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J. Biol. Chem., Vol. 266, Issue 14, 8907-8915, 05, 1991

The CACC box upstream of human embryonic epsilon globin gene binds Sp1 and is a functional promoter element in vitro and in vivo

CY Yu, K Motamed, J Chen, AD Bailey and CK Shen
Department of Genetics, University of California, Davis 95616.

DNA sequences 179 base pairs upstream and 23 base pairs downstream of the cap site of human embryonic epsilon globin gene exhibit promoter activity in transfected cell cultures. The nuclear factor binding in vitro of this epsilon promoter region was studied by DNase I foot- printing, methylation interference, and gel mobility shift assay. Four major nuclear factor-binding sites are detected in complexes formed with unfractionated nuclear extracts: NF-E1 at -163, epsilon F1 at - 143, CACC box at -111, and CBF at -81, respectively. Of these, NF-E1 is an erythroid-specific factor. epsilon F1 is probably a ubiquitous factor because it is present in both erythroid K562 cells and nonerythroid HeLa cells. The epsilon F1-binding site exhibits sequence similarity to that of the cAMP response element-binding protein family of transcription factors. Finally, the CCAAT box-binding protein (CBF)- binding site centers around the CCAAT promoter box. Comparative binding studies with unfractionated nuclear extracts and affinity purified HeLa Sp1 demonstrated that the epsilon-globin CACC box at -111 is a binding site of Sp1. The spatial arrangements of the NF-E1-binding site, the CACC box, and CCAAT box, with respect to their mutual separations by approximately integral numbers of helical turns, is well conserved in all mammalian embryonic epsilon globin promoters. Transient expression assay, using human growth hormone gene as the receptor, demonstrated that similar to the other two human beta-like globin genes (beta and gamma), the CACC box of epsilon globin gene is an essential promoter element for epsilon globin gene expression in vivo in K562 cells. This CACC promoter box also functions in vitro in nuclear extracts prepared from K562 cells. These data, together with Sp1-binding studies of the human beta and gamma globin CACC boxes, suggest that the general transcription factor Sp1, through its differential interactions with different forms of CACC promoter boxes, is an essential component of the machinery that controls the developmental program of mammalian globin gene regulation.
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