Abstract
Erythroid Kruppel-like factor (EKLF) is an erythroid-specific transcription factor that binds a CACCC motif found in the human beta-globin gene promoter. We have studied the promoter of the EKLF gene and identified binding sites for the transcription factors GATA-1 and CCAAT-binding Protein 1 (CP1). We show that both types of binding sites are required for full activity, and that the GATA motif at -60 is essential. The EKLF promoter can be directly activated in nonerythroid cells in cotransfection experiments by forced expression of GATA-1. These results suggest that EKLF is dependent on GATA-1 for its expression and lies downstream of, or coincident with, GATA-1 in a regulatory hierarchy in erythroid development.
Highlights
MATERIALSANDMETHODS binding sites for the transcription factors GATA-1 and Cloning of the EIUF Genomic Sequences and Construction of Pro
CCAAT-bindingProtein 1 (CP1).We show that both typesmoter Plasmids-Radiolabeled Erythroid Kruppel-like factor (EKLF) cDNA[1] was usedsctoreen [8]a of binding sites are required for full activity, and that mouse genomiclibrary prepared in A FIX I1 (Stratagene, La Jolla, CA)
We demonstrated that thceomplex can be competed with the CP1 sitefrom the Eagene promoter [6] and that it comigrates with the complex formed on and Doug Engel, was added, where indicated, during the binding reaction
Summary
ATATCGCACA strand), which does not conform to thepreferred GATA-1binding site((T/A)GATA(A/G)() ).To test whether GATA-1. ~ ~ CCCCTC CTTTGCCGTTTT GCTTTG~CTGG G T C G~G G ~ GACAG recognizes this site, an oligonucleotide containing site 3 was used asa cold competitor in the above assay. CCAATCAGAT G ~ GGCAGA CAGGAGCCCTC CAAGAAACTT TCCTAGCCTC show, site 3 competes for binding but toa lesser extent than site 2. Site 3 binds GATA-1, albeit somewhat weakly
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