The alcohol dehydrogenase (Adh) gene of D. melanogaster is transcribed from two different promoters during fly development: the distal (adult) and the proximal (embryonic-larval). Certain aspects of Adh gene regulation are represented in Drosophila continuous cell lines. We have used Drosophila tissue culture cells in an in vivo transient expression assay to delimit cis-acting sequences affecting Adh expression, and to investigate the role of chromatin structure in Adh gene regulation. These studies show that positive cis-acting elements of the distal promoter can exist in at least 2 alternative chromatin configurations. There is a close correlation between specific transcriptional activity of the Adh distal promoter and a defined, localized chromatin structural change that indicates altered DNA-protein interactions. Thus, chromatin structure appears to play a role in regulating the accessibility of defined positive cis-acting regulatory sequences of Adh to transcription factors and the transcription machinery.
Chromatin footprinting in Drosophila tissue culture cells has detected the binding of a non-histone protein at +8 of the distal Adh RNA start site, on a 10-bp direct repeat motif abutting a nucleosome positioned over the inactive Adh distal promoter. Alternatively the active promoter is bound by a transcription initiation complex. We have characterized and purified a protein Adf-2 that binds specifically to this direct repeat motif 5'TCTCAGTGCA3', present at +8 and -202 of the distal RNA start site. DNase I footprinting, methylation interference, and UV-crosslinking analyses showed that both direct repeats interact in vitro with a nuclear protein of approximately 120 kilodaltons (kDa). We purified Adf-2 through multiple rounds of sequence-specific DNA affinity chromatography. Southwestern analysis showed that the purified 120 KDa polypeptide binds the Adf-2 motif efficiently as a monomer or homomultimer. In vivo titrations of Adf-2 activity with the Adf-2 motif by transient co-transfection competitions in different Drosophila cell lines suggested that Adf-2 is a cell-specific repressor. Adf-2 has been detected ubiquitously in vitro, but is functional in vivo as a sequence-specific DNA binding protein and repressor only in the cells that have the inactive distal promoter. We discuss the possibility that an activation process is required for Adf-2 protein to bind DNA and function in vivo.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.