CHARACTERIZATION OF A MYELOID-SPECIFIC ENHANCER OF THE CHICKEN LYSOZYME GENE - MAJOR ROLE FOR AN ETS TRANSCRIPTION FACTOR-BINDING SITE

Expression of the lysozyme gene in chicken macro phages is regulated b y an enhancer located 2.7 kilobases upstream of the transcription star t site. The organization of this enhancer was analyzed by in vitro ass ays (DNase I footprinting, dimethyl sulfate methylation protection, an d band shift assays) and in vivo footprinting experiments. The results show that the enhancer contains four regions (I-IV) of protein-DNA in teractions. First, transient gene transfer experiments demonstrate tha t region II contributes most to the enhancer function. This region con tains a recognition site for the macrophage- and B cell-specific trans cription factor PU.1, a member of the Ets transcription factor family. This site plays a major role in the enhancer since a mutation of this site abolishes enhancer activity. Second, in competition band shift e xperiments, we show that the only myeloid-specific complexes in region s I and II with nuclear factors are formed on the PU.1 recognition sit e by a member of the Ets transcription factor family. Third in in vivo footprinting experiments, the only sign of a protein-DNA interaction is a dimethyl sulfate-hyperreactive guanine 3'-adjacent to the PU.1 re cognition site.