EKLF is an erythroid specific transcription factor that is expressed early in erythroid development. It binds and transactivates a sequence element """"""""CACCC"""""""" in the beta-globin promoter. Point mutations in some of these residues, including those known to lead to beta-thalassemia, abolish the transactivating ability of EKLF. Genetic disruption of EKLF in mice causes a severe beta-thalassemia that leads to embryonic lethality. This renewal proposal will investigate the molecular mechanism of action of EKLF by assessing whether phosphorylation regulates EKLF activity and by identifying proteins that interact with EKLF. Further, an evaluation of EKLF mutations in previously undefined beta-thalassemia patients will be performed. Previous studies from the principal investigator's laboratory have determined the DNA binding site of EKLF and have shown that EKLF functions with promoter and cell type specificity. Deletion mutants in the transactivating region of EKLF demonstrate an inhibitory domain and a domain that may be required for interaction with a positive acting factor. The author proposes that one of the CAC binding activities in MEL extracts, CAC-D is EKLF. Treatment of extracts with CIP disrupts binding of this activity as analyzed by gel shift. The CAC-D band could be disrupted with an EKLF antibody. However, gel shifts from EKLF- deficient mice have been used to show at least that CAC-D is EKLF. An inducible, epitope tagged EKLF has been successfully been introduced into MEL cells for purification experiments. Further, a poly-his tagged version of EKLF has been expressed in bacteria for antibody production (data not shown). Portions of EKLF have been expressed in a yeast two hybrid vector. Finally, the human EKLF has been cloned.
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