8288615

pubmed:Citation

2

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.

eng

IM

MEDLINE

0021-9258

Print

269

NLM

1493-500

pubmed-meshheading:8288615-Amino Acid Sequence, pubmed-meshheading:8288615-Base Sequence, pubmed-meshheading:8288615-Binding Sites, pubmed-meshheading:8288615-Computer Graphics, pubmed-meshheading:8288615-DNA-Binding Proteins, pubmed-meshheading:8288615-Early Growth Response Protein 1, pubmed-meshheading:8288615-Globins, pubmed-meshheading:8288615-Humans, pubmed-meshheading:8288615-Hydrogen Bonding, pubmed-meshheading:8288615-Immediate-Early Proteins, pubmed-meshheading:8288615-Kruppel-Like Transcription Factors, pubmed-meshheading:8288615-Models, Molecular, pubmed-meshheading:8288615-Molecular Sequence Data, pubmed-meshheading:8288615-Promoter Regions, Genetic, pubmed-meshheading:8288615-Sequence Alignment, pubmed-meshheading:8288615-Sequence Homology, Amino Acid, pubmed-meshheading:8288615-Transcription Factors, pubmed-meshheading:8288615-Zinc Fingers, pubmed-meshheading:8288615-beta-Thalassemia

Analyses of beta-thalassemia mutant DNA interactions with erythroid Krüppel-like factor (EKLF), an erythroid cell-specific transcription factor.

Journal Article, Comparative Study, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't