Characterization and purification of Adh distal promoter factor 2, Adf-2, a cell-specific and promoter-specific...

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.