Analyses of beta-thalassemia mutant DNA interactions with erythroid Krüppel-like factor (EKLF), an erythroid cell-specific transcription factor.
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.
Feng
W C
WC
Department of Physiology, Mount Sinai School of Medicine, New York, New York 10029.
Southwood
C M
CM
Bieker
J J
JJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003196:Computer Graphics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D051766:Early Growth Response Protein 1; D005914:Globins / Q000235:genetics*; D006801:Humans; D006860:Hydrogen Bonding; D017874:Immediate-Early Proteins; D051741:Kruppel-Like Transcription Factors; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry / Q000502:physiology*; D016335:Zinc Fingers; D017086:beta-Thalassemia / Q000235:genetics*
D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D051741:Kruppel-Like Transcription Factors; D014157:Transcription Factors; C080774:erythroid Kruppel-like factor; D005914:Globins