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.
Sabath
D E
DE
Department of Laboratory Medicine, University of Washington School of Medicine, Seattle 98195, USA. dsabath@u.washington.edu
Koehler
K M
KM
Yang
W Q
WQ
Phan
V
V
Wilson
J
J
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D005914:Globins / Q000235:genetics*; D006801:Humans; D020014:K562 Cells; D008322:Mammals / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D005914:Globins