INTERACTION BETWEEN PU.1 AND ANOTHER ETS FAMILY TRANSCRIPTION FACTOR PROMOTES MACROPHAGE-SPECIFIC BASAL TRANSCRIPTION INITIATION

Numerous macrophage-restricted promoters lack TATA boxes or other conv entional initiation motifs but contain high affinity binding sites (PU boxes) for the macrophage-restricted Ets family transcription factor PU.1, In RAW264 murine macrophages, multimerized PU boxes were not act ive as enhancers when placed upstream of a minimal promoter. To model their role in basal promoters, we inserted PU boxes into a promoterles s luciferase reporter plasmid, Two sites, regardless of orientation, w ere necessary and sufficient to direct reporter gene expression in tra nsient transfections of the RAW264 macrophage-like cell line, This act ivity was absent in transfected 3T3 fibroblasts but could be induced b y PU.1 coexpression. Both the model promoter and the macrophage-specif ic mouse and human c-fms promoters were activated in RAW264 cells by o ther Ets family transcription factors, Ets-2 and Elf-1. In fibroblasts , the effects of PU.1 and Ets-a were multiplicative, whereas overexpre ssion of PU.1 in RAW264 cells reduced activation of c-fms or model pro moters by the other Ets factors, The PU.1 and Ets-a binding sites of t he mouse c-fms promoter have been located by DNase footprinting. A con served Ets-like motif at the transcription site, CAG-GAAC, that bound only weakly to PU.1, was identified as an additional critical basal c- fms promoter element, Comparison of studies on the model promoter, c-f ms and other myeloid promoters provides evidence for a conserved mecha nism that involves three separate and functionally distinct Ets-like m otifs.