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

C. Y. Yu, K. Motamed, J. Chen, A. D. Bailey, C. K.J. Shen

Research output: Contribution to journalArticle

59 Citations (Scopus)

Abstract

DNA sequences 179 base pairs upstream and 23 base pairs downstream of the cap site of human embryonic ε globin gene exhibit promoter activity in transfected cell cultures. The nuclear factor binding in vitro of this ε promoter region was studied by DNase I footprinting, 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, εF1 at -143, CACC box at -111, and CBF at -81, respectively. Of these, NF-E1 is an erythroid-specific factor. εF1 is probably a ubiquitous factor because it is present in both erythroid K562 cells and nonerythroid HeLa cells. The εF1-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 ε-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 ε globin promoters. Transient expression assay, using human growth hormone gene as the receptor, demonstrated that similar to the other two human β-like globin genes (β and γ), the CACC box of ε globin gene is an essential promoter element for ε 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 β and γ 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.

Original languageEnglish
Pages (from-to)8907-8915
Number of pages9
JournalJournal of Biological Chemistry
Volume266
Issue number14
Publication statusPublished - 1991
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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