Decreased transcriptional activity of promoter activity of promoter fragments containing mutated PU.1 and/or Sp1 sites. (A) Diagramatic representation of reporter gene constructs of the murine MR 5′ flanking region. The wild-type (WT) MR construct depicting the four PU.1 sites (P177, P164, P32, and P22) as well as the single Sp1 site and the initiator element (Inr) overlapping the transcription start site at +1 (large arrow) are presented. The translation start site (ATG) at +100 and the fused luciferase reporter gene (luc) are also indicated. (B) The mutations introduced at the various transcription factor sites are portrayed above the wild-type sequence in the diagrams of each construct. (C) Constructs containing 300 bp of upstream promoter sequence fused to a luciferase reporter gene were developed in which either of the PU.1 sites at −177 or −164, respectively, were mutated or the single Sp1 site at −104 was mutated (see Fig 2). In the P177 (6 bp) and the P164 (6 bp) constructs, 6 bp of the PU.1 consensus were mutated (GAGGAA to CCATGG), whereas in the P164 (2 bp) construct, 2 bp of the PU.1 consensus were mutated (GAGGAA to GACTAA). In the Sp1 (2 bp) construct, 2 bp of the consensus sequence were mutated (CCGCCC to CCGAGC). In the P177/P164/Sp1 (6 bp) construct, 6 bp of the PU.1 consensus were mutated (GAGGAA to CCTGG) and 2 bp of the Sp1 consensus were mutated (GC to TA). The constructs were transiently transfected by electroporation and the luciferase activity was determined as described in the Materials and Methods.