DNA-PROTEIN INTERACTIONS IN THE PROXIMAL ZETA-GLOBIN PROMOTER - IDENTIFICATION OF NOVEL CCACCC-BINDING AND CCAAT-BINDING PROTEINS

The human zeta-globin gene is expressed in a tissue-and developmental- specific pattern, with expression confined to primitive erythroid cell s of the embryonic yolk sac blood islands. Transgenic mouse studies ha ve shown that the proximal zeta-globin promoter contains sequences tha t contribute to the stage-specificity of expression, but no systematic functional studies of the cis elements in the proximal zeta-globin pr omoter have been reported. In this paper, we show that a number of con served sequence elements in the zeta-globin promoter are important for promoter activity in transiently transfected K562 erythroleukemia cel ls, which constitutively express zeta-globin. These include a GATA sit e at -105, a CCACC site at -93, a CCAAT box at -65, and a TATA box at -29, A highly conserved CCTCC sequence at -78 is not important for zet a-globin promoter activity in this system. Mutations at these sites do not result in increased promoter activity in OCIM1 cells, an erythroi d line that does not express zeta-globin, suggesting none of these sit es is a developmental silencer. Electrophoretic mobility shift assays show that K562 and OCIM1 nuclear extracts contain DNA-binding activiti es that interact with the -105 GATA, -65 CCAAT, and -29 TATA sites. In addition K562 cells, but not OCIM1 cells, have an activity that binds the -93 CCACC site. GATA-1 interacts with the GATA site. The K562 CCA CC-binding protein is distinct from Sp1, Sp2, Sp3, Sp4, EKLF, and BKLF , A specific -65 CCAAT-binding activity is present in K562 and OCIM1 n uclear extracts that is distinct from other CCAAT-binding proteins inc luding CBF/NF-Y, C/EBP, NF-1, and CP2. Thus, we have identified two no vel factors that may contribute to the tissue or developmental stage-s pecific expression of zeta-globin. (C) 1998 Academic Press.