Characterization and purification ofAdhdistal promoter factor 2, Adf-2, a cell-specific and promoter-specific repressor inDrosophila

Abstract

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.

Nucleotide sequence of the genomic region encompassing Adh and Adh-Dup genes of D. lebanonensis (Scaptodrosophila): Gene expression and evolutionary relationships

“…The alignment of the 5'region flanking the distal promoter of the Adh gene of D. melanogaster and D. lebanonensis is very poor, although some motifs that have been shown to bind transcription factors Benyajati 1990, 1992;Benyajati et al 1992) have been conserved (Fig. 10).…”

“…The positive element DEP1-DEP2 (Ayer and Benyajati 1992) is 95% conserved. The repeats Adf2a and Adf2b which are sites for the binding of Adf2 repressor factor in D. melanogaster Adh-negative cells (Benyajati et al 1992) are partially conserved in D. lebanonensis where Adf2a and Adf2b show 90% and 80% identity, respectively. The sequence from position 601 to 642 only is 46% identical to the Adfl site described in D. melanogaster…”

Expression of Drosophila melanogaster gene encoding transcription factor GAGA is tissue‐specific and temperature‐dependent

“…The GAF gene, present in the Drosophila genome in a single copy [4,5], was shown to generate multiple developmentally regulated transcripts [5]. To date at least two cDNA copies have been identified to encode proteins with different carboxy termini [18], since we used different portions of GAF cDNA as probes generated for Northern blot hybridizations.…”

“…The GAF factor binds to (GA)n-rich sites in promoter regions of several D. melanogaster genes. These include such well-known genes as Ultrabithorax (Ubx) [3], Adh [4], engrailed, fushi tarazu [5], Kruppel [6], heat shock genes (hsp26 [7], hsp70 [8]) and those encoding histones H3 and H4 [9]. The binding of GAF to specific elements leads to chromatin structure remodeling in the promoter region (dynamic disruption of nucleosomes, formation of DNase I-hypersensitive regions) [10], thus the GAF is suggested to allow other transcription factors access to DNA [11] and to help keep on target genes by maintaining a 'open" configuration in the local chromatin [12].…”

A composite intragenic silencer domain exhibits negative and positive transcriptional control of the bone-specific osteocalcin gene: promoter and cell type requirements.

“…Some examples of exonic transcriptional regulatory sequences are positive cis-acting elements in the first exon of housekeeping genes (34,35). Others are negative exonic regulatory elements in genes associated with specialized functions (25,32,36,37). The evolutionary significance of silencers residing within exonic and particularly within protein-coding regions (this report; ref.…”