ANALYSES OF BETA-THALASSEMIA MUTANT-DNA INTERACTIONS WITH ERYTHROID KRUPPEL-LIKE FACTOR (EKLF), AN ERYTHROID CELL-SPECIFIC TRANSCRIPTION FACTOR

被引:0
作者
FENG, WC
SOUTHWOOD, CM
BIEKER, JJ
机构
[1] CUNY MT SINAI SCH MED,BROOKDALE CTR MOLEC BIOL,BOX 1126,1 GUSTAVE L LEVY PL,NEW YORK,NY 10029
[2] CUNY MT SINAI SCH MED,DEPT PHYSIOL & BIOPHYS,NEW YORK,NY 10029
[3] CUNY MT SINAI SCH MED,DEPT BIOCHEM,NEW YORK,NY 10029
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中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We describe functional tests and molecular modeling of erythroid Kruppel-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 sequenee 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 contaets 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 EKLFs proposed role in erythroid-specific transcriptional activation through the CACCC elements.
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页码:1493 / 1500
页数:8
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