Multiple Proteins Binding to a GATA-E Box-GATA Motif Regulate the Erythroid Krüppel-like Factor (EKLF) Gene*
- From the Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, Ohio 45267-0524
Abstract
Erythroid Krüppel-like factor (EKLF) is a zinc finger transcription factor required for β-globin gene expression and is implicated as one of the key factors necessary for the fetal to adult switch in globin gene expression. In an effort to identify factors involved in the expression of this important erythroid-specific regulatory protein, we have isolated the mouse EKLF gene and systematically analyzed the promoter region. Initially, a reporter construct with 1150 base pairs of the EKLF 5′-region was introduced into transgenic mice and shown to direct erythroid-specific expression. We continued the expression studies in erythroid cells and have identified a sequence element consisting of two GATA sites flanking an E box motif. The three sites act in concert to elevate the transcriptional activity of the EKLF promoter. Each site is essential for EKLF expression indicating that the three binding sites do not work additively, but rather function as a unit. We further show that GATA-1 binds to the two GATA sites and present evidence for binding of another factor from erythroid cell nuclear extracts to the E box motif. These results are consistent with the formation of a quaternary complex composed of an E box dimer and two GATA-1 proteins binding at a combined GATA-E box-GATA activator element in the distal EKLF promoter.
Footnotes
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↵* This work was supported by National Institutes of Health Grant DK39585.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) .
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↵‡ To whom correspondence should be addressed: Dept, of Molecular Genetics, Microbiology, and Biochemistry, University of Cincinnati, 231 Bethesda Ave., ML 524, Cincinnati, OH 45267. Tel.: 513-558-5458; Fax: 513-558-1885.
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↵1 The abbreviations used are: EKLF, erythroid Krüppel-like factor; TAL, T-cell acute lymphoblastic leukemia; bHLH, basic helix-loop-helix; Lmo, LIM domain only; CAT, chloramphenicol acetyltransferase; MEL, murine erythroleukemia; EMSA, electrophoretic mobility shift assay; kb, kilobase pair(s); PCR, polymerase chain reaction.
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↵2 K. P. Anderson, unpublished results.
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- Received November 14, 1997.
- Revision received February 23, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
