Characterization and purification of Adh distal promoter factor 2, Adf-2, a cell-specific and promoter-specific repressor in Drosophila.
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.
Benyajati
C
C
Department of Biology, University of Rochester, NY 14627.
Ewel
A
A
McKeon
J
J
Chovav
M
M
Juan
E
E
D000426:Alcohol Dehydrogenase / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012097:Repressor Proteins / Q000302:isolation & purification / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D000426:Alcohol Dehydrogenase