DNA-protein interactions in the proximal zeta-globin promoter: identification of novel CCACCC- and CCAAT-binding proteins.

The human zeta-globin gene is expressed in a tissue- and developmental-specific pattern, with expression confined to primitive erythroid cells of the embryonic yolk sac blood islands. Transgenic mouse studies have shown that the proximal zeta-globin promoter contains sequences that contribute to the stage-specificity of expression, but no systematic functional studies of the cis elements in the proximal zeta-globin promoter have been reported. In this paper, we show that a number of conserved sequence elements in the zeta-globin promoter are important for promoter activity in transiently transfected K562 erythroleukemia cells, which constitutively express zeta-globin. These include a GATA site 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 zeta-globin promoter activity in this system. Mutations at these sites do not result in increased promoter activity in OCIM1 cells, an erythroid line that does not express zeta-globin, suggesting none of these sites is a developmental silencer. Electrophoretic mobility shift assays show that K562 and OCIM1 nuclear extracts contain DNA-binding activities 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 CCACC-binding protein is distinct from Sp1, Sp2, Sp3, Sp4, EKLF, and BKLF. A specific -65 CCAAT-binding activity is present in K562 and OCIM1 nuclear extracts that is distinct from other CCAAT-binding proteins including CBF/NF-Y, C/EBP, NF-1, and CP2. Thus, we have identified two novel factors that may contribute to the tissue or developmental stage-specific expression of zeta-globin.