CHARACTERIZATION OF THE MOLECULARLY CLONED MURINE ALPHA-GLOBIN TRANSCRIPTION FACTOR CP2

We recently cloned human and murine cDNAs that encode CP2, a transcrip tion factor that interacts with the murine alpha-globin promoter. In t his report, we exploited our ability to express CP2 in bacteria and eu karyotic cells to further investigate factor activities in vitro and i n vivo. CP2 expressed in bacteria was significantly enriched and used in a series of DNase I footprinting and electrophoretic gel shift assa ys. The results suggest that CP2 binds a hyphenated recognition sequen ce motif that spans one DNA helix turn. In addition, the enriched bact erial protein activated transcription of alpha-globin promoter templat es approximately 3- to 4-fold in vitro. We then tested the effect of e levating CP2 levels 2.5- to 5.5-fold in vivo using both transient and stable transformation assays. When a reporter construct comprised of t he intact murine alpha-globin promoter driving the bacterial chloramph enicol acetyltransferase (CAT) gene was introduced into these overexpr essing cells, we observed a 3- to 6-fold increase in CAT activity when compared to cells expressing normal levels of CP2. These results defi ne the CP2 factor binding site in more detail and help characterize th e activities of the factor in vivo.