Nucleic Acids Research, 2003, Vol. 31, No. 4 1292-1301
© 2003 Oxford University Press
Characterization of the human ß-globin downstream promoter region
Center for Mammalian Genetics, Powell Gene Therapy Center, Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, 1600 SW Archer Road, PO 100245, Gainesville, FL 32610, USA and 1 Biochemistry and Molecular Biology, Rockefeller University, New York, NY, USA
*To whom correspondence should be addressed. Tel: +1 352 392 0121; Fax: +1 352 392 2953; Email: jbungert{at}college.med.ufl.edu
The human ß-globin gene is abundantly expressed specifically in adult erythroid cells. Stage-specific transcription is regulated principally by promoter proximal cis-regulatory elements. The basal promoter contains a non-canonical TATA-like motif as well as an initiator element. These two elements have been shown to interact with the TFII-D complex. Here we show that in addition to the TATA and initiator elements, conserved E-box motifs are located in the ß-globin downstream promoter. One of the E-box motifs overlaps the initiator and this composite element interacts with USF1 and TFII-I in vitro. Another E-box, located 60 bp 3' to the transcription initiation site, interacts with USF1 and USF2. Mutations of either the initiator or the downstream E-box impair transcription of the ß-globin gene in vitro. Mutations of a putative NF-E2-binding site in the downstream promoter region do not affect transcription in vitro. USF1, USF2, TFII-I and p45 can be crosslinked to a ß-globin promoter fragment in MEL cells in vivo, whereas only TFII-I and USF2 crosslink to the ß-globin gene in K562 cells. The summary data demonstrate that in addition to the well-characterized interactions of the TFII-D complex with the basal promoter, E-box motifs contribute to the efficient formation of transcription complexes on the adult ß-globin gene.
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