Analyses of beta-thalassemia mutant DNA interactions with erythroid Krüppel-like factor (EKLF), an erythroid cell-specific transcription factor
| Autor: | W C, Feng, C M, Southwood, J J, Bieker |
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| Rok vydání: | 1994 |
| Předmět: |
Models
Molecular Binding Sites Base Sequence Sequence Homology Amino Acid Molecular Sequence Data beta-Thalassemia Kruppel-Like Transcription Factors Hydrogen Bonding Zinc Fingers Globins Immediate-Early Proteins DNA-Binding Proteins Computer Graphics Humans Amino Acid Sequence Promoter Regions Genetic Sequence Alignment Early Growth Response Protein 1 Transcription Factors |
| Zdroj: | The Journal of biological chemistry. 269(2) |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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