decrease its ability to activate transcription. Some of these carboxy-terminal Adf-1 mutants are also deficient for binding to the holo-TFIID complex. Similar experiments using individual TAFs and other Adf-1 mutants were performed to examine these results in more detail. Paralleling the TFIID-binding results, mutants 5A214 and N228 bind with low efficiency to full-length hTAFII250 and TAFII110 as well as to an amino-terminal fragment of hTAFII250 (amino acids 1 to 414). These results provide a good correlation between TAF binding and transcriptional activation by the carboxy-terminal region of Adf-1. However, because activation by Adf-1 requires sequences outside of the carboxy-terminal region, it is suspected that binding may be necessary but not sufficient to stimulate transcriptional activation by Adf-1. Instead, these results suggest that sequences outside of the TAF interaction region contribute additional activities essential for transactivation (Cutler, 1998).
A distinct DBD is located in the amino-terminal 100 amino acids of Adf-1. This domain contains homologies to the Myb DNA-binding motif and Drosophila protein Stonewall. Adf-1 dimerizes in solution through a domain found near the carboxy terminus. Although dimerization increases the affinity of Adf-1 for DNA, its activity is not essential for binding, as mutants unable to dimerize retain sequence-specific DNA-binding activity. Since most Myb proteins contain two or three tandem repeats of the Myb motif, it is possible that the dimerization by Adf-1 compensates for the presence of only one DNA-binding Myb motif in the protein. Although monomeric forms of Adf-1 can still bind DNA, they do so with a lower affinity. In addition to being required for dimerization, the carboxy terminus of Adf-1 possesses TAF-binding activity. TFIID cannot be replaced by TBP for Adf-1-directed transcriptional activation, consistent with the notion that TAF binding is likely to be functionally important for the activity of Adf-1. All mutations that disrupt Adf-1 dimerization also disrupt TAF binding, while mutants of Adf-1 that are competent for dimerization are also competent for TAF binding. While these data suggest that dimerization may be required for TAF binding, the observation that a carboxy-terminal fragment of Adf-1 containing the dimerization domain is sufficient for TAF binding indicates that dimerization and TAF binding activities are likely due to closely adjacent or overlapping sequences within Adf-1 (Cutler, 1998).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.