TRANSCRIPTIONAL REGULATION OF HEMOGLOBIN SWITCHING

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Hemoglobinopathies (such as sickle cell disease) and thalassemias are defects of red blood cell function that are manifested as moderate to life-threatening anemias. Reactivation of fetal globin provides a therapeutic benefit to these adult patients by compensating for absent beta-globin chains (in beta-thalassemia) or by interfering with polymerization or mutant hemoglobin(in sickle cell disease). Such a course is naturally found under conditions of hereditary persistence of fetal hemoglobin (HPFH) or by pharmacological intervention, whereby fetal hemoglobins are expressed at abnormally high levels in the audit. Studies on the control of beta-like globin synthesis have successfully focused on deciphering the mechanisms by which each gene within the cluster is maximally expressed. One of the critically important transcriptional activators is Erythroid Kruppel Factor (EKLF). EKLF is a zinc finger protein that binds to the adult beta-globin CACCC element and is required for preferentially establishing high levels of beta-globin expression. Genetic ablation of EKLF results in a profound beta- thalassemia and embryonic death at the time of the genetic switch to adult globin synthesis. Molecular data indicates that EKLF does not bind well to the fetal gamma-globin CAC site due to a subtle difference compared to the beta globin CAC site) in the portion of the sequence that is predicted to interact with finger-1 of EKLF. As a result, the question remains as to the identity of the protein(s) that occupies the fetal CACCC element. Apart from its intrinsic relevance to illuminating mechanistic details of globin developmental regulation, identification of the relevant protein raises the possibility that it can be used to transcriptionally reactivate the embryonic and/or fetal globin gene within the adult erythroid cell. Ameliorating the life-threatening effects of sickle cell disease and beta- thalassemias provides a considerable clinical rationale for pursuing not just this goal, but also for examining a way to utilize already available reagents to achieve the same end. This proposal addresses these issues by the following specific aims: 1. A modified EKLF protein will be designed such that it will recognize, with high affinity, the gamma-globin CAC site by mutagenesis of residues with EKLF finger-1 whose changes will correctly changes the target specificity; 2) The gamma-globin CAC-site binding protein will be identified by isolation of EKLF-like finger proteins from sources that are expressing the embryonic/fetal globin genes. The end results of these aims will be to make available a transcriptional reagent that will be tested for its ability to reactivate the fetal globin gene within the adult erythroid environment. These studies will be aided immensely by the services of the Hematopoiesis Core Facility and collaborations with the other co-investigators in the Center.