Abstract
We describe functional tests and molecular modeling of erythroid Krüppel-like factor (EKLF) interactions with its DNA binding site. EKLF, a zinc finger-containing, erythroid-specific transcription factor, binds and transactivates from the CACCC element, an evolutionarily conserved DNA sequence present within a large number of erythroid-specific promoters and enhancers. This DNA binding element is the site of naturally occurring point mutations that give rise to beta-thalassemia. We have directly tested whether CAC site point mutations (including two of the beta-thalassemia mutants) affect EKLF transactivation and DNA binding function. In vivo analyses demonstrate that EKLF is unable to transactivate a reporter plasmid that contains these mutations. In vitro analyses reveal a 40-100-fold decrease in binding affinity for these sites that accounts for the in vivo observations. The homology between the three EKLF and Zif268 zinc fingers and their conserved sequence-specific contacts to their target site allowed us to formulate a molecular model of the EKLF/CAC site complex, based primarily on energy minimization/refinement of the Zif268/DNA co-crystal structure. These models suggest that both specific and nonspecific hydrogen bonding play a critical role in the ability of EKLF to prefer binding to its cognate site. Analysis of sequence-specific contacts by EKLF to its target site within the beta-globin promoter verified the residues predicted to be important by the functional and modeling data. Together these results demonstrate that EKLF displays a strong discriminatory ability among potential DNA target sites consistent with the beta-thalassemia data. They also suggest that lack of EKLF binding to these sites may play a determining role in its phenotype, and they strengthen the evidence in favor of EKLF's proposed role in erythroid-specific transcriptional activation through the CACCC elements.
Highlights
@-globin promoter verifiethde residues predictedto be important by the functionalandmodeling data. Together these results demonstrate that Erythroid Kfiippel-like factor (EKLF) displays a strong discriminatory ability among potentiDaNl A target sites consistent with the P-thalassemia data
EKLF is proposed to be intimately involved in generation of erythroid-gene specificity, as the CACCC sequence and its derivatives are required for optimal transcription of a large number of erythroid-specific promoters
A gel shift assay wasused to separate the EKLFDNA complex from free DNA after incubation of EKLF with a partially methylated murine P-globin promoter fragment that was radiolabeled on the non-coding strand (*)
Summary
By the manufacturer), precipitated, cleaved with piperidine [47], and resolved on 8% polyacrylamide/ureasequencinggels.Formicacid-. Under theconditions of the assay, an 11-fold excess of wild type CACCC sites was required tocompete the EKLF-CAC shift by 50% (Fig. 2) This value provided the basis against which all other oligonucleotides were compared. Because the detailed analysis of the sequence-specific arginine and histidine contacts with the guanine bases in Zit268 indicated a striking consistency in the molecular geometry in which the guanidinium or imidazole side chain interacts with the guanine bases among all the Zif268 zinc fingers, we modified the XYZ histidinelarginine side-chain dihedral angles of EKLF in order to form Zif268-like contacts Both aspartates in fingers 1 and 3 do not makeany significant base contacts in FIG..
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