Identification and functional characterization of the murine Rac2 gene promoter

Rac2, a member of the Rho family of GTPases, is highly expressed in myeloid cells and is a regulator of the NADPH-oxidase complex. A murine genomic cl one was isolated that contains the 5' end and putative promoter region of t he Rac2 gene. Ribonuclease protection experiments detected 13 transcription initiation sites scattered 50 to 130 bp upstream of the translation initia tion site. Transient transfection studies revealed that -7 kb to +31 bp (re lative to the strongest transcription initiation site) of the Rac2 gene 5'- flanking region exhibited strong promoter activity in both RAW 264.7 macrop hage cells that express the endogenous Rac2 gene and NIH-3T3 fibroblast cel ls that do not express the endogenous gene, Truncated Rac2 promoter fragmen ts containing as little as the -74 to +31 bp sequence produced full transcr iptional activity, However, a -57 to +31 promoter fragment directed signifi cantly less transcription, and a -39 to +31 promoter fragment was transcrip tionally inactive. In vitro binding assays revealed sequence-specific and w idely expressed DNA-binding activities that interacted within the -74 to -5 8 Rac2 promoter cis element. Oligonucleotide competition and antibody disru ption studies indicated that these complexes contained the transcription fa ctors Spl and Sp3. Specific ablation of the Sp1/Sp3 binding site significan tly decreased Rac2 promoter activity in both RAW 264.7 and NIH-3T3 cells. A dditional cis elements may be required to restrict Rac2 promoter activity t o hematopoietic cells expressing the endogenous gene.